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https://openalex.org/W2967233312
https://aguassubterraneas.abas.org/asubterraneas/article/download/29583/19113
Portuguese
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Química ambiental: monitoramento físico-químico da água de um poço artesiano na cidade de Remígio-PB
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 de dados e estudos de interesse local, sem caráter científico. Sendo assim, a Revista Águas Subterrâneas não se responsabiliza pelo conteúdo publicado. Estudos de Caso e Notas Técnicas Estudos de Caso e Notas 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. Estudos de Caso e Notas Técnicas Estudos de Caso e Notas Técnicas Estudos de Caso e Notas Estudos de Caso e Notas 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 Técnicas Disclaimer: Articles published in this section are not peer-reviewed and are not indexed.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 Therefore, Revista Águas Subterrâneas is not r Disclaimer: Articles published in this section are not peer-reviewed and are not indexed.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. 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https://openalex.org/W4368240950
https://www.mechanics-industry.org/10.1051/meca/2023003/pdf
English
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Static reinforcement and vibration reduction of structures using topology optimization
Mechanics & industry
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cc-by
11,052
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: ˆ 2140 elements such that xe > 0.99 ˆ 6420 elements such that xe < 0.01 ˆ 80 elements such that 0.01 < xe < 0.99 In the final shape for solution ˜xd, there are: ˆ 2140 elements such that xe > 0.99 ˆ 6400 elements such that xe < 0.01 ˆ 100 elements such that 0.01 < xe < 0.99 ˆ 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. ˆ 7064 elements such that xe < 0.01 ˆ 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. ˆ 2368 elements such that xe > 0.99 ˆ 7072 elements such that xe < 0.01 ˆ 64 elements such that 0.01 < xe < 0.99 ˆ 2368 elements such that xe > 0.99 ˆ 7072 elements such that xe < 0.01 ˆ 64 elements such that 0.01 < xe < 0.99 ˆ 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. 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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. 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Effect of finasteride on lipid profile in individuals with androgenetic alopecia
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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
Energies
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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
IFIP advances in information and communication technology
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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 destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 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. 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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. 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Team leadership assessment after advanced life support courses comparing real teams vs. simulated teams
Frontiers in psychology
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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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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. 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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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Comparative phylotranscriptomics reveals ancestral and derived root nodule symbiosis programmes
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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 https://hal.inrae.fr/hal-04187925v1 Submitted on 25 Aug 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 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 6 6 100 200 200 100 100 200 100 200 20 40 Density 0 500 0 500 0 0.05 0.10 0.15 1,000 1,500 0 0.05 0.10 0.15 0.20 1,000 1,500 2,000 40 Length of SignalP containing proteins 0 500 1,000 1,500 2,000 0 0.01 0.02 0.03 0.04 Density 0 0.1 0.2 0.3 0.4 0 0.05 0.10 0.15 0.20 0.25 40 50 Density 0 500 0 500 1,000 1,500 1,000 1,500 2,000 0 5 10 15 0 5 10 15 0 5 6 10 15 0 10 20 30 0 10 20 30 0 10 20 30 0 5 10 15 0 10 20 30 40 50 Length of SignalP containing proteins 0 500 1,000 1,500 2,000 0 0.0025 0.0050 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 0.05 0.10 0.15 0 5 10 15 Cysteine proportion Cysteine proportion Medtru FIId a 20 Density 2,000 20 Length of SignalP containing proteins 6 100 100 200 0 500 0 0.05 0.10 0.15 1,000 1,500 Length of SignalP containing proteins 0 500 1,000 1,500 2,000 0 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 0.05 0.10 0.15 0.20 1,000 1,500 2,000 40 0 10 20 30 0 10 20 30 a Proline proportion Density Density Cysteine proportion Length of SignalP containing proteins 20 2,000 6 100 100 200 20 0 500 1,000 1,500 0 5 10 15 0 5 6 10 15 Length of SignalP containing proteins 0 500 1,000 1,500 2,000 0 0.0025 0.0050 0.0075 Length distribution of SignalP containing proteins of M. pudica 100 200 Length of SignalP containing proteins 0 500 1,000 1,500 2,000 0 0.0025 0.0050 0.0075 Density Length 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. 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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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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. 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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. 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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. 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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 License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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Plant Peroxisomes: A Factory of Reactive Species
Frontiers in plant science
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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. 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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). 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An exploration of changes in plantar pressure distributions during walking with standalone and supported lateral wedge insole designs
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. 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Correlates of weapon carrying among high school students in the United States
Annals of general psychiatry
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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. 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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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The Use of Social Media to Combat Research-Isolation
Annals of the Entomological Society of America
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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- stricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 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. 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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.
https://openalex.org/W2940315859
https://europepmc.org/articles/pmc6510322?pdf=render
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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. 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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
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English
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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
https://www.qeios.com/read/2800Z4/pdf
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RASSF1 wt Allele
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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
https://openalex.org/W2280323653
https://europepmc.org/articles/pmc4766703?pdf=render
English
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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
BMC cancer
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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. 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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 and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit p y y p 34. 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https://openalex.org/W4391688517
https://link.springer.com/content/pdf/10.1007/s40520-023-02645-y.pdf
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Analysis of intestinal flora and cognitive function in maintenance hemodialysis patients using combined 16S ribosome DNA and shotgun metagenome sequencing
Aging Clinical and Experimental Research
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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=​biosa​mple_​sra&​from_​uid=​29625​227 under accession code BioProject PRJNA860849. The shotgun metagenomic sequencing data can be found at [https://​www.​ncbi.​nlm.​nih.​ gov/​biopr​oject/​PRJNA​849628 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-​ proje​ct.​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. 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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/​circr​esaha.​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 Testing the Influence of Self-Efficacy and Demo- graphic Characteristics among International Students on Entrepre- neurial Intention in the Context of Hungary (Wu et al. 2022) Sustainability; Basel ProQuest Entrepreneurial Intention of Chinese Students Studying at Universities in the Community of Madrid (Lin et al. 2022) Sustainability; Basel ProQuest Entrepreneurship or Employment? A Survey of College Students’ Sustainable Entrepre- neurial Intentions (Zhu et al. 2022) Sustainability; Basel ProQuest Drivers of Student Entre- preneurial Intention and the Moderating Role of Entrepreneurship Educa- tion: Evidence from an Indian University (Akhtar et al. Appendix 2022) Discrete Dynamics in Nature and Society ProQuest STUDIES IN HIGHER EDUCA- TION Taylor and Francis International Journal of Research in Business and Social Science ProQuest Eskisehir Osmangazi Universitesi Sosyal Bilimler Dergisi ProQuest Sustainability; Basel ProQuest Determinants of Somali Student’s Entrepreneur- ial Intentions: The Case Study of University Students in Mogadishu 1 (Alin and Dil 2022) Testing the Influence of Self-Efficacy and Demo- graphic Characteristics among International Students on Entrepre- neurial Intention in the Context of Hungary (Wu et al. 2022) 1 3 Factors affecting students’ entrepreneurial intentions:… 1931 Title of the paper Name of authors Name of Journals Database Sustainability at Universi- ties as a Determinant of Entrepreneurship for Sustainability (Fanea-Ivanovici and Baber 2022) Sustainability; Basel ProQuest Impact of attitude towards entrepreneurship educa- tion and role models on entrepreneurial intention (Amofah and Saladrigues 2022) Journal of Innovation and Entre- preneurship ProQuest People-Centered Entrepreneurship: The Impact of Empathy and Social Entrepreneurial Self-efficacy for Social Entrepreneurial Inten- tion (Kim 2022) Global Business & Finance Review ProQuest The Impact of Personal Values and Attitude toward Sustainable Entrepreneurship on Entrepreneurial Inten- tion to Enhance Sustain- able Development: Empirical Evidence from Pakistan (Yasir et al. 2022) Sustainability; Basel ProQuest Entrepreneurship and Sustainable Develop- ment Goals: A Multi- group Analysis of the Moderating Effects of Entrepreneurship Educa- tion on Entrepreneurial Intention (Ashari et al. 2022) Sustainability; Basel ProQuest Entrepreneurial Intention of Students (Managers in Training): Personal and Family Character- istics (Dragin et al. 2022) Sustainability; Basel ProQuest The Role of Psycho- logical Factors on Entrepreneurial Inten- tions among Business Students (Qudus et al. 2022) Journal of Behavioural Sciences ProQuest Sustainability orientation and sustainable entre- preneurship intention: the mediating role of entrepreneurial opportu- nity recognition (Bapoo et al. 2022) Academy of Entrepreneurship Journal ProQuest Technopreneurial Inten- tions: The Effect of Innate Innovativeness (Salhieh and Al-Abdallat 2022) Sustainability; Basel ProQuest 3 1932 G. Maheshwari et al. Title of the paper Name of authors Name of Journals Database “Is Old Gold?” the Role of Prior Experience in Exploring the Determi- nants of Islamic Social Entrepreneurial Inten- tions: Evidence from Bangladesh (Ashraf 2021) Journal of Social Entrepreneurship Scopus Bridging the gap in real estate enterprise: the impact of mentoring on entrepreneurial intentions of real estate students in Nigeria (Ayodele et al. Appendix 2021) Property Management Scopus Determinants of entre- preneurial intention in Mexican university stu- dents [Determinantes de la intención emprende- dora en estudiantes uni- versitarios mexicanos] (Virginia Guadalupe et al. 2021) Revista de Ciencias Sociales Scopus Does the Support System Mediate the Relation- ship between University Roles and Entrepreneur- ial Intentions among University Students? (Shamsudin et al. 2021) Journal of Information Technology Management Scopus Effect of entrepreneurship education on entrepre- neurial intention among university students (Abdullahi et al. 2021) Journal of Technical Education and Training Scopus Effect of gender role identity on the entre- preneurial intention of university students (Datta et al. 2021) Journal of Small Business and Entrepreneurship Scopus Entrepreneurial ecosys- tem, entrepreneurial self-efficacy, and entre- preneurial intention in higher education: Evidence from Saudi Arabia (Elnadi and Gheith 2021) International Journal of Manage- ment Education Scopus Entrepreneurial Self- Efficacy Mediates the Impact of the Post-pan- demic Entrepreneurship Environment on College Students’ Entrepreneur- ial Intention (Zhang and Huang 2021) Frontiers in Psychology Scopus Determinants of entre- preneurial intention in Mexican university stu- dents [Determinantes de la intención emprende- dora en estudiantes uni- versitarios mexicanos] Effect of entrepreneurship education on entrepre- neurial intention among university students (Abdullahi et al. 2021) Effect of gender role identity on the entre- preneurial intention of university students (Datta et al. 2021) 1 3 Factors affecting students’ entrepreneurial intentions:… Factors affecting students’ entrepreneurial intentions:… 1933 Title of the paper Name of authors Name of Journals Database Entrepreneurship educa- tion and its influence on higher education students’ entrepreneurial intentions and motiva- tion in Portugal (Mónico et al. 2021) BAR—Brazilian Administration Review Scopus Entrepreneurship educa- tion, academic major, and university students’ social entrepreneurial intention: the perspec- tive of Planned Behavior Theory (Chang et al. 2021) Studies in Higher Education Scopus Exploring the impact of studying abroad in hungary on entrepre- neurial intention among international students (Wu and Rudnák 2021) Sustainability (Switzerland) Scopus Gender, risk-taking and entrepreneurial inten- tions: assessing the impact of higher educa- tion longitudinally (Gurel et al. 2021) Education and Training Scopus Impacts of architectural education on entrepre- neurial intention: a case study of senior archi- tects from six universi- ties in Turkey (İlerisoy et al. Appendix 2021) Archnet-IJAR Scopus The Antecedents of Sav- ing Behavior and Entre- preneurial Intention of Saudi Arabia University Students (Alshebami and Seraj 2021) Educational Sciences: Theory and Practice Scopus The differential impact of the experiential- entrepreneurial learning method on the entre- preneurial intentions of higher education students (Ali and Negasi 2021) International Journal of Learn- ing, Teaching and Educational Research Scopus The impact of entre- preneurial passion on the entrepreneurial intention; moderating impact of perception of university support (Anjum et al. 2021b) Administrative Sciences Scopus 1 3 1934 G. Maheshwari et al. Title of the paper Name of authors Name of Journals Database The impact of narrow personality traits on entrepreneurial intention in developing coun- tries: A comparison of Turkish and Iranian undergraduate students using ordered discrete choice models (Çelik et al. 2021) European Research on Manage- ment and Business Economics Scopus The Impact of University Innovation and Entre- preneurship Education on Entrepreneurial Intention From the Per- spective of Educational Psychology (Wang et al. 2021) Frontiers in Psychology Scopus Visual Thinking Boosting Spanish Higher Educa- tion Students’ Entrepre- neurial Intentions (Gismera Tierno et al. 2021) Journal of the Knowledge Economy Scopus Youth entrepreneurial intentions: an integrated model of individual and contextual factors (Gulzar and Fayaz 2021) International Journal of Organiza- tional Analysis Scopus Determinants of entre- preneurial intentions of university students in selected post-communist countries in Europe: investigating cross- cultural differences (Tomal and Szromnik 2021) Journal of Business Economics and Management ProQuest Drivers of sustainable entrepreneurial inten- tions among university students: an integrated model from a develop- ing world context (Agu et al. 2021) International Journal of Sustain- ability in Higher Education Scopus Motivational and Atti- tudinal Determinants of Entrepreneurial Intention: Hospitality and Tourism Students’ Perspectives (Al-Jubari et al. Appendix 2021) Journal of Hospitality and Tourism Education Scopus The moderating role of self-efficacy on the cognitive process of entrepreneurship: An empirical study in Vietnam (Doanh 2021a) International Journal of Entre- preneurship and Innovation Management Scopus 1 3 1935 Factors affecting students’ entrepreneurial intentions:… Title of the paper Name of authors Name of Journals Database Effects of Psychological and Cognitive Factors on the Relation between Entrepreneurial Inten- tion and Academic Hazing: Case of the New Students in the Faculty of Social and Human Sciences at the University of Beira— Portugal (Garcez and Franco 2021) Entrepreneurship Research Journal Scopus An examination of the entrepreneurial intent of MBA students in Australia using the entrepreneurial intention questionnaire (Lee-Ross 2017) Journal of Management Develop- ment ProQuest The Impact of Self- efficacy on International Student Entrepreneur Intention (Akadiri et al. 2017) International Review of Manage- ment and Marketing ProQuest The influence of the dark triad on the relationship between entrepreneurial attitude orientation and entrepreneurial inten- tion: A study among students in Taiwan University (Do and Dadvari 2017) Asia Pacific management review Scopus Entrepreneurial self- efficacy and intention among vietnamese students: a meta-analytic path analysis based on the theory of planned behavior (Doanh and Bernat 2019) Management Science Letters Scopus Determinants of indi- viduals’ entrepreneurial intentions: a gender- comparative study (Arshad et al. 2016) Career Development International Scopus Determinants Influencing Entrepreneurial Inten- tion among Undergradu- ates in Universities of Vietnam (Bui et al. 2020) Journal of Asian Finance, Econom- ics and Business Scopus Entrepreneurial Intentions among University Stu- dents: The Moderating Role of Creativity (Entrialgo and Iglesias 2020) European Management Review Scopus Factors affecting students’ entrepreneurial intentions:… 1935 Journal of Management Develop- ment ProQuest 3 1936 G. Maheshwari et al. Title of the paper Name of authors Name of Journals Database Determinants of entre- preneurial intentions: Technical-vocational education and training students in Ethiopia (Buli and Yesuf 2015) Education and Training Scopus Investigating entrepre- neurial intention among public sector university students of Pakistan (Shah and Soomro 2017) Education and Training Scopus Sports university educa- tion and entrepreneurial intentions: A compari- son between Spain and Lithuania (González-Serrano et al. 2018) Education and Training Scopus Gender and university degree: a new analysis of entrepreneurial intention (López-Delgado et al. Appendix 2019) Education and Training Scopus Analyzing the Moderating Effect of Entrepreneur- ship Education on The Antecedents of Entre- preneurial Intention (Bhat and Singh 2018) Journal of entrepreneurship educa- tion Scopus How does the public and private university environment affect students’ entrepreneurial intention? (Canever et al. 2017) Education and Training Scopus Entrepreneurial intention of international business students in Viet Nam: a survey of the country joining the Trans-Pacific Partnership (Nguyen 2017) Journal of Innovation and Entre- preneurship Scopus Entrepreneurial Intentions of Agricultural Students: Levels and Determinants (Pouratashi 2015) Journal of Agricultural Education and Extension Scopus Tool for measuring the influence of the field of knowledge on entrepre- neurial intention among university students (Díez-Echavarría et al. 2020) Periodica Polytechnica Social and Management Sciences Scopus Entrepreneurial intentions of university students: An international comparison between African, European and Canadian students (St-Jean et al. 2014) International Journal of Entre- preneurship and Innovation Management Scopus 1 3 1 3 Factors affecting students’ entrepreneurial intentions:… 1937 Title of the paper Name of authors Name of Journals Database Short-Term and Long- Term Entrepreneurial Intention Comparison between Pakistan and Vietnam (Nasar et al. 2019) Sustainability (Switzerland) Scopus The impact of entrepre- neurial alertness on entrepreneurial inten- tion among university students in Malaysia: Theory of planned behaviour (Lim et al. 2017) Journal of Engineering and Applied Sciences Scopus Determinants of entrepre- neurial intention among engineering students based on structural equation modeling (Arango-Botero et al. 2020) Pertanika Journal of Social Sci- ences and Humanities Scopus Determinants of personal attitudes dimensions on entrepreneurial inten- tions (Ashokan et al. 2019) International Journal of Engineer- ing and Advanced Technology Scopus Determinants of stu- dents’ entrepreneurial intention—a compara- tive study in Poland and Hong Kong (Law and Jakubiak 2020) International Journal of Innovation and Learning Scopus Family education? Unpacking parental factors for tourism and hospitality students’ entrepreneurial intention (Liu and Zhao 2021) Journal of Hospitality, Leisure, Sport and Tourism Education Scopus Factors affecting entrepre- neurial intention among tourism undergraduate students in Vietnam (Phuc et al. 2020) Management science letters Scopus Academic, family, and peer influence on entre- preneurial intention of engineering students (Lingappa et al. 2020) SAGE Open Scopus Determinants of social entrepreneurial inten- tions for educational programs (Asma et al. 2019) Journal of Public Affairs Scopus The validity and reli- ability of thriving scale in academic context: Mindfulness, GPA, and entrepreneurial inten- tion among university students (Ozcan et al. Appendix 2021) Current Psychology Scopus 3 1938 G. Maheshwari et al. 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The role of entrepreneurial skills, propensity to take risks, and innovativeness in open business models (Shahzad et al. 2021) Journal of Open Innovation: Tech- nology, Market, and Complexity Scopus Pathways toward entrepre- neurial intention among Malaysian universities’ students (Bazkiaei et al. 2021) Business Process Management Journal Scopus Factors influencing entre- preneurial intentions (Maheshwari 2021) Education and Training Scopus Entrepreneurial Business and Economics Review Scopus 1 3 Factors affecting students’ entrepreneurial intentions:… Factors affecting students’ entrepreneurial intentions:… 1943 f g p Title of the paper Name of authors Name of Journals Database Influence of educational programs oriented toward entrepreneurship on the entrepreneurial intention of university students: the case of Chile [La influencia de programas de aprendi- zaje orientados al emprendimiento en la intención emprendedora de estudiantes universi- tarios: el caso de Chile] (Silva et al. 2021) Academia Revista Latinoameri- cana de Administracion Scopus Do entrepreneurial education and big-five personality traits predict entrepreneurial inten- tion among universities students? (Bazkiaei et al. 2020) Cogent Business & Management Scopus Relationship between entrepreneurship educa- tion and innovative start- up intentions among university students (Hien and Cho 2018) International journal of entrepre- neurship Scopus Determinants of students’ entrepreneurial inten- tion: An empirical research [Empirijsko istraživanje odrednica studentske poduzetničke namjere] (Zovko et al. 2020) Management (Croatia) Scopus Integrating and extend- ing competing intention models to understand the entrepreneurial intention of senior university students (Eid et al. 2019) Education and Training Scopus Factors impacting entre- preneurial intentions among university stu- dents in Saudi Arabia: testing an integrated model of TPB and EO (Al-Mamary et al. 2020) Education and Training Scopus University entrepreneurial push strategy and students’ entrepreneurial intention (Wegner et al. Appendix 2020) International journal of entrepre- neurial behaviour & research Scopus An investigation of factors influencing entrepreneurial intention amongst university (Kör et al. 2020) Journal of Higher Education Theory and Practice Scopus Relationship between entrepreneurship educa- tion and innovative start- up intentions among university students (Hien and Cho 2018) Determinants of students’ entrepreneurial inten- tion: An empirical research [Empirijsko istraživanje odrednica studentske poduzetničke namjere] (Zovko et al. 2020) 1 3 1944 G. Maheshwari et al. Title of the paper Name of authors Name of Journals Database Exploring the link between entrepreneur- ship education and entrepreneurial inten- tions: the moderating role of educational fields (Duong 2021) Education and Training Scopus The impact of individual and environmental char- acteristics on students’ entrepreneurial intention (Duong et al. 2020) Management science letters Scopus Influence of the link between resources and behavioural factors on the entrepreneurial intentions of electrical installation and mainte- nance work students (Ohanu and Shodipe 2021) Journal of Innovation and Entre- preneurship Scopus Social entrepreneurial intentions and its influential factors: A comparison of students in Taiwan and Hong Kong (Hsu and Wang 2019) Innovations in Education and Teaching International Scopus The role of structural sup- port in predicting entre- preneurial intention: Insights from Vietnam (Van Trang and Doanh 2019) Management science letters Scopus The Impact of Access to Finance and Envi- ronmental Factors on Entrepreneurial Intention: The Mediator Role of Entrepreneurial Behavioural Control (Nguyen 2020) Entrepreneurial Business and Economics Review Scopus A conceptual study on factors of entrepre- neurial potentiality and their impact on entrepreneurial intention with the moderating role of entrepreneurship education (Aggarwal 2019) Prabandhan: Indian Journal of Management Scopus Perceived employability and entrepreneurial intentions across university students and job seekers in Togo: The effect of career adapt- ability and self-efficacy (Atitsogbe et al. 2019) Frontiers in Psychology Scopus 1 3 1 3 Factors affecting students’ entrepreneurial intentions:… 1945 1945 Factors affecting students entrepreneurial intentions:… Title of the paper Name of authors Name of Journals Database Entrepreneurial intention of engineering students and associated influence of contextual factors/ Intención emprendedora de los estudiantes de ingeniería e influencia de factores contextuales (Morales-Alonso et al. 2016) Revista de Psicologia Social Scopus Factors influencing entre- preneurial intentions of undergraduate agricul- tural students in Nigeria [Nijerya’da ziraat fakültesi öğrencilerinin girişimciliklerini etkileyen faktörler] (Adeyonu et al. Appendix 2019) Yuzuncu Yil University Journal of Agricultural Sciences Scopus The impact of network ties, shared languages and shared visions on entrepreneurial inten- tions of online univer- sity students (Pérez-Macías et al. 2020) Studies in Higher Education Scopus The effect of psycho- logical and contextual factors on the entre- preneurial intention of university students in South Africa (Dzomonda et al. 2015) Corporate Ownership and Control Scopus An exploratory study of entrepreneurial inten- tion among university students in Ghana (Amanamah et al. 2018) International Journal of Scientific and Technology Research Scopus Gender and Social Legitimacy of Entre- preneurship: Contribu- tion to Entrepreneurial Intention in University Students from Chile and Colombia (Soria et al. 2016) Journal of technology management & innovation Scopus Factors influencing e-entrepreneurial intention among female students in Saudi Arabia (Alzamel et al. 2020) International Journal of Criminol- ogy and Sociology Scopus The influence of gender on the entrepreneurial intentions of journalism students (Caro-González et al. 2017) Intangible Capital Scopus The influence of culture on entrepreneurial intentions: a Nigerian university graduates’ perspective (Chukwuma-Nwuba 2018) Transnational Corporations Review Scopus The impact of network ties, shared languages and shared visions on entrepreneurial inten- tions of online univer- sity students The effect of psycho- logical and contextual factors on the entre- preneurial intention of university students in South Africa (Amanamah et al. 2018) International Journal of Scientific and Technology Research Scopus (Soria et al. 2016) Journal of technology management & innovation Scopus An exploratory study of entrepreneurial inten- tion among university students in Ghana Transnational Corporations Review Scopus 1 3 1946 G. 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Title of the paper Name of authors Name of Journals Database Entrepreneurial inten- tion among online and face-to-face university students: The influ- ence of structural and cognitive social capital dimensions (Pérez-Macías et al. 2021) Journal of International Entrepre- neurship Scopus Entrepreneurial intentions of university students: a case-based study (Palalić et al. 2017) Journal of Enterprising Communi- ties Scopus A psychosocial study of self-perceived creativity and entrepreneurial intentions in a sample of university students (Laguía et al. 2019) Thinking Skills and Creativity Scopus Model of the entrepre- neurial intention of university students in the Pearl River Delta of China (Hou et al. 2019) Frontiers in Psychology Scopus Determinants of university students’ entrepreneurial intention: GUESSS Colombia study (Cano and Tabares 2017) Espacios Scopus Exploring the factors responsible in predicting entrepreneurial intention among nascent entrepre- neurs: A field research (Tiwari et al. Appendix 2020) South Asian Journal of Business Studies Scopus Relationship among influential factors of entrepreneurial intention in terms of gender: Case of postgraduate students in Malaysia (Yaghmaei et al. 2015) Mediterranean Journal of Social Sciences Scopus Validating the Entre- preneurial Intention Model on the University Students in Saudi Arabia (Hoda et al. 2020) Journal of Asian Finance, Econom- ics and Business Scopus Entrepreneurial intentions of private university students in the kingdom of Bahrain (Al-Shammari and Waleed 2018) International Journal of Innovation Science Scopus Entrepreneurial intention among Latin American university students [Intención emprende- dora entre estudiantes universitarios en Lati- noamérica] (Leiva et al. 2021) Academia Revista Latinoameri- cana de Administracion Scopus Journal of Enterprising Communi- ties Scopus Thinking Skills and Creativity Scopus Frontiers in Psychology Scopus 1 3 1 3 Factors affecting students’ entrepreneurial intentions:… Factors affecting students’ entrepreneurial intentions:… 1947 Title of the paper Name of authors Name of Journals Database Comparing the Entre- preneurial Intention between Female and Male Engineering Students (Lo et al. 2017) Journal of Women’s Entrepreneur- ship and Education (JWEE) Google Scholar How University Entre- preneurship Support Affects College Stu- dents’ Entrepreneurial Intentions: An Empirical Analysis from China (Lu et al. 2021) Sustainability (Switzerland) Scopus Factors influencing entrepreneurial intention of university students in china: Integrating the perceived university support and theory of planned behavior (Su et al. 2021) Sustainability (Switzerland) Scopus A structural model for predicting entrepre- neurial intention of university students (Tassawa 2019) Humanities, Arts and Social Sci- ences Studies Scopus Fostering sustainable businesses: understand- ing sustainability-driven entrepreneurial intention among university stu- dents in Pakistan (Waris et al. 2021) Social Responsibility Journal Scopus A Model of Factors Affecting Entrepre- neurial Intention among Information Technology Students in Vietnam (Vuong et al. 2020) Journal of Asian Finance, Econom- ics and Business Scopus Entrepreneurial intention among female university students: Examining the moderating role of entrepreneurial educa- tion (Anwar et al. 2020) Journal for International Business and Entrepreneurship Develop- ment Scopus Determinants of entrepre- neurial intention among undergraduates in a Muslim community (Ezeh et al. 2020) Management Research Review Scopus Entrepreneurial Intention: Creativity, Entrepre- neurship, and University Support (Anjum et al. 2021a) Journal of open innovation Scopus Effect of memorial university’s environment & support system in shaping entrepreneurial intention of students (Bazan et al. Appendix 2019) Journal of Entrepreneurship Education Scopus Journal of Asian Finance, Econom- ics and Business Scopus A Model of Factors Affecting Entrepre- neurial Intention among Information Technology Students in Vietnam (Vuong et al. 2020) 1 3 1948 G. Maheshwari et al. Title of the paper Name of authors Name of Journals Database Entrepreneurship educa- tion and entrepreneurial intentions of university students in Vietnam: the mediating roles of self- efficacy and learning orientation (Hoang et al. 2021) Education and Training Scopus The impact of entre- preneurial education on entrepreneurial intention: The case of Vietnamese (Doan and Phan 2020) Management science letters Scopus Influence of university- related factors on students’ entrepreneurial intentions (Lopez and Alvarez 2019) International Journal of Entrepre- neurial Venturing Scopus The relationship between higher education and entrepreneurial intention among Vietnamese students (Kim et al. 2020) Management science letters Scopus The analysis of the effect of entrepreneurship education, perceived desirability, and entre- preneurial self-efficacy on university students’ entrepreneurial intention (Suratno and Kusmana 2019) Universal Journal of Educational Research Scopus Factors affecting the entrepreneurial inten- tions among university students of Thailand (Tamprateep et al. 2019) Journal of Computational and Theoretical Nanoscience Scopus Role of entrepreneurial education in shaping entrepreneurial inten- tion among university students: Testing the hypotheses using media- tion and moderation approach (Anwar et al. 2022) Journal of Education for Business Scopus Perceived university support, entrepreneurial self-efficacy, heteroge- neous entrepreneurial intentions in entrepre- neurship education: The moderating role of the Chinese sense of face (Shi et al. 2020) Journal of Entrepreneurship in Emerging Economies Scopus 1 3 1 3 Factors affecting students’ entrepreneurial intentions:… 1949 Title of the paper Name of authors Name of Journals Database Characterization of entrepreneurial intention in university students as from Systemic Entre- preneurship Intention Model: A case study (Velásquez et al. 2018) Cuadernos de Gestion Scopus Entrepreneurial intentions of university students in Colombia: Exploration based on the theory of planned behavior (Rueda Barrios et al. 2022) Journal of Education for Business Scopus University entrepreneurial intentions: mainland and insular regions – are they different? (Lopes et al. 2020) Education and Training Scopus Entrepreneurial intention and obstacles of under- graduate students: the case of the universities of Andalusia (Arranz et al. 2019) Studies in Higher Education Scopus Entrepreneurial intention of Indian university students: the role of opportunity recognition and entrepreneurship education (Hassan et al. Appendix 2020) Education and Training Scopus Entrepreneurial intention models as applied to Latin America (Guzmán-Alfonso and Guzmán-Cuevas 2012) Journal of Organizational Change Management Scopus The role of autonomy as a predictor of entrepre- neurial intention among university students in Yemen (Al-Jubari et al. 2017) International Journal of Entrepre- neurship and Small Business Scopus The Role of Personal Values in Forming Stu- dents’ Entrepreneurial Intentions in Developing Countries (Karimi and Makreet 2020) Frontiers in Psychology Scopus University Students’ Behaviour towards Entrepreneurial Inten- tion in Ecuador: Testing for the Influence of Gender (Rodriguez-Gutierrez et al. 2020) International Journal of Environ- mental Research and Public Health Scopus Contextual Motivations for Undergraduates’ Entrepreneurial Inten- tions in Emerging Asian Economies (Looi 2020) The Journal of Entrepreneurship Scopus 3 1950 G. Maheshwari et al. Title of the paper Name of authors Name of Journals Database Entrepreneurial Intention: A Gender Study in Business and Economics Students from Chile (Contreras-Barraza et al. 2021) Sustainability (Switzerland) Scopus Impact of behavioural factors as related to available resources on entrepreneurial inten- tions of electrical instal- lation and maintenance works students (Benedictohanu et al. 2020) International Journal of Engineer- ing Education Scopus Factors Affecting the Entrepreneurial Intention of Students Pursuing Journalism and Media Studies: Evidence from Spain (Goyanes 2015) JMM International Journal on Media Management Scopus Linking entrepreneurial intentions and mindset models: A compara- tive study of public and private universities in Vietnam (Cao and Ngo 2019) Gadjah Mada International Journal of Business Scopus Social and economic factors affecting the entrepreneurial intention of university students (Belas et al. 2017) Transformations in Business and Economics Scopus Determinants of entrepre- neurial intention among Sudanese university students (Ali and Abou 2020) Management science letters Scopus Determinants of entre- preneurial intention of pharmacy students in Chennai (Sankar and Irin Sudha 2016) International Journal of Pharma- ceutical Sciences Review and Research Scopus Do Islamic Values Impact Social Entrepreneurial Intention of University Students in Malaysia? An Empirical Investiga- tion Into The Mediating Role of Empathy (Mohammadi et al. 2020) International Journal of Economics and Management Scopus Entrepreneurial inten- tions—theory and evidence from Asia, America, and Europe (Paul et al. 2017) Journal of International Entrepre- neurship Scopus Exploring entrepreneurial intentions in Latin American university students (Torres et al. (Gangwani and Ballout 2020) Appendix 2017) International Journal of Psycho- logical Research Scopus 1 3 1 3 Factors affecting students’ entrepreneurial intentions:… Factors affecting students’ entrepreneurial intentions:… 1951 Title of the paper Name of authors Name of Journals Database The role of social and psychological factors on entrepreneurial intention among islamic college students in Indonesia (Ahamed and Rokhman 2015) Entrepreneurial Business and Economics Review Scopus Research on the influenc- ing factors of entrepre- neurial intentions based on mediating effect of self-actualization (Dong et al. 2019) International Journal of Innovation Science Scopus Do Entrepreneurship Students Have an Intention to Become an Entrepreneur? (Kurniawan et al. 2019) Journal of entrepreneurship educa- tion Scopus Planned behavior and religious beliefs as ante- cedents to entrepreneur- ial intention: A study with university students [Comportamento plane- jado e crenças religiosas como antecedentes da intenção empreende- dora: Um estudo com universitários] (Paiva et al. 2020) Revista de Administracao Mac- kenzie Scopus Factors influencing the entrepreneurial intention of business graduate students of Saudi Arabia University (Gangwani and Ballout 2020) Academy of Entrepreneurship journal Scopus Determinants of students’ entrepreneurial intention to compete in a fast- pitch competition (Jang et al. 2019) Journal of Education for Business Scopus Determinants of entrepre- neurial intention among generation y students within the Johannesburg Metropolitan Area of South Africa (Maziriri et al. 2019) African Journal of Business and Economic Research Scopus Assessing the university students’ entrepreneurial intention: Entrepre- neurial education and creativity (Saptono et al. 2019) Humanities and Social Sciences Reviews Scopus Entrepreneurial inten- tions among university students: A case study of Durban University of Technology (Jwara and Hoque 2018) Academy of Entrepreneurship journal Scopus universitários] Factors influencing the entrepreneurial intention of business graduate students of Saudi Arabia University (Gangwani and Ballout 2020) Academy of Entrepreneurship journal Scopus Determinants of students’ entrepreneurial intention to compete in a fast- pitch competition (Jang et al. 2019) Journal of Education for Business Scopus Determinants of entrepre- neurial intention among generation y students within the Johannesburg Metropolitan Area of South Africa (Maziriri et al. 2019) African Journal of Business and Economic Research Scopus Assessing the university students’ entrepreneurial intention: Entrepre- neurial education and creativity (Saptono et al. 2019) Humanities and Social Sciences Reviews Scopus Entrepreneurial inten- tions among university students: A case study of Durban University of Technology (Jwara and Hoque 2018) Academy of Entrepreneurship journal Scopus Academy of Entrepreneurship journal Scopus 3 1952 G. Maheshwari et al. Appendix Title of the paper Name of authors Name of Journals Database A critical comparison of factors affecting science and technology students’ entrepreneurial intention: a tale of two genders (Roy and Das 2020) International Journal for Educa- tional and Vocational Guidance Scopus The mediating role of entrepreneurial passion in the relationship between entrepreneur education and entrepre- neurial intention among university students in Thailand (Sriyakul and Jermsitti- parsert 2019) International Journal of Innova- tion, Creativity and Change Scopus Determinants of entre- preneurial intention: Empirical insights from Malaysian undergradu- ate business students (Ali et al. 2017) International Journal of Economic Research Scopus Entrepreneurial education and entrepreneurial intention: The mediating role of creativity dispo- sition among University Students in Thailand (Rungsrisawat and Sut- duean 2019) International Journal of Innova- tion, Creativity and Change Scopus Effects of entrepreneurial knowledge on entre- preneurial intentions: a longitudinal study of selected South-east Asian business students (Roxas 2014) Journal of Education and Work Scopus Factors influencing entrepreneurial inten- tion: A case of students in a South African University (Nsahlai et al. 2020) Academy of Entrepreneurship journal Scopus Determinants of entrepre- neurial intentions: An institutional embedded- ness perspective (Shahid et al. 2018) Journal of Small Business and Entrepreneurship Scopus Psycho-demographic Factors and Entrepre- neurial Intention Among University Students (Ojewumi et al. 2020) Open Journal for Psychological Research Google Scholar Factors affecting entrepre- neurial intention among UniSZA students (Ghazali et al. 2012) Asian Social Science Scopus Effect of demographic fac- tors on entrepreneurial intention of manage- ment students in Nagpur university, India (Uike 2019) International Journal of Scientific and Technology Research Scopus (Sriyakul and Jermsitti- parsert 2019) International Journal of Innova- tion, Creativity and Change Scopus International Journal of Economic Research Scopus Factors influencing entrepreneurial inten- tion: A case of students in a South African University (Nsahlai et al. 2020) 1 3 Factors affecting students’ entrepreneurial intentions:… Factors affecting students’ entrepreneurial intentions:… 1953 Title of the paper Name of authors Name of Journals Database What impact entrepre- neurial intention? Cultural, environmental, and educational factors (Ao and Liu 2014) Journal of Management Analytics Scopus Understanding the entre- preneurial intention in the light of contextual factors: Gender analysis (Rahaman et al. 2020) Journal of Asian Finance, Econom- ics and Business Scopus Symmetric and asymmet- ric modeling of entre- preneurial ecosystem in developing entre- preneurial intentions among female university students in Saudi Arabia (Ali et al. Appendix 2019) International journal of gender and entrepreneurship Scopus Students’ Attitudes toward Entrepreneurship at the Arab Open University- Lebanon (Hendieh et al. 2019) Journal of entrepreneurship educa- tion Scopus Determinants of entrepre- neurial intention among millennial generation in emerging countries (Wibowo et al. 2019) Journal of Legal, Ethical and Regulatory Issues Scopus Determinants of eco entrepreneurial intention among students: Study in the entrepreneurial education practices (Nuringsih and Pus- pitowati 2017) Advanced Science Letters Scopus Factors affecting entre- preneurial intention of Senior University Students (Duque Aldaz et al. 2018) Espacios Scopus Indonesian university students’ entrepreneurial intention: A conceptual study (Pradana et al. 2020) Journal of Critical Reviews Scopus A comparative analysis of entrepreneurial intention and migration attitudes of students in Vietnam and Poland (Le Trung et al. 2020) Management science letters Scopus Social values as determi- nants of entrepreneurial intentions among uni- versity students in Cape Town—South Africa (Kalitanyi and Visser 2016) Problems and perspectives in management Scopus Sociological and theory of planned behaviour approach to understand- ing entrepreneurship: (Nguyen et al. 2020) Cogent business & management Scopus 1 3 1954 G. Maheshwari et al. Title of the paper Name of authors Name of Journals Database Determinants of Entre- preneurial Intentions Among Romanian Students (Popescu et al. 2016) Transformations in Business and Economics Scopus How does the perception of entrepreneurial eco- system affect entrepre- neurial intention among university students in Saudi Arabia? (Elnadi et al. 2020) International Journal of Entrepre- neurship Scopus Factors influencing entre- preneurial intentions among arab students (Al Bakri and Mehrez 2017) International Journal of Entrepre- neurship Scopus Relationships between students’ work values and entrepreneurial intention among Viet- namese students (Van Trang et al. 2020) Academy of Entrepreneurship journal Scopus Corruption shock in Mex- ico: FsQCA analysis of entrepreneurial intention in university students (Castelló-Sirvent and Pinazo-Dallenbach 2021) Mathematics Scopus Influence of institutional environment on entre- preneurial intention: a comparative study of two countries university students (Díaz-Casero et al. 2012) International Entrepreneurship and Management Journal Scopus Parental and gender effects on the entre- preneurial intention of university students in South Africa (Fatoki 2014) Mediterranean Journal of Social Sciences Scopus Influence of demographic factors on the entrepre- neurial intentions of university students in Oman (Uddin et al. 2016) Investment Management and Financial Innovations Scopus The effect of educational background on entrepre- neurial intention (Xuan et al. Appendix 2020) Management science letters Scopus A decade of entrepreneur- ship education in the Asia Pacific for future directions in theory and practice (Wu and Wu 2017) Management decision Scopus Contextual factors and their effects on future entrepreneurs in China: A comparative study of entrepreneurial inten- tions (Bernhofer and Han 2014) International Journal of Technol- ogy Management 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) Advanced Science Letters Scopus University Students’ Entrepreneurial Inten- tions: A Comparative Analysis of Hong Kong and Guangzhou (Bickenbach et al. 2017) China and World Economy Scopus Determinants of entrepre- neurial intention among university business students in Kenya: Lessons from Kenyatta University (Kilonzo and Nyambegera 2014) International Journal of Entrepre- neurship and Small Business Scopus Tracking the cyber entrepreneurial intention of private universities students in Malaysia (Ismail et al. 2012) International Journal of Entrepre- neurship and Small Business Scopus Descriptive research study on factors influencing entrepreneurial intention among engineering stu- dents in Virudhunagar District (Sumathi et al. 2018) Journal of Advanced Research in Dynamical and Control Systems Scopus An examination of univer- sity student entrepre- neurial intentions by type of venture (Carey et al. 2010) Journal of Developmental Entre- preneurship Scopus Impact of academic majors on entrepre- neurial intentions of Vietnamese students: An extension of the theory of planned behavior (Dao et al. 2021) Heliyon Scopus Funding  Open Access funding enabled and organized by CAUL and its Member Institutions. No funds, grants, or other support was received. Data availability  Data sharing is not applicable as no datasets are generated in this current study. All the articles used for the analysis are already provided in the article (in Table 2 and Appendix table). Declarations Conflict of interest  The authors declare that they have no conflict of interest. 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 3 3 G. Maheshwari et al. 1956 you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons 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. 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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 ISSN: 2055-8198 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 ISSN: 2055-8198 URL: http://press-start.gla.ac.uk 78 2015 | Volume 2 | Issue 2 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 ISSN: 2055-8198 URL: http://press-start.gla.ac.uk Press Start ISSN: 2055-8198 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 ISSN: 2055-8198 URL: http://press-start.gla.ac.uk Press Start ISSN: 2055-8198 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 ISSN: 2055-8198 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 ISSN: 2055-8198 URL: http://press-start.gla.ac.uk 2015 | Volume 2 | Issue 2 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 ISSN: 2055-8198 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 ISSN: 2055-8198 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 ISSN: 2055-8198 URL: http://press-start.gla.ac.uk Press Start ISSN: 2055-8198 URL: http://press-start.gla.ac.uk 85 Games-based Interdisciplinary Learning in CfE 2015 | Volume 2 | Issue 2 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 ISSN: 2055-8198 URL: http://press-start.gla.ac.uk 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 ISSN: 2055-8198 URL: http://press-start.gla.ac.uk 2015 | Volume 2 | Issue 2 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 ISSN: 2055-8198 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 ISSN: 2055-8198 URL: http://press-start.gla.ac.uk 88 2015 | Volume 2 | Issue 2 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 ISSN: 2055-8198 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 ISSN: 2055-8198 URL: http://press-start.gla.ac.uk 2015 | Volume 2 | Issue 2 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 ISSN: 2055-8198 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 ISSN: 2055-8198 URL: http://press-start.gla.ac.uk Press Start ISSN: 2055-8198 URL: http://press-start.gla.ac.uk 2015 | Volume 2 | Issue 2 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 98 98
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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
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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. 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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. 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Integrated solutions for sustainable fall prevention in primary care, the iSOLVE project: a type 2 hybrid effectiveness-implementation design
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. 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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. 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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. 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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
Ekonomi, politika ve finans araştırmaları dergisi
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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. 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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). 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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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Clinical Significance and Genetic Characteristics in HER2 Low Expression Tumors of Hormone Receptor Positive Breast Cancer Patients
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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. 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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. 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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 C O E M A S. E C To Tä L. M A E E B C C R S. M Se E U A K F. Fr M M E K C M Z P A Le B D O G B C Pe K A a C 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. 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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. 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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. 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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 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. y 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
Energies
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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 bi 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. 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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. 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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
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 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (a) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (b) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (a) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (b) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (c) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (d) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 (a) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 (b) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (c) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (d) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (c) 00: 00: (d) 20 ⁎ ⁎ ⁎ 18 16 14 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 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (a) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (b) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (c) 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 00:00 06:00 06:00 12:00 18:00 12:00 18:00 00:00 00:00 ⁎ (d) 20 ⁎ ⁎ ⁎ 18 16 14 12 Days to reentrain 10 08 06 04 02 0 WT-PBS 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]. 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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)
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. 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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
MATEC web of conferences
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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. 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https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2024.1343411/pdf
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Novel FOXL2 variants in two Chinese families with blepharophimosis, ptosis, and epicanthus inversus syndrome
Frontiers in genetics
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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. 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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
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 Ativo ou passivo T. Su Ce Su Su Su Su Su Ce Su Su Su Ce Su Su Su Su Su o (2014) Ce ento. (a) Tra 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. 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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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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. 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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 institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. 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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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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 i Thi i t fi t i C d ill l t id th i k d b fit f t hf l iti d di 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 (page number not for citation purposes) XSL•FO 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 (page number not for citation purposes) http://www.researchprotocols.org/2018/11/e10926/ XSL•FO 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 (page number not for citation purposes) JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 4 (page number not for citation purposes) XSL•FO 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 (page number not for citation purposes) http://www.researchprotocols.org/2018/11/e10926/ XSL•FO 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 Research Protocols (http://www.researchprotocols.org), 20.11.2018. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on http://www.researchprotocols.org, as well as this copyright and license information must be included. http://www.researchprotocols.org/2018/11/e10926/ XSL•FO RenderX XSL•FO RenderX
https://openalex.org/W2019435250
https://www.scielo.br/j/rbz/a/zjSZQPGWrtt6ydpbm84XMmH/?lang=pt&format=pdf
Portuguese
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Feno de maniçoba em dietas para ovinos: consumo de nutrientes, digestibilidade aparente e balanço nitrogenado
Revista Brasileira de Zootecnia
2,007
cc-by
5,499
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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Selective attention for affiliative and agonistic interactions of dominants and close affiliates in macaques
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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 d 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. 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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 institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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SEAFLOOR POSITIONING MODEL FOR SIMULTANEOUS ESTIMATION OF SOUND VELOCITY
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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. 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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. 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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
BMC cardiovascular disorders
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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. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 27. 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Liver Histopathological Analysis of 24 Postmortem Findings of Patients With COVID-19 in China
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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). 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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. 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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
Knee surgery, sports traumatology, arthroscopy
2,020
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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. 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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 DEAL. 1 3 691 Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693 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://creat​iveco​mmons​.org/licen​ses/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. 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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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Conjugalidades e parentalidades de gays, lésbicas e transgêneros no Brasil
Revista Estudos Feministas
2,006
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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 pp Салимов Б.Л. Илмий билиш жараёнида тасодифнинг ўрни. Фалсафа фанлари Ў р д д ф ўр ф ф р 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
https://openalex.org/W2018781231
https://www.cdc.gov/pcd/issues/2014/pdf/14_0047.pdf
English
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Characteristics of the Built Environment in Relation to Objectively Measured Physical Activity Among Mexican Adults, 2011
Preventing chronic disease
2,014
public-domain
10,229
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. www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 7 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. p Sports Exerc 2007;39(8):1423. www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147 AUGUST 2014 PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147 AUGUST 2014 www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 7 www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Preventio 7 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. 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
Molecules/Molecules online/Molecules annual
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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 • 8 of 18 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 Molecules 2018, 23, 2238 9 of 18 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. Molecules 2018, 23, 2238 10 of 18 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 • • • • • • 11 of 18 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 Molecules 2018, 23, 2238 12 of 18 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 Molecules 2018, 23, 2238 13 of 18 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. Molecules 2018, 23, 2238 14 of 18 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. 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Toxicol. Lett. 2014, 228, 207–215. [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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Planar Microwave Sensor for Theranostic Therapy of Organic Tissue Based on Oval Split Ring Resonators
Sensors
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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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Thiopurine methyltransferase and treatment outcome in the <scp>UK</scp> acute lymphoblastic leukaemia trial <scp>ALL</scp>2003
British journal of haematology
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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. 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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. 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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. 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(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: sagepub.com/journals-permissions 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. 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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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On leptonic width of X(4260)
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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. 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2-Complex Symmetric Composition Operators on H2
Axioms
2,022
cc-by
5,870
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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Boosting with AIDSVAX B/E Enhances Env Constant Region 1 and 2 Antibody-Dependent Cellular Cytotoxicity Breadth and Potency
Journal of virology
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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 Volume 94 Issue 4 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 Volume 94 Issue 4 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 Volume 94 Issue 4 e01120-19 Easterhoff et al. February 2020 Volume 94 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 Volume 94 Issue 4 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 Volume 94 Issue 4 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 7 February 2020 Volume 94 Issue 4 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 Volume 94 Issue 4 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 Volume 94 Issue 4 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 8 February 2020 Volume 94 Issue 4 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 9 February 2020 Volume 94 Issue 4 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 Volume 94 Issue 4 e01120-19 jvi.asm.org 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 Volume 94 Issue 4 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 Volume 94 Issue 4 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 12 February 2020 Volume 94 Issue 4 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 Volume 94 Issue 4 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 Volume 94 Issue 4 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 Volume 94 Issue 4 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. 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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. 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https://link.springer.com/content/pdf/10.1007/s13384-023-00653-5.pdf
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Academic work from the perspectives of aspiring academics: a study of doctoral candidates and recent graduates
Australian educational researcher
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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) 3 456789) 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. 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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)
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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 g p g 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
https://openalex.org/W2332769882
https://zenodo.org/records/2186268/files/article.pdf
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.
https://openalex.org/W3197887526
https://link.springer.com/content/pdf/10.1007/s12975-021-00939-9.pdf
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://​creat​iveco​mmons.​org/​licen​ses/​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. 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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.​elsev​ier.​com/​retri​eve/​pii/​00134​69461​900086. 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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APPROACHES OF NATIONAL 3D MAPPING: RESEARCH RESULTS AND STANDARDISATION IN PRACTICE
ISPRS annals of the photogrammetry, remote sensing and spatial information sciences
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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
https://openalex.org/W4378233715
https://discovery.ucl.ac.uk/id/eprint/10170955/1/Roatti%20et%20al%202023%20RSOS.pdf
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Social network inheritance and differentiation in wild baboons
Royal Society open science
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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 0 10 20 30 40 50 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 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 –0.1 –0.2 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 –0.1 –0.2 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 –0.1 –0.2 –0.1 –0.2 0 10 20 30 40 50 60 sex F M 0 10 20 30 40 50 60 age (months) 0 10 20 30 40 50 60 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 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 –0.1 –0.2 0 10 20 30 40 50 60 gr. given 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 –0.1 –0.2 0 10 20 30 40 50 60 royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230219 16 16 gr. total 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 –0.1 –0.2 (months) 0 10 20 30 40 50 60 gr. received mother–offspring n 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 –0.1 –0.2 0 10 20 30 40 50 60 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 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 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 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 sex F M 0 10 20 30 40 50 60 age (months) 0 10 20 30 40 50 60 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 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 gr. given—MP1c.1 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 s 17 royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230219 17 gr. total—MP1c.2 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 months) 0 10 20 30 40 50 60 gr. given—MP1c.2 odds network s 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 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 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 –2.0 0 10 20 30 40 50 60 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 –2.0 0 10 20 30 40 50 60 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 –2.0 0 10 20 30 40 50 60 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 –2.0 0 10 20 30 40 50 60 sex F M age (months) 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 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 –2.0 0 10 20 30 40 50 60 gr. given 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 –2.0 0 10 20 30 40 50 60 s 18 royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230219 18 gr. total 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 –2.0 0 10 20 30 40 50 60 F M (months) gr. received social networ 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 –2.0 0 10 20 30 40 50 60 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 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 0 10 20 30 40 50 60 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 0 10 20 30 40 50 60 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 0 10 20 30 40 50 60 age (months) 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 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 0 10 20 30 40 50 60 (months) gr. total—MP2b.1 centrality social n 2.0 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 0 10 20 30 40 50 60 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 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 10 20 30 proximity—MP2c.1 proximity—MP2c.3 age (months) immature 40 50 60 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 gr. given—MP2c.1 gr. received—MP2c.1 immature 40 50 60 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 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 gr. total—MP2c.1 gr. given—MP2c.2 gr. received—MP2c.2 gr. total—MP2c.2 gr. given—MP2c.3 gr. received—MP2c.3 gr. 4. Discussion total—MP2c.3 40 50 60 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 sex F M 40 50 60 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 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 odds being with social partner peer 40 50 60 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 same-sex 40 50 60 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 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 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 same-sex 40 50 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. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230219 20 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 proximity—MP2c.1 immature 40 50 60 proximity—MP2c.3 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 same-sex 40 50 60 20 royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230219 20 gr. received—MP2c.1 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 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 gr. given—MP2c.1 immature 40 50 60 h social partner gr. total—MP2c.2 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 F M 40 50 60 gr. received—MP2c.2 gr. total—MP2c.2 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 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 gr. received—MP2c.2 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 gr. 4. Discussion given—MP2c.2 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 odds being with peer 40 50 60 age (months) gr. received—MP2c.3 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 gr. total—MP2c.3 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 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. 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https://zenodo.org/records/3524311/files/GLO%20OSA%20-%20optica-4-2-209.pdf
English
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Ultralow transmission loss in inhibited-coupling guiding hollow fibers
Optica
2,017
cc-by
8,720
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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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.
https://openalex.org/W2726018660
https://journals.eco-vector.com/uroved/article/download/6772/5404
Russian
null
The infertility overcoming in men with retrograde ejaculation and anejaculation
Urologičeskie vedomosti
2,017
cc-by
3,067
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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Relationship between Different Anaerobic Digestion Parameters in a Pig-dung Aided Water Hyacinth Digestion Process
European journal of engineering and technology research
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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
Reproductive health
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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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​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/​rimed​icalj​ournal/​2017/​12/​2017-​12-​14-​pov-​parri​llo.​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.​teenv​ogue.​com/​story/​homel​essne​ss-​and-​perio​ds. 12. McNamara B. How periods perpetuate homelessness. Teen. 2017. https://​ www.​teenv​ogue.​com/​story/​homel​essne​ss-​and-​perio​ds. 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.​jadoh​ealth.​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/​journ​al.​pmed.​10019​62. 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/​resou​rce/​resmgr/​LegCl​inic/​2018_​perio​ ds_​policy_​report.​pdf. References 1. VanLeeuwen C, Torondel B. 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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.​biome​dcent​ral.​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.​coali​tionf​orthe​homel​ess.​org/​wp-​conte​nt/​uploa​ds/​2019/​04/​State​ OfThe-​Homel​ess20​19.​pdf. Accessed 17 Dec 2020. 32. Specia M. Scotland is 1st nation to make period products free. The New York Times. 2020. https://​www.​nytim​es.​com/​2020/​11/​24/​world/​europe/​ scotl​and-​free-​period-​produ​cts.​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.​latim​es.​com/​calif​ornia/​story/​2019-​12-​28/​la-​moves-​to-​provi​de-​free-​ tampo​ns-​and-​pads-​in-​public-​restr​ooms. Accessed 21 Aug 2020. 24. Romero AP, Goldberg SK, Vasquez LA. LGBT people and housing afford- ability, and homelessness. 2020. https://​willi​amsin​stitu​te.​law.​ucla.​edu/​ wp-​conte​nt/​uploa​ds/​LGBT-​Housi​ng-​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.​chica​gotri​bune.​com/​subur​bs/​highl​ and-​park/​ct-​hpn-​free-​tampo​ns-​femin​ine-​hygie​ne-​tl-​0919-​20190​912-​24sfi​ he3pn​gsjei​t6pg4​qdbptu-​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/​secti​ons/​coron​ avirus-​live-​updat​es/​2020/​04/​01/​82549​0269/​save-​those-​pharm​acy-​recei​ pts-​new-​payme​nt-​flexi​bility-​court​esy-​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://​creat​iveco​mmons.​ org/​licen​ses/​by/4.​0/ 28. The New York City Council. File #: Int 1123-2016. 2016. https://​legis​tar.​ counc​il.​nyc.​gov/​Legis​latio​nDeta​il.​aspx?​ID=​26371​12&​GUID=​31322​AF8-​ 376A-​4D7F-​93B2-​4243B​A5E41​81. 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. 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Rapid Eocene diversification of spiny plants in subtropical woodlands of central Tibet
Nature communications
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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 DY1 b 0 10 20 30 40 50 60 70 80 90 100 DY Depth (m) 0 50 100 150 200 250 300 XD Shale Plant fossil Mudstone Sandstone Muddy shale Silty mudstone Siltstone d c XDA1 XDB3 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 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. 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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. 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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/. 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
Substance abuse treatment, prevention, and policy
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12,256
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. 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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. 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Genetic variability of Pantaneiro horse using RAPD-PCR markers
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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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
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6,699
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 http://adc.bmj.com/ 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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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. 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(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. 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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 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 2 4 6 8 10 12 14 16 18 2.5 2.0 1.5 1.0 0.5 0 1 2 3 4 5 6 7 8 9 10 11 5 10 15 20 25 30 35 5 10 15 20 25 30 35 40 45 Data set: Voice Data set: TIC2000 Data set: Adult Data set: Default 10 20 30 40 (a) Voice (b) TIC2000 (d) Default (c) Adult 50 60 2 4 6 8 10 12 2 4 6 8 10 12 14 16 18 20 0.6 0.5 0.4 0.3 0.2 0.1 0 0.30 0.25 0.20 0.15 0.10 0.05 0 3.0 2.5 2.0 1.5 1.0 0.5 0 ℓ1 Fig. 3     The weight matrix (absolute value) of the learned model w.r.t. the   regularization 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 2 4 6 8 10 12 14 16 18 2.5 2.0 1.5 1.0 0.5 0 1 2 3 4 5 6 7 8 9 10 11 5 10 15 20 25 30 35 Data set: Voice Data set: TIC2000 (a) Voice (b) TIC2000 3.0 2.5 2.0 1.5 1.0 0.5 0 1 2 3 4 5 6 7 8 9 10 11 5 10 15 20 25 30 35 Data set: TIC2000 (b) TIC2000 3.0 2.5 2.0 1.5 1.0 0.5 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 2 4 6 8 10 12 14 16 18 2.5 2.0 1.5 1.0 0.5 0 Data set: Voice (a) Voice 5 10 15 20 25 30 35 40 45 Data set: Adult Data set: Default 10 20 30 40 (d) Default (c) Adult 50 60 2 4 6 8 10 12 2 4 6 8 10 12 14 16 18 20 0.6 0.5 0.4 0.3 0.2 0.1 0 0.30 0.25 0.20 0.15 0.10 0.05 0 ℓ1 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 0.80 0.78 0.955 0.950 0.945 0.940 0.67 0.66 0.65 0.64 0.105 0.100 0.095 0.090 0.30 0.29 0.28 0.01 0.1 1 10 100 The trade-off parameter λ 0.01 0.1 1 10 100 The trade-off parameter λ 0.01 0.1 1 10 100 The trade-off parameter λ 0.01 0.1 1 10 100 The trade-off parameter λ 0.01 0.1 1 10 100 The trade-off parameter λ 0.01 0.1 1 10 100 The trade-off parameter λ (a) Flare1 (b) WQplants Data set: WQplants Data set: Flare1 EM HS SEM EM HS SEM 0.65 0.64 0.63 0.62 0.065 0.060 0.055 0.050 0.25 0.24 0.23 0.22 0.45 0.40 0.35 0.035 0.030 0.025 0.020 0.16 0.14 0.12 0.10 0.01 0.1 1 10 100 The trade-off parameter λ 0.01 0.1 1 10 100 The trade-off parameter λ 0.01 0.1 1 10 100 The trade-off parameter λ 0.01 0.1 1 10 100 The trade-off parameter λ 0.01 0.1 1 10 100 The trade-off parameter λ 0.01 0.1 1 10 100 The trade-off parameter λ (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 0.80 0.78 0.01 0.1 1 10 100 The trade-off parameter λ 0.01 0.1 1 10 100 The trade-off parameter λ Data set: Flare1 EM HS 0.67 0.66 0.65 0.64 0.105 0.100 0.095 0.090 0.01 0.1 1 10 100 The trade-off parameter λ 0.01 0.1 1 10 100 The trade-off parameter λ 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. 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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. 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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
English
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Ringed fluorodeoxyglucose uptake predicted poor prognosis after resection of pulmonary pleomorphic carcinoma
Journal of cardiothoracic surgery
2,022
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. 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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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Electrolyte and renal disorders in patients with newly diagnosed glioblastoma
Future oncology
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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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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. 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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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Label-Free Quantitative Proteomics Combined with Biological Validation Reveals Activation of Wnt/β-Catenin Pathway Contributing to Trastuzumab Resistance in Gastric Cancer
International journal of molecular sciences
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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 u u , J a gbe ua , e o g u , J a u a g a d J a C a g 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 y q 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 g 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 p y g 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 t C ll 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 l ti th 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. 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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. 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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. 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Synthesis, Affinity for Complementary RNA and DNA, and Enzymatic Stability of Triazole-Linked Locked Nucleic Acids (t-LNAs)
ACS omega
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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 (CCH), 78.0 (CCH), 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, CCH), 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 (CCH), 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, CH2CCH), 4.29 (dd, J = 16.0, 2.4 Hz, 1H, CH2CCH), 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, CCH), 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
https://openalex.org/W2122163840
https://bmcproc.biomedcentral.com/track/pdf/10.1186/1753-6561-9-S3-A65
English
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Is there a relation between carpal tunnel syndrome and trigger finger?
BMC proceedings
2,015
cc-by
1,274
© 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 and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: 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%).
https://openalex.org/W2588906900
https://www.jtit.pl/jtit/article/download/978/967
English
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Intrusion Detection in Heterogeneous Networks of Resource-Limited Things
Journal of Telecommunications and Information Technology
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4,213
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
BMC neuroscience
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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. 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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 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 Page 14 of 14 (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
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
https://openalex.org/W3082753900
https://hal.science/hal-03103329/file/1482.full.pdf
English
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Phospholipase pPLAIIIα Increases Germination Rate and Resistance to Turnip Crinkle Virus when Overexpressed
Plant physiology
2,020
cc-by
14,651
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 Submitted on 11 Mar 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. 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 Downloaded on March 11, 2021. - Published by 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. https://plantphysiol.org Downloaded on March 11, 2021. - Published by 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 Downloaded on March 11, 2021. - Published by 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 Downloaded on March 11, 2021. - Published by 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 Downloaded on March 11, 2021. - Published by 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 Downloaded on March 11, 2021. - Published by 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 Downloaded on March 11, 2021. pPLAIIIa Expression Level Affects the Size and Shape of Many Cells and Organs - 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. 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 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 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 Downloaded on March 11, 2021. - Published by 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 Downloaded on March 11, 2021. - Published by 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 https://plantphysiol.org Downloaded on March 11, 2021. - Published by 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 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. 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 Downloaded on March 11, 2021. - Published by 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 Downloaded on March 11, 2021. - Published by 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 Downloaded on March 11, 2021. - Published by 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 Downloaded on March 11, 2021. - Published by 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. 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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 Downloaded on March 11, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
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Agricultural Real Property as a Subject of Limitations on Transactions Transferring Ownership
Studia Iuridica Lublinensia
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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 ( 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. 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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https://research-repository.st-andrews.ac.uk/bitstream/10023/19469/1/Schnor_2020_DR_Remain_CC.pdf
en
Remain, leave, or return? Mothers’ location continuity after separation in Belgium
Demographic research
2,020
cc-by
17,449
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. 246 http://www.demographic-research.org 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 247 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). 248 http://www.demographic-research.org 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 http://www.demographic-research.org 249 Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium 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 250 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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 http://www.demographic-research.org 251 Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium 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). 252 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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 http://www.demographic-research.org 253 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). 254 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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). http://www.demographic-research.org 255 Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium 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. 256 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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. http://www.demographic-research.org 257 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. 258 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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 http://www.demographic-research.org 259 Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium 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. 260 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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. http://www.demographic-research.org 261 Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium 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. 262 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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 http://www.demographic-research.org -.1 -.05 0 .05 Average marginal effect .1 .15 .2 263 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 264 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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 http://www.demographic-research.org 265 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. 266 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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 http://www.demographic-research.org -.15 -.1 -.05 0 .05 Average marginal effect .1 .15 .2 .25 267 Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium 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 268 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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 http://www.demographic-research.org 269 Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium 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. 270 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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 http://www.demographic-research.org 271 Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium 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 272 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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. http://www.demographic-research.org 273 Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium 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. 274 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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). 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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 http://www.demographic-research.org Demographic Research: Volume 42, Article 9 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%) http://www.demographic-research.org 283 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 http://www.demographic-research.org 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 http://www.demographic-research.org 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 http://www.demographic-research.org 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). 292 http://www.demographic-research.org
https://openalex.org/W4248482301
https://europepmc.org/articles/pmc8479974?pdf=render
English
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Emotional Reactivity to Binge Food and Erotic Cues in Women With Bulimia Nervosa Symptoms
Research Square (Research Square)
2,021
cc-by
9,627
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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​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. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​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. 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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. 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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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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. „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. 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https://figshare.com/articles/thesis/Restoring_the_Soils_of_Nauru_Plants_as_Tools_for_Ecological_Recovery/16992997/1/files/31431556.pdf
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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. 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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 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 4 5 3 2 4 3 2 3 2 4 4 4 1 2 1 1 5 2 1 3 2 5 4 2 3 3 5 1 5 1 3 4 1 1 2 5 3 4 1 3 1 2 1 3 4 2 2 4 5 1 4 5 3 5 3 5 2 4 3 2 2 3 5 1 4 5 5 4 1 1 4 2 3 3 2 1 5 4 5 1 3 5 5 2 2 1 1 3 5 4 1 3 4 3 2 4 4 3 4 4 5 2 4 5 1 1 3 1 1 5 3 2 2 2 2 5 3 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 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 4 5 3 2 4 3 2 3 2 4 4 4 1 2 1 1 5 2 1 3 2 5 4 2 3 3 5 1 5 1 3 4 1 1 2 5 3 4 1 3 1 2 1 3 4 2 2 4 5 1 4 5 3 5 3 5 2 4 3 2 2 3 5 1 4 5 5 4 1 1 4 2 3 3 2 1 5 4 5 1 3 5 5 2 2 1 1 3 5 4 1 3 4 3 2 4 4 3 4 4 5 2 4 5 1 1 3 1 1 5 3 2 2 2 2 5 3 4 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 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 4 5 3 2 4 3 2 3 2 4 4 4 1 2 1 1 5 2 1 3 2 5 4 2 3 3 5 1 5 1 3 4 1 1 2 5 3 4 1 3 1 2 1 3 4 2 2 4 5 1 4 5 3 5 3 5 2 4 3 2 2 3 5 1 4 5 5 4 1 1 4 2 3 3 2 1 5 4 5 1 3 5 5 2 2 1 1 3 5 4 1 3 4 3 2 4 4 3 4 4 5 2 4 5 1 1 3 1 1 5 3 2 2 2 2 5 3 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. 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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 4 5 3 2 4 3 2 3 2 4 4 4 1 2 1 1 5 2 1 3 2 5 4 2 3 3 5 1 5 1 3 4 1 1 2 5 3 4 1 3 1 2 1 3 4 2 2 4 5 1 4 5 3 5 3 5 2 4 3 2 2 3 5 1 4 5 5 4 1 1 4 2 3 3 2 1 5 4 5 1 3 5 5 2 2 1 1 3 5 4 1 3 4 3 2 4 4 3 4 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 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 4 5 3 2 4 3 2 3 2 4 4 4 1 2 1 1 5 2 1 3 2 5 4 2 3 3 5 1 5 1 3 4 1 1 2 5 3 4 1 3 1 2 1 3 4 2 2 4 5 1 4 5 3 5 3 5 2 4 3 2 2 3 5 1 4 5 5 4 1 1 4 2 3 3 2 1 5 4 5 1 3 5 5 2 2 1 1 3 5 4 1 3 4 3 2 4 4 3 4 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). 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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. 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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 12:468. 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https://openalex.org/W2777021866
https://jurnal.unimed.ac.id/2012/index.php/school/article/download/8093/6871
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PENGARUH PENGGUNAAN STRATEGI QUANTUM LEARNING TERHADAP KEMAMPUAN SISWA DALAM SPEAKING
School Education Journal PGSD FIP Unimed
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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
https://openalex.org/W3015203085
https://europepmc.org/articles/pmc7323539?pdf=render
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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Related content Influence of bone morphological properties on a new expandable orthopaedic fastener M Oldakowski, I Oldakowska, T B Kirk et al. - Study of post annealing effects on structural and optical properties of sol-gel derived ZnO thin films grown on n-Si substrate Aniruddh Bahadur Yadav, C Periasamy and S Jit - Mechanical activation influence on the morphological properties of La2O3-TiO2-B O Dolmatov, V Zakusilov, M Kuznetsov et al. - Influence of bone morphological properties on a new expandable orthopaedic fastener M Oldakowski, I Oldakowska, T B Kirk et al. - Study of post annealing effects on structural and optical properties of sol-gel derived ZnO thin films grown on n-Si substrate Aniruddh Bahadur Yadav, C Periasamy and S Jit - Mechanical activation influence on the morphological properties of La2O3-TiO2-B O Dolmatov, V Zakusilov, M Kuznetsov et al. - Structural and morphological properties of sol-gel ZnO:Ni films To cite this article: T Ivanova et al 2018 J. Phys.: Conf. Ser. 992 012044 To cite this article: T Ivanova et al 2018 J. Phys.: Conf. Ser. 992 012044 View the article online for updates and enhancements. Mechanical activation influence on the morphological properties of La2O3-TiO2-B O Dolmatov, V Zakusilov, M Kuznetsov et al. - This content was downloaded from IP address 130.238.141.100 on 06/12/2018 at 15:05 Journal of Physics: Conference Series Journal of Physics: Conference Series 1 Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 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.] 2degrees 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.] 2degrees 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
https://openalex.org/W4309900092
https://link.springer.com/content/pdf/10.1007/s12187-022-09993-7.pdf
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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
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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. 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To view a copy of this licence, visit http://​creat​iveco​\nmmons.​org/​licen​ses/​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. 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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
Frontiers in microbiology
2,018
cc-by
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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. 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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. 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Successful en bloc venous resection with reconstruction and subsequent radiotherapy for 2 consecutive recurrences of intravenous leiomyoma—a case report
BMC cancer
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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. 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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
Medical engineering & physics
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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. 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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
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 Submitted on 15 Feb 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 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. 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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 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
https://openalex.org/W3193708091
https://periodicos.ufmg.br/index.php/aletria/article/download/26194/27558
Portuguese
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"Combinação possível de palavras"
Aletria
2,021
cc-by
9,722
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 ƒ! while ~H is the oppositely directed C3 ƒ. 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 ƒ! and C3 ƒ. Con- sider the bipartite representations BðC3 ƒ!Þ and BðC3 ƒÞ, and take their symmetric difference r. It contains exactly one alternating cycle (the edges come alternately from BðC3 ƒ!Þ and BðC3 ƒÞ), 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 ƒ!Þ with all edges coming from BðC3 ƒÞ 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; V00Š 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; V00Š 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; VŠ contains exactly |U@| of . The right-hand side can be estimated from below as since bM0½U00; VŠ 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  2e2p2n=9 þ n  2e2ð1pÞ2m=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
https://openalex.org/W2950822047
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An extreme halophilic xylanase from camel rumen metagenome with elevated catalytic activity in high salt concentrations
AMB express
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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://snapg​ene.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′-AAGAA​TTC​CAA​GAA​ TGG​CAT​TTT​AAA​GAAAC-3′) and reverse (5′-AACTC​ GAG​TTC​GCC​CTC​AGC​GC-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/clust​alo/). The structural analyses were per- formed using SignalP-5.0 Server (http://www.cbs.dtu.dk/ servi​ces/Signa​lP/) and the NCBI conserved domain data- base (CDD) (https​://www.ncbi.nlm.nih.gov/Struc​ture/ cdd/wrpsb​.cgi). The prediction of the enzyme structure was performed using RaptorX web server (http://rapto​ rx.uchic​ago.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?
JAMA
1,907
public-domain
8,731
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