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https://openalex.org/W2765184011	https://revistaseletronicas.pucrs.br/index.php/iberoamericana/article/download/28882/16041	Portuguese	null	Para além do bem e do mal: um novo mundo nos trópicos	Estudos ibero-americanos/Estudos Ibero-Americanos	1,995	cc-by	5,066	• Aula inaugural do Instituto de Filosofia e Ciências Humanas da PUCRS, proferida em 6-3-95. •• Professora do Departamento de Ciências Sociais e do Curso de Pós-Graduação em História do Instituto de Filosofia e Ciências Humanas da PUCRS. ! TODOROV, Tzvetan.La conquête de l'Amérique. La question de L' autre. 1991. Paris, Seuil, p. 306. 2 VEYNE, Paul. O inventário das diferenças. História e Sociologia. 1983. São Paulo, Brasiliense, p. 54,55. Estudos Ibero-Americanos. PUCRS, v.Xx], n.l, p. 49-59, julho, 1995 2 VEYNE, Paul. O inventário das diferenças. História e Sociologia. 1983. São Paulo, Brasiliense, p. 54,55. • Aula inaugural do Instituto de Filosofia e Ciências Humanas da PUCRS, proferida em 6-3-95. PARA ALÉM DO BEM E DO MAL: UM NOVO MUNDO NOS TRÓPICOS* Léa Freitas Perez* * Duas observações expressam o tom de minha fala. A primeira é de Tzvetan Todorov, quando ele refere preferir assumir abertamente sua visão das coisas sem a travestir em descrição das coisas elas mesmas.' Ela faz eco às observações de Paul Veyne, segundo as quais "é mais importante ter idéias do que conhecer verdades" , pois" ter idéias significa também dispor de uma tópica, tomar consciência do que existe, explici- tá-Io, conceituá-Io, arrancá-Io à mesmice", mais ainda, "é deixar de ser inocente, e perceber que o que é poderia não ser", uma vez que "o real está envolto numa zona indefinida de compossíveis não-realizados; a verdade não é o mais elevado dos valores do conhecimento.' No percurso que tenho seguido nos últimos anos minha intenção tem sido a de dialogar com o Brasil, de olhar diferentemente nosso país, de tentar responder de outro modo às questões que nos inquietam desde sempre. Dito de outro modo: tento mudar o registro analítico e fazer uma releitura da história a partir da reflexão sociológica, tomando como ponto de partida a crítica de certas verdades estabelecidas, tentando ultrapassar a oposição entre o intelectual e o sensível, típica da compartimentação Estudos Ibero-Americanos, XXi( 1) - julho, 1995 50 de origem positivista ainda preponderante nas chamadas ciências huma- nas. Realizo, assim, um exercício de reflexão e uma tomada de posição no que diz respeito às relações da sociedade brasileira com a moderni- dade e, mais particularmente, no que diz respeito a uma certa concepção do Brasil para a qual, em nosso país, a modernidade é um projeto inacabado. Conforme já discuti em outra ocasião, segundo essa visão, o Brasil seria ainda incompleto, devendo se "modernizar" para "conquistar seu lugar no concerto das nações desenvolvidas".' É o que a doxa chama de "dilemas brasileiros". O país seria ainda uma potencialidade - "o gigante adormecido", "o país do futuro" - e não uma realidade acabada. Um "país dual" formado por contrastes e por contradições. Os "dois Brasis", eterna e irremediavelmente divorciados, de costas um para o outro. Uma "sociedade a duas velocidades", cindida entre o moderno e o tradicional, entre a ordem e a desordem. Essa maneira de ver o Brasil articula-se em torno de uma lógica da falta e opera a partir de oposições binárias, sobretudo das oposições tradicional-moderno e centro-perife- ria. O centro -o moderno - é a vanguarda. 3 PEREZ, Léa Freitas. "Por uma poética do sincretismo tropical". 1992. In: Estudos Ibero-Americanos. Porto Alegre, PUCRS, 18(2): 43-52, dezembro. PARA ALÉM DO BEM E DO MAL: UM NOVO MUNDO NOS TRÓPICOS* Lá, tudo é criado - de forma original - por isso o seu poder de tudo comandar. A periferia - o tradicional - não passa de uma retarguarda. Aqui, tudo é cópia e, portanto, sem originalidade. A modernização, sinônimo de crescimento econômico segundo os padrões do centro, é o único caminho para a periferia sair da marginalidade e da dependência. O ponto focal da mudança é a industrialização que, bem implantada, agiria no conjunto da sociedade subdesenvolvida de maneira a aproximá-Ia das sociedades desenvolvidas, conforme uma visão messiânica de que o poder da economia resolve todos os problemas e uma lógica claramente evolucio- nista: os mesmos processos levariam ao mesmo fim. Essa concepção do Brasil e de seus dilemas de país dividido aponta para uma forma de tradicionalização do moderno. Vivemos uma sorte de nostalgia do que jamais existiu, segundo um raciocínio do tipo "se tivesse sido diferente" e de uma postura teórica míope, para a qual a persistência da tradição é, ao mesmo tempo, um sintoma e uma prova da execução apenas parcial do projeto moderno. O di ã i d d d d j Ora, como a tradição que temos - independente de nossos desejos - é a da colonização portuguesa, é a ela que se deve atacar. Ah, se o Brasil tivesse sido colonizado pelos ingleses, tal como os Estados Unidos! Ah, Para além do bem e do mal: .. 51 se o holandeses ou os franceses não tivessem sido expulsos! O Brasil seria hoje uma grande potência! Resumindo: a colonização portuguesa é a causa de todos os problemas - os ditos males e paradoxos do país. O raciocínio implícito é o seguinte: é preciso destruir completamente esse passado colonial que é uma mancha e um entrave à Ordem e ao Progresso e, em seu lugar, construir um país e uma sociedade "autenticamente nacionais" e "uma nova ordem em sintonia com o mundo contemporâ- neo". Em nome da modernidade redentora, uma série interminável de operações é posta em marcha com o objetivo de pôr em seu verdadeiro e legítimo lugar as coisas e os homens. Num tal campo de idéias, o trabalho social desenvolvido pela colonização lusitana é mal compreen- dido e malíssimamente analisado. 4 FREYRE, Gilberto. Casa grande e senzala. Formação da família brasileira sob o regime da economia patriarcal. 1984. Rio de Janeiro, José Olympio, 23. ed., p. 15, 24. Oliveira Vianna. Apud FREYRE Casa grande e senzala. Op. cit., p. 24. 5 FREYRE. Casa grande e senzala. Op. cit., p. 4. 6 FREYRE. Casa grande e senzala. Op. cit., p. 4, 5. 7 Citando apenas dois exemplos: a bula Inter Coetera (1494) instituiu o patronato das terras conquistadas, colocadas sob a autoridade da Ordem de Cristo. Em 1551, pela bula Praeclara carissimi, a Ordem de Cristo é incorporada à Coroa de modo que o rei substituía o Santo-padre na administração religiosa dos territórios, percebendo os impostos correspondentes. 8 FREYRE. Casa grande e senzala. Op. cit., p. 8. PARA ALÉM DO BEM E DO MAL: UM NOVO MUNDO NOS TRÓPICOS* Retém-se unicamente a dimensão colonizadora do processo, tomada num sentido negativo, mais ainda, pejorativo, esquecendo-se - evidentemente por um processo de hiposta- sia - de seu caráter de efetiva construção, pouco importando, nesse sentido, o objetivo visado. Não se trata aqui de fazer uma apologia da empresa colonial, longe disso. Meu objetivo é simplesmente ver o Brasil português a partir de um olhar, ao mesmo tempo, mais objetivo e mais generoso. Para traçar um quadro global do trabalho social implicado na criação do Brasil e para situar o contraste entre a realidade inicial com que se depararam os colonizadores e o resultado obtido após três séculos de colonização proponho retomar alguns elementos. Os portugueses encontraram terra e homens em "estado bruto": uma realidade onde tudo era - para eles - desequilíbrio, do excessivo e do lacunar. A terra, ao contrário do sonho de Caminha, era rebelde ao trabalho agrícola, com matagais de difícil aproveitamento e com rios quase impróprios à navegação. Nem ouro, nem seda. Os homens? Selvagerís. Nos trópicos, aos olhos dos conquistadores, parecia faltar tanto a "riqueza organizada", quanto uma base para a atividade comercial tal como havia nas Índias. 4 Foi preciso preencher esse vazio absoluto. A promessa foi, para além do bem e do mal, cumprida. Seja como for, um mundo foi criado, uma sociedade complexa foi organizada. A América foi feita. Em torno de 1570, as bases do Brasil colonial estavam estabelecidas, a ordem Estudos Ibero-Americanos, XXI( 1) - julho, 1995 52 portuguesa inteiramente implantada. A sociedade organizada com a particularidade de ter sido forjada "menos pela ação oficial do que pelo braço e pela espada do particular".' Na base, a agricultura, a estabilidade dada pela família patriarcal e a regularidade do trabalho repousando na escravatura. Uma sociedade que se articulava em torno das grandes famílias e de suas casas. No comando, os potentados - senhores de terras e de escravos - "com altar e capelão dentro de casa e índios de arco e flecha ou negros armados de arcabuses às suas ordens". Homens inde- pendentes "que dos senados das Câmaras falaram sempre grosso aos representantes d'el Rei e pela voz liberal dos filhos padres ou doutores clamaram contra toda espécie de abuso da Metrópole e da própria Madre Igreja"." A colonização do Brasil foi uma obra de envergadura: empreendi- mento moderno que combinou ações comerciais e militares e cruzada civilizadora. 8 FREYRE. Casa grande e senzala. Op. cit., p. 8. Para além do bem e do mal: .. Para além do bem e do mal: .. 53 o Brasil considerado" a Nazaré das colônias portuguesas", um vazio onde não havia nem ouro, nem prata, cedo encontrados pelos espanhóis no México, mas "somente pau-de-tinta e almas para Jesus Cristo." o Brasil considerado" a Nazaré das colônias portuguesas", um vazio onde não havia nem ouro, nem prata, cedo encontrados pelos espanhóis no México, mas "somente pau-de-tinta e almas para Jesus Cristo." p p Capitais e técnicas foram investidos na colônia a fim de criar um fluxo de bens destinado ao mercado europeu. Sob a pressão das circuns- tâncias, o colonizador português foi o primeiro a transformar a coloni- zação tropical de pura extração de riquezas naturais em criação local de bens, cuja primeira expressão é a economia açucareira." O sucesso do empreendimento é atestado, entre outros fatores, pelo fato de que em 1560 já havia, entre Porto Seguro e Pernambuco, 60 engenhos de açúcar em pleno funcionamento e de que a partir de 1570 o Brasil tornou-se o principal produtor mundial de açúcar. A colônia de plantação "caracteri- zada pela base agrícola e pela fixação do colono na terra inaugurou uma nova fase na exploração colonial, dando origem à formação de uma sociedade singular. A realização da colonização em um território tão imenso quanto vazio, como parecia ser o caso do Brasil, sobretudo para um empreen- dedor que, como era o caso de Portugal, dispunha de um pequeno contingente populacional, era um tarefa de peso. A solução mais razoá- vel, diante desse quadro nada favorável, era alocar o grosso do trabalho nas mãos da iniciativa privada, responsável pelos gastos de instalação e pela defesa militar do empreendimento. As dificuldades, sabidas em grande número, seríam recompensadas através da concessão de privilé- gios relativos ao comando e à jurisdição das terras. Dessa maneira, a astuta metrópole criava um poderoso e atrativo elo entre possessão territorial e poder de mando, característica de base do sistema patriarcal brasileiro, cuja encarnação paradigmática é a figura do senhor de enge- nho. Paralelamente à implantação da base econômica era preciso orga- nizar o espaço. A coroa implantou o sistema de capitanias hereditárias (1532), segundo o modelo já utilizado nas ilhas Madeira, no arquipélago dos Açores, em Cabo Verde e em São Torné. 9 FREYRE. Casa grande e senzala. Op. cit., p. 242. 10 FREYRE. Casa grande e senzala. Op. cit., p. 17. PARA ALÉM DO BEM E DO MAL: UM NOVO MUNDO NOS TRÓPICOS* A terra foi consolidada nas mãos portuguesas pela via da força armada, uma conquista militar. O próprio empreendimento ultra- marino foi feito sob as graças papais, como uma cruzada moderna, cujas bulas reconheciam e aprovavam os primeiros passos.' Em que pesem esses aspectos, a expansão portuguesa jamais implicou a aplicação de regras imperativas e rígidas. A coroa sempre soube ceder face às conve- niências e às necessidades. O "realismo econômico" dos portugueses corrigiu, desde o começo, os excessos do espírito militar e religioso presentes na formação da sociedade colonial." Os acontecimentos pós 1530 são um bom exemplo. A partir de 1530 ocorreu uma grande mudança nas relações entre a metrópole lusitana e a colônia tropical. O Brasil deixou de ser um mero empreendimento de exploração de produtos naturais para transformar-se em parte ativa da economia de reprodução em escala mundial como retaguarda agrícola de Portugal. A mudança foi ainda mais admirável se levarmos em conta que no começo a colonização foi feita, como observa Gilberto Freyre, "quase sem vontade, com um sobej o apenas de homens", Para além do bem e do mal: .. Fracassado o sistema das capitanias hereditárias, a metrópole introduziu um novo sistema admi- nistrativo, o Governo Geral (1549), tomando o êxito de São Vicente e Pernambuco como modelos. Uma tal reorganização administrativa mos- tra o sucesso da exploração econômica, em que pese o insucesso do Estudos Ibero-Americanos, XXI( 1) - julho, 1995 54 sistema administrativo: "fracassaram as capitanias, mas prosperava a terra; malograva-se o sistema, mas vingava o negócio"." Ao findar o século XVII, nova mudança de situação para o reino ibérico. Não tinha mais o monopólio do comércio do açúcar, nem das especiarias orientais. Pouco a pouco se tornava uma colônia inglesa. A queda do preço do açúcar devido à concorrência antilhana implicava graves problemas financeiros para a coroa. No Brasil, a economia açucareira encontrava-se desorganizada e a população dedicava-se uni- camente a atividades de subsistência. Os colonos não podiam importar como faziam antes, pois exportavam pouco. O sistema estava em crise profunda; mas intervém o milagre: o ouro é descoberto, salvando todos. Uma vez mais a terra dos rios de mel mostrou sua generosidade. O paraíso sobre a terra tornou-se de novo factível. O ouro tudo mudou, mesmo que por um fugaz período. A época gloriosa da colonização portuguesa teve fim no século XVIII. A mineração aurífera, geradora de grandes riquezas, após um período áureo entre 1740-1760, deixava de ser economicamente rentá- vel. A colônia rica e poderosa exigia reformas e mudanças. Tanto em Portugal quanto no Brasil, desde o fim do século XVIII surgiram uma multiplicidade de reformadores e de projetos. A necessidade de reformar o sistema é consensual. O marquês de Pombal é a expressão mais desenvolvida da tendência modernizante que despontou na. metrópole. Sob a égide do pombalismo reformador, a metrópole tomou medidas que tendiam a uma certa modernização, como a liberação da implantação de manufaturas de ferros (1795) e mudanças no regime de exploração das minas (1803). Fato é, portanto, que desde cedo a questão da moderniza- ção foi posta em cena no Brasil. Uma modernização que significava, antes de mais nada, reformismo, sem pôr em causa a ordem estabeleci da. Os ventos do contexto internacional impulsionavam ainda para mais Ionge. A independência dos Estados Unidos (1776) e o pensamento iluminista causaram forte impressão nos jovens brasileiros que estuda- vam na Europa. Emergiram algumas idéias de autonomia em relação metrópole, sendo a Inconfidência Mineira uma de suas manifestações. 11 FAORO, Raymundo. Os Donos do Poder. Formação do patronato político brasilei- ro. 1989, v. 1 e 2. Rio de Janeiro, Globo, 8. ed., p. 143. 12 BASTIDE, Roger. Brésil. Terre des contrastes. 1957, Paris, Hachette, p. 13. 13 Um universo plurivocal é caracterizado pela plural idade de vozes e de consciências eqüipolentes e distintas pela polifonia. Diferentes vozes cantam diferentemente um mesmo tema. BAKHTINE, Mikhail. La poétique de Dostoievski. 1970. Paris, Edi- tions du Seuil, p. 32, 33, 79. p 14 ELIADE, Mircea. Le mythe de l'éternel retour. 1963. Paris, Gallimard, p. 134 15 SIMMEL, Georg. Philosophie de Ia modernité. Tomme J. La femme, Ia ville, l'indi- vidualisme. 1989. Paris, Payot, p. 307, 308. 2 BASTIDE, Roger. Brésil. Terre des contrastes. 1957, Paris, Hachette, p. 13. 12 BASTIDE, Roger. Brésil. Terre des contrastes. 1957, Paris, Hachette, p. 13. 13 Um universo plurivocal é caracterizado pela plural idade de vozes e de consciências eqüipolentes e distintas pela polifonia. Diferentes vozes cantam diferentemente um mesmo tema. BAKHTINE, Mikhail. La poétique de Dostoievski. 1970. Paris, Edi- tions du Seuil, p. 32, 33, 79. 14 ELIADE, Mircea. Le mythe de l'éternel retour. 1963. Paris, Gallimard, p. 134. 15 SIMMEL, Georg. Philosophie de Ia modernité. Tomme J. La femme, Ia ville, l'indi- vidualisme. 1989. Paris, Payot, p. 307, 308. Para além do bem e do mal: .. Após 1792, de modo crescente, explodiram os conflitos estruturais do sistema colonial. O senhor de engenho e o grande fazendeiro brasi- leiros se viram submetidos às pressões de seus credores, os comerciantes Para além do bem e do mal: .. 55 portugueses. Os mulatos, um grupo já importante da população, recla- mavam sua exclusão social. O pequeno colono vivia com mais dificul- dade a exploração dos grandes senhores. O comerciante não fazia senão seu trabalho quando exigia o pagamento das dívidas. O escravo queria a liberdade. Os brancos e os senhores, cônscios de sua "natural supe- rioridade", aplicavam as regras estabelecidas "desde sempre". O açúcar já não era mais o centro da riqueza econômica. O ouro encontrava-se em queda livre. O café, apenas começava a aparecer. Em resumo: todas as partes do sistema tinham sua parte de razão e eram complementares. Diria que eram complementares por causa mesmo dos conflitos que os opunham. No Brasil, como observou judiciosamente Roger Bastide, "se a harmonia existe até no contraste, o contraste continua até a reconcilição dos antagonismos"." Esta mistura de vozes nos coloca face a um univer- 1· 113 so p unvoca. No fim a criatura havia suplantado o criador. No começo do século XIX, a ordem colonial tinha seus dias contados. Em uma palavra: o paraíso mítico havia sido perdido pelo simples fato de que havia sido realizado, havia tomado forma e durava." Na aventura, e a construção de um mundo novo nos trópicos o é, existe uma estreita relação entre o destino exterior e as fontes interiores da vida, de modo a produzir uma impressão de contraste, pois na aventura "a continuidade da vida é rejeitada por princípio, uma vez que pré-existe uma qualidade de estran- geiro, de intocável, de fora-de-série". Vale dizer que a aventura é independente de qualquer tipo de linearismo, de antes e de depois, ela determina seus próprios limites, ou seja, "a aventura não termina porque uma outra coisa começa, mas porque sua forma temporal, seu ato radical de terminar é exatamente a realização de seu sentido interior" .15 Se a ação colonizadora portuguesa foi calcada na tradição, na continuidade, isso não impossibilitou alterações de percurso quando necessárias. 16 Dando apenas um exemplo das dificuldades de comunicação entre Portugal e sua colônia tropical: durante o século XIX, e com um bom navio à vela, eram necessários trinta e cinco dias para percorrer o trecho Lisboa-Bahia; no século XVI podia se prolongar até dois ou três meses. Para além do bem e do mal: .. Mesmo que a maneira inicial de organizar a colônia tropical seguisse os mesmos princípios já testados na África e no Oriente, a Estudos Ibero-Americanos, XXI(1) - julho, 1995 56 conquista e o domínio de uma realidade nova, no caso do Brasil tão radicalmente diversa, foi uma situação que exigiu improvisação e adap- tação. A política geral, as grandes direções de intenção foram acompa- nhadas de gestos particulares, relativos ao caso concreto. O ajustamento da regra global ao caso particular se impunha. A plasticidade e a flexibilidade tornaram-se obrigatórias. Não existia possibilidade para uma única via. A mistura era intrínseca, imperativa mesmo. A metrópole se adaptava às contingências, sabendo manter a tradição de continuidade onde era preciso, do mesmo modo que era capaz de introduzir inovações, conio também misturar, com sucesso, o novo e o antigo. Da mistura, ou ainda melhor da mestiçagem, entre espírito de cruzada, exploração comercial e aventura, resultou um sistema social rico, que compõe um vasto campo para a análise sociológica. p p g Os eixos de compreensão do sistema colonial luso-brasileiro são, no plano econômico, a monocultura, o escravismo e a grande propriedade e, no plano sócio-cultural, o patriarcalismo. A monocultura associada ao escravismo nos deu um perfil de sociedade agrícola em termos de estrutura e de técnica de exploração econômica. Se a economia agrícola compunha a ossatura da sociedade colonial brasileira, sua carne era dada, sem a menor sombra de dúvida, pelo patriarcalismo. Se o patriarcalismo podia ser encontrado como possibilidade na ordem patrimonial portu- guesa e, assim, nos princípios que norte aram a colonização dos trópicos, foi o meio local brasileiro que determinou seu florescimento. A autori- dade pública, para além dos problemas provocados pelas distâncias entre a metrópole e a colônia, tinha que compor com o forte poder enraizado dos senhores de terra e de escravos; ela precisava contar com ele para poder agir no conjunto do território." Quem detinha efetivamente o poder, a autoridade e o prestígio era o grande proprietário; este, não o esqueçamos, observação aparentemente simples, mas freqüentem ente esquecida nas análises sobre o período colonial, se, no começo, era um português, ou seja, um colonizador, em uma ou duas gerações - não mais - era um homem da terra, isto é, um brasileiro. 17 FREYRE. Casa grande e senzala. Op. cit., p. 190. 18 HOLANDA, Sérgio Buarque de. Raizes do Brasil. 1991. Rio de Janeiro, José Olympio, 22 ed., p. 18. 19 Os movimentos cíclicos são definidos como aqueles onde "a economia, no fim, retoma a seu ponto de partida". Assim, eles se opõem aos movimentos de longa duração, que "levam a mudanças estruturais". MAURO, Frédéric. Le Brésil du XV" à ia fin du XVIII" siêcle. 1977. Paris, Société d'Edition d'Enseignement Supérieur, p.37. OLANDA, Sérgio Buarque de. Raizes do Brasil. 1991. Rio de Janeiro, José lympio, 22 ed., p. 18. 17 FREYRE. Casa grande e senzala. Op. cit., p. 190. 20 Cf. PRADO JUNIOR, Caio. Formação do Brasil contemporâneo. Colônia. 1969. São Paulo, Brasiliense, 9. ed., p. 286. 21 ELIADE. Le mythe de l'éternel retour. Op. cit., p. 107. 22 ELIADE. Le mythe de l'éternel retour. Op. cit., p. 105, 161, 162, 164. 23 ELIADE. Le mythe de l'éternel retour. Op. cit., p. 106, ] 19,152. Para além do bem e do mal: .. É moeda corrente dizer-se que a expansão portuguesa foi uma sucessão de ciclos: o desenvolvimento até o esgotamento de uma certa estrutura econômica centrada em torno de um único produto comercial." De acordo com essa lógica econômica, o Brasil teria sido concebido e acordo com essa lógica econômica, o Brasil teria sido concebido e Estudos Ibero-Americanos, XXI(1) - julho, 1995 58 organizado como uma empresa de exploração de produtos naturais: o pau-brasil, o açúcar, o ouro e o café. A economia brasileira teria se desenvolvido por saltos, isto é, por ciclos que se sucederiam uns aos outros, seja no tempo, seja no espaço e que teriam sempre nos levado da prosperidade à ruína." Não se poderia ver, para além desse olhar negativo da história, essencialmente moderno, uma outra concepção do tempo, da história e das relações com a natureza e com o espaço que guarda semelhanças com as chamadas sociedades tradicionais (não modernas)? Se não vejamos. Nas concepções cíclicas do tempo e da história, a noção de retorno é uma maneira de anular a irreversibilidade do tempo. No tempo cíclico tudo recomeça em seu começo a cada instante. Nenhum acontecimento é irreversível e nenhuma transformação é definitiva." Não se pode negar que a concepção cíclica do tempo e da história, típica das sociedades tradicionais, manteve-se nas chamadas sociedades modernas, sobretudo até o século XVII. É somente a partir dessa época que, pouco a pouco, "o linearismo e a concepção progressiva da história se afirmam, instaurando a fé em um progresso infinito", até sua vulgari- zação no século XIX, com o triunfo das idéias evolucionistas. A econo- mia política moderna, em sua versão evolucionista, reabilita as noções de ciclo, de flutuação, de oscilação periódica características das concep- ções tradicionais. É justo, pois, recolocar a questão, formulada por Mircea Eliade, se no fundo não seria somente nas teorias modernas que o sentido do mito arcaico da eterna repetição receberia todo seu alcance.f Não se pode negar que a concepção cíclica do tempo e da história, típica das sociedades tradicionais, manteve-se nas chamadas sociedades modernas, sobretudo até o século XVII. É somente a partir dessa época que, pouco a pouco, "o linearismo e a concepção progressiva da história se afirmam, instaurando a fé em um progresso infinito", até sua vulgari- zação no século XIX, com o triunfo das idéias evolucionistas. Para além do bem e do mal: .. O produto sócio-econômico da ordem patriarcal brasileira foi a grande exploração agrícola, sob a forma do engenho de açúcar e da fazenda (criação de gado e de café), conduzida pela mão forte do todo Para além do bem e do mal: .. 57 poderoso patriarca, senhor da vida e da morte de seus sujeitos - as mulheres, os agregados e os escravos. Colocado no centro da vida econômica e social da colônia, o grande proprietário tornava-se um aristocrata. Ele possuía tudo o que efetivamente importava possuir: o poder, a autoridade e a tradição, esta última ancorada na família. O sistema de colonização deixava campo livre à ação individual: o colono fez-se no Brasil "senhor de terras mais vastas, dono de homens mais numerosos, que qualquer outro colonizador da América"." Tanto em Portugal como no Brasil, a ascensão social através da prestação de "bons serviços" ao Estado sempre permitiu um amplo desenvolvimento das relações pessoais, centradas em laços de consentimento, de fidelida- de, de simpatia e de obediência. Qualquer um podia tornar-se nobre. Em Portugal, dizia-se mesmo que todo mundo era nobre, se não de fato, ao menos de direito e, mais ainda, em modo de ser. A contrapartida da nobreza generalizada, que caracterizava o mundo colonial, era uma profunda aversão pelo trabalho ordinário, de onde a opção pelo escravis- mo - um dos centros do complexo colonial. Na mentalidade ibérica, o ideal em relação ao trabalho era uma digna ociosidade, uma vida de grande senhor, sem preocupações e sem o menor esforço. O que o português procurava não Brasil era a riqueza que custava audácia, mas jamais trabalho." Relembro, de passagem, uma referência a essa mora- lidade presente em nosso hino: o Brasil seria um país deitado eternamente em berço esplêndido. Certamente polêmica, de sentido dúbio, todavia reveladora, no plano sociológico, de uma certa maneira de ser e de se relacionar com o mundo que se afasta do paradigma burguês clássico do homem racional- trabalhador, disciplinado, organizado, poupador, etc. - e que se aproxima mais do modelo de homem da corte do século XVIII. Para além do bem e do mal: .. A econo- mia política moderna, em sua versão evolucionista, reabilita as noções de ciclo, de flutuação, de oscilação periódica características das concep- ções tradicionais. É justo, pois, recolocar a questão, formulada por Mircea Eliade, se no fundo não seria somente nas teorias modernas que o sentido do mito arcaico da eterna repetição receberia todo seu alcance.f A lógica luso-brasileira não seria solidária a um certo "caráter otimista da vida? A uma visão cuja idéia central é a de que nada é jamais completamente definido, que "tudo tem lugar de uma maneira cíclica, a morte é seguida inevitavelmente de uma ressureição, o cataclisma de uma nova criação"? O caráter otimista é marcado por uma consciência da normalidade da catástrofe cíclica, isto é, a certeza de que ela tem um sentido e sobretudo de que ela não é jamais definitiva. A regeneração necessariamente acontece." Uma perpétua renovação. Essa organização por ciclos não seria uma das particularidades da modernidade portugue- sa? p ç A lógica luso-brasileira não seria solidária a um certo "caráter otimista da vida? A uma visão cuja idéia central é a de que nada é jamais completamente definido, que "tudo tem lugar de uma maneira cíclica, a morte é seguida inevitavelmente de uma ressureição, o cataclisma de uma nova criação"? O caráter otimista é marcado por uma consciência da normalidade da catástrofe cíclica, isto é, a certeza de que ela tem um sentido e sobretudo de que ela não é jamais definitiva. A regeneração necessariamente acontece." Uma perpétua renovação. Essa organização por ciclos não seria uma das particularidades da modernidade portugue- sa? Para além do bem e do mal: .. Para além do bem e do mal: .. 59 A colonização do Brasil é solidária ao espírito de uma época plena de transformações, a um "tempo tão novo e com nenhum outro pareci- do" .24 A ação colonial portuguesa é tributária da modernidade ocidental e de seu projeto civilizador. 24 Las casas Apud. TODOROV. La conquête de l'Amérique. Op. cit., p. 13. 25 Tomo de empréstimo a Sérgio Buarque de Holanda as idéias de ética da aventura e concepção espaciosa do mundo. HOLANDA. Raizes do Brasil. Op. cit., p. 13, 15, 16. 26 PEREZ. "Por uma poética do sincretismo tropical". Op. cit., p. 51. Para além do bem e do mal: .. 24 Las casas Apud. TODOROV. La conquête de l'Amérique. Op. cit., p. 13. 16. 26 PEREZ. "Por uma poética do sincretismo tropical". Op. cit., p. 51. Para além do bem e do mal: .. Todavia, ela foi orientada por uma ética da aventura e por uma concepção espaciosa e otimista do mundo, cujos principais elementos são a aspiração de obter sem custo tanto a prospe- ridade e a riqueza, quanto os títulos e as posições sociais privilegiadas, o que não é de ordem completamente moderna, no sentido de racional, impessoal, planificado, etc." Nas motivações que impulsionaram o pequeno reino lusitano em direção ao vasto oceano se faziam presentes, de modo ainda vivo, os sonhos dos cavalheiros do medievo. Éjustamente essa combinação entre tradição e modernidade que possibilitou a expan- são colonial e a criação de um novo mundo nos trópicos. ç p Na lógica seguida por este trabalho nada permite pensar que as analogias que traço entre as sociedades portuguesa e brasileira baseiam- se em uma perspectiva segundo a qual a origem condiciona o futuro. É justamente a crítica a esse tipo de olhar, típico da lógica da falta, que tento empreender. Meu objetivo é mostrar que, se quisermos melhor compreender esse mundo novo nos trópicos, é preciso levar em conta o realismo e o sonho, o velho e o novo, o espírito romântico e o espírito histórico, a mistura de elementos de organização social e de códigos de relação, num entrelaçamento particular de modernidade e de tradição. Repito aqui o que já disse antes, seguindo uma inspiração de Jean Baudrillard: paremos de procurar a "Revolução que não existe, a Grande Transformação que não tem sentido! No tombemos na armadilha dos "utópicos nostálgicos dilacerados pelo ideal, mas no fundo repugnando 1· -" 26 sua rea lzaçao .
https://openalex.org/W2963133454	https://www.nature.com/articles/s41467-019-11275-w.pdf	English	null	KLF-1 orchestrates a xenobiotic detoxification program essential for longevity of mitochondrial mutants	Nature communications	2,019	cc-by	17,722	1 Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD) and Institute for Mitochondrial Diseases and Ageing, Medical Faculty, University of Cologne, D-50931 Cologne, Germany. 2 Center for Molecular Medicine Cologne (CMMC), D-50931 Cologne, Germany. Correspondence and requests for materials should be addressed to A.T. (email: aleksandra.trifunovic@uk-koeln.de) ARTICLE ARTICLE ARTICLE In worms, klf-1 is expressed in the intestine, hypodermis and a handful of neurons (Supplementary Fig. 2b). Recently, KLF-1 has been implicated in the regulation of the longevity phenotype upon dietary restriction and insulin signalling, together with KLF- 3, through its role in the regulation of autophagy12,13. However, the exact function of KLF-1 and its downstream targets remains largely unknown. j g Interestingly, analyses of expression proﬁles from long-lived mice, including calorically restricted mice, different dwarf mice or mice treated with rapamycin, revealed that many CYPs are upregulated and positively correlate with increased longevity4,5. Moreover, increased expression of multiple cyp genes was reported in diverse long-lived C. elegans models, including mitochondrial mutants6,7. Although interesting, these ﬁndings provided just a correlative connection to longevity. Importantly, little is known about the activation and regulation of xenobiotic responses in respect to longevity, since most studies to date have focused only on describing changes in the DME expression in different mutants and conditions1. g y To understand the role of KLF-1 in mitomutants longevity, we analysed it in the context of the previously reported isp-1;ctb-1 phenotypes9. While KLF-1 depletion did not affect the delayed development and the smaller brood size of the isp-1;ctb-1, the lower movement rates (at the ﬁfth day of adulthood—D5) were further decreased (Supplementary Fig. 3a–c). Likewise, klf-1 knockdown at D5 caused a further decrease in the respiration rate of isp-1;ctb-1 (Supplementary Fig. 3d). Nevertheless, this decrease is unlikely to contribute to the loss-of-longevity phenotype since a much stronger drop in oxygen consumption after cco-1 knock- down, a subunit of respiratory complex IV, in the isp-1;ctb-1 animals further prolongs lifespan (Supplementary Fig. 3e). Here we identify Krüppel-like factor 1 (KLF-1) as a major regulator of the detoxiﬁcation response involved in longevity assurance in Caenorhabditis elegans. We show that upon mild mitochondrial dysfunction and/or oxidative stress, KLF-1 trans- locates to the nucleus and activates cyp genes that in different organisms often encode enzymes involved in the xenobiotic detoxiﬁcation process. We further show that a redox-regulated KLF-1 activation coincides with the peak of somatic mitochon- drial biogenesis that occurs between L3 and D1 and is essential for the mitohormetic response that dictates longevity. ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w Remarkably, klf-1 knockdown only during adulthood (Fig. 1b) completely suppressed the long-lived phenotype in the isp-1;ctb-1 mutant, whereas knockdown during developmental stages did not have an effect (Fig. 1c), even when performed over two generation (Supplementary Fig. S1d). Similarly, klf-1 deﬁciency during adulthood decreased the longevity induced by the developmental knockdown of different mitochondrial respiratory chain (MRC) subunits (Supplementary Fig. 1e). KLF-1 activity in the early adulthood (ﬁrst day of adulthood—D1) seems to be crucial for longevity assurance, since the klf-1 deﬁciency starting from third or ﬁfth day of adulthood, had much milder effect on the phenotype (Supplementary Fig. 1f). This is further complemented with the observed 2.5-fold increase in the klf-1 expression in isp-1; ctb-1 mutant at D1, levels that are reached in WT animals only later in the adulthood (Fig. 1d, e). L L ifespan extension in different species can be achieved by various genetic manipulations and treatments, such as dis- ruption of insulin/IGF1 signalling, decrease in mitochon- drial respiration, suppression of translation or caloric restriction. Despite very different origins of these longevity programmes, they all warrant increased resistance to various stresses like heat, oxidative stress or radiation1,2. Although the concept that lifespan might depend on the capacity to withstand external stress cues is very old, little is currently known about signalling pathways underlying these cytoprotective responses and their ability to affect lifespan. Furthermore, how much an individual cytopro- tective mechanism contributes to the lifespan extension induced by different manipulations is a key question that remains to be answered2. Transcription proﬁling of many long-lived mutants from worm to mouse has recently revealed that upregulation of a number of genes involved in xenobiotic detoxiﬁcation is common to longevity-assurance pathways across different phyla1. Xenobiotic detoxiﬁcation includes activation of drug-metabolizing enzymes (DMEs), which are classiﬁed in two main groups: phase I—mainly cytochrome P450 oxidases (CYPs) and phase II—mainly UDP- glucuronosyltransferases (UGTs), glutathione-S-transferases (GSTs), sulfotransferases, and acetyltransferases, coupled to the activity of phase III transporters that mediate the efﬂux of meta- bolic end products out of the cells after the completion of phase II conjugation3. KLF-1 does not act through UPRmt or HIF-1 pathway. C. ele- gans klf-1 encodes a protein of the Krüppel-like transcription factor family (KLF) that shares high homology to the Zn-ﬁnger domain of other KLF proteins (Supplementary Fig. 2a) and its closest mammalian homologues are KLF2, KLF4 and KLF5. ARTICLE p g p pp y g To further elucidate the KLF-1 role in longevity assurance of isp-1;ctb-1 mutants, we analysed pathways previously shown to be important for lifespan extension by mitochondrial dysfunction, like the mitochondrial unfolded protein response (UPRmt) and HIF-1 pathway11,14. Loss of KLF-1, however, did not show any signiﬁcant effect on the highly activated UPRmt in isp-1;ctb-1 animals (Fig. 2a, b). Similarly, our results imply that the HIF-1 pathway is not directly regulated by KLF-1 as we did not detect a change in the level of its bona ﬁde targets (nhr-57 and egl-9) in young isp-1;ctb-1 mutants (Fig. 2c). Remarkably, the HIF-1 pathway seems to be suppressed in isp-1;ctb-1 mutants at D5 of adulthood, a feature that is lost upon KLF-1 depletion (Fig. 2c). Thus, our ﬁndings strongly imply that (i) both the UPRmt and HIF-1 pathways are only indirectly affected by the hormetic response later in life (D5) by the KLF-1 depletion; (ii) although these pathways could still contribute to the observed phenotypes, they are most likely not the main source of lifespan reduction caused by the klf-1 depletion. KLF-1 orchestrates a xenobiotic detoxiﬁcation program essential for longevity of mitochondrial mutants Marija Herholz1, Estela Cepeda1, Linda Baumann1, Alexandra Kukat1, Johannes Hermeling1, Sarah Maciej1, Karolina Szczepanowska1, Victor Pavlenko1, Peter Frommolt1 & Aleksandra Trifunovic 1,2 Most manipulations that extend lifespan also increase resistance to various stress factors and environmental cues in a range of animals from yeast to mammals. However, the underlying molecular mechanisms regulating stress resistance during aging are still largely unknown. Here we identify Krüppel-like factor 1 (KLF-1) as a mediator of a cytoprotective response that dictates longevity induced by reduced mitochondrial function. A redox-regulated KLF-1 activation and transfer to the nucleus coincides with the peak of somatic mitochondrial biogenesis that occurs around a transition from larval stage L3 to D1. We further show that KLF-1 activates genes involved in the xenobiotic detoxiﬁcation programme and identiﬁed cytochrome P450 oxidases, the KLF-1 main effectors, as longevity-assurance factors of mitochondrial mutants. Collectively, these ﬁndings underline the importance of the xenobiotic detoxiﬁcation in the mitohormetic, longevity assurance pathway and identify KLF-1 as a central factor in orchestrating this response. 1 Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD) and Institute for Mitochondrial Diseases and Ageing, Medical Faculty, University of Cologne, D-50931 Cologne, Germany. 2 Center for Molecular Medicine Cologne (CMMC), D-50931 Cologne, Germany. Correspondence and requests for materials should be addressed to A.T. (email: aleksandra.trifunovic@uk-koeln.de) 1 NATURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications TURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w EV Percent survival a klf-1 RNAi Percent survival Days N2 wild type EV Egg L1 L2 L3 L4 Adult EV b klf-1 RNAi Percent survival Days 0 0 20 60 100 20 40 60 EV EV_klf-1 RNAi Percent survival Days 0 0 20 60 100 20 40 EV_klf-1 RNAi c EV klf-1 RNAi_EV N2 wild type EV klf-1 RNAi_ EV Percent survival Days 0 0 20 60 100 20 40 60 Percent survival Days 0 0 20 60 100 20 40 isp-1(qm150);ctb-1(qm189) N2 wild type N2 wild type Egg L1 L2 L3 L4 Adult EV klf-1 RNAi N2 wild type isp-1(qm150);ctb-1(qm189) isp-1(qm150);ctb-1(qm189) Percent survival Days 0 0 20 60 100 20 40 N2 wild type klf-1(tm1110) EV N2 wild type klf-1 RNAi Days 0 20 40 0 50 100 0 50 100 0 20 40 EV Percent survival a klf-1 RNAi Percent survival Days Egg L1 L2 L3 L4 Adult EV klf-1 RNAi isp-1(qm150);ctb-1(qm189) N2 wild type Percent survival Days 0 0 20 60 100 20 40 N2 wild type klf-1(tm1110) EV N2 wild type klf-1 RNAi Days 0 20 40 0 50 100 0 50 100 0 20 40 EV klf-1 RNAi Percent survival Days N2 wild type 0 20 40 0 50 100 Percent survival a isp-1(qm150);ctb-1(qm189) EV N2 wild type klf-1 RNAi Days 0 50 100 0 20 40 a EV Percent survival a klf-1 RNAi Percent survival Days N2 wild type EV Egg L1 L2 L3 L4 Adult EV b klf-1 RNAi Percent survival Days 0 0 20 60 100 20 40 60 EV EV_klf-1 RNAi Percent survival Days 0 0 20 60 100 20 40 EV_klf-1 RNAi c EV klf-1 RNAi_EV N2 wild type EV klf-1 RNAi_ EV Percent survival Days 0 0 20 60 100 20 40 60 Percent survival Days 0 0 20 60 100 20 40 L4 D1 D5 0 1 2 3 4 Relative expression (fold change) e pklf-1::gfp Wild type isp-1;ctb-1 N2 wild type isp-1;ctb-1 klf-1 d isp-1(qm150);ctb-1(qm189) N2 wild type N2 wild type Egg L1 L2 L3 L4 Adult EV klf-1 RNAi N2 wild type isp-1(qm150);ctb-1(qm189) isp-1(qm150);ctb-1(qm189) Percent survival Days 0 0 20 60 100 20 40 N2 wild type klf-1(tm1110) EV N2 wild type klf-1 RNAi Days 0 20 40 0 50 100 0 50 100 0 20 40 Fig. 1 KLF-1 mediates the longevity of mitochondrial isp-1;ctb-1 mutant. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w a Lifespan curves of isp-1(qm150);ctb-1(qm189) mutant (left) and wild-type animals (middle) grown on control (empty vector, EV) or klf-1 RNAi plates. Right panel shows lifespan curve of klf-1(tm1110) mutant animals. b isp-1(qm150);ctb-1 (qm189) mutants (left) and wild type animals (right) were grown on control (EV) RNAi plates until L4 developmental stage and then transferred to klf-1 RNAi. c Lifespan curve of isp-1(qm150);ctb-1(qm189) mutant (left) and wild-type animals (right) grown on klf-1 RNAi plates until L4 developmental stage and then transferred to control (EV) RNAi. d klf-1 expression level was assessed by qPCR at the L4 larval stage, the ﬁrst (D1) and the ﬁfth day (D5) of adulthood, in the wild-type and isp-1(qm150);ctb-1(qm189) mutants. Data are presented as mean ± SEM. p < 0.01, p < 0.001, one-way ANOVA with Tukey post hoc test. n = 5 samples per condition. e Fluorescent images of gfp expression under klf-1 promoter in wild type and isp-1(qm150);ctb-1(qm189) at the ﬁrst day of adulthood. Scale bar 100 µm Percent survival Days 0 0 20 60 100 20 40 N2 wild type klf-1(tm1110) N2 wild type b Percent survival Days 0 0 20 60 100 20 40 60 EV EV_klf-1 RNAi isp-1(qm150);ctb-1(qm189) b c N2 wild type EV klf-1 RNAi_ EV Percent survival Days 0 0 20 60 100 20 40 60 Egg L1 L2 L3 isp-1(qm150);ctb-1(qm189) c EV klf-1 RNAi_EV Percent survival Days 0 0 20 60 100 20 40 N2 wild type L4 D1 D5 0 1 2 3 4 Relative expression (fold change) N2 wild type isp-1;ctb-1 klf-1 d d e pklf-1::gfp Wild type isp-1;ctb-1 e Fig. 1 KLF-1 mediates the longevity of mitochondrial isp-1;ctb-1 mutant. a Lifespan curves of isp-1(qm150);ctb-1(qm189) mutant (left) and wild-type animals (middle) grown on control (empty vector, EV) or klf-1 RNAi plates. Right panel shows lifespan curve of klf-1(tm1110) mutant animals. b isp-1(qm150);ctb-1 (qm189) mutants (left) and wild type animals (right) were grown on control (EV) RNAi plates until L4 developmental stage and then transferred to klf-1 RNAi. c Lifespan curve of isp-1(qm150);ctb-1(qm189) mutant (left) and wild-type animals (right) grown on klf-1 RNAi plates until L4 developmental stage and then transferred to control (EV) RNAi. d klf-1 expression level was assessed by qPCR at the L4 larval stage, the ﬁrst (D1) and the ﬁfth day (D5) of adulthood, in the wild-type and isp-1(qm150);ctb-1(qm189) mutants. Data are presented as mean ± SEM. Results KLF-1 regulates the mitomutant longevity during adulthood. We performed a genome-wide RNAi screen starting with chro- mosome III of the existing feeding library8 in isp-1(qm150);ctb-1 (qm189), a long-lived mitochondrial mutant that carries two mutations in complex III subunits9. We focused ultimately on transcription factors that suppress longevity upon knockdown, but have no effect on wild-type (WT) lifespan. Only klf-1 fulﬁlled these criteria (Fig. 1a). KLF-1 depletion in the isp-1(qm150) single mutant or the short-lived mitochondrial mutants, gas-1(fc21) and mev-1(kn1), also led to a lifespan decrease (Supplementary Fig. 1a–c) in agreement with our previous observation that both long- and short-lived mutants activate the same longevity assurance responses10. KLF-1 is vital for a response to the oxidative stress. Increased longevity in mitochondrial mutants has been correlated to the increased resistance to oxidative stress and ROS-mediated signalling15,16. Hence, we investigated the antioxidant capacity of the isp-1;ctb-1 mutant by exposing them chronically (from L4) to the ROS-producing reagent paraquat. As expected, isp-1;ctb-1 p Mitochondrial dysfunction needs to occur during late C. elegans developmental stages to assure the longevity phenotype11. NATURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w 0 1 2 3 2 4 6 8 0 0 5 10 15 N2 wild type isp-1; ctb-1 EV D1 D5 klf-1 RNAi EV phsp-6::gfp a b N2 D1 Relative expression (fold change) Relative expression (fold change) c nhr-57 isp-1; ctb-1 N2 D5 isp-1; ctb-1 egl-9 N2 D1 isp-1; ctb-1 N2 D5 isp-1; ctb-1 klf-1 RNAi N2 D1 isp-1; ctb-1 N2 D5 isp-1; ctb-1 Normalized to N2 D1 isp-1;ctb-1 N2 isp-1;ctb-1 N2 + + + + + – + – – – – – HSP-6 GFP Tubulin HSP-60 D1 D5 klf-1 RNAi phsp-6::gfp EV klf-1 RNAi EV klf-1 RNAi EV klf-1 RNAi 70 60 27 50 Fig. 2 KLF-1 mildly affects the mitochondrial UPR and does not directly affect the HIF-1 pathway. a Mitochondrial UPR was assayed by activation of gfp expression under the control of hsp-6 promoter. Confocal images (left) were taken at the ﬁrst (D1) and the ﬁfth (D5) day of adulthood in wild type and isp-1 (qm150);ctb-1(qm189) mutant grown on control (EV) or klf-1 RNAi. Scale bar 100 µm. Quantiﬁcation is shown on the right. Data are presented as mean ± SEM. p < 0.05, **p < 0.001, one-way ANOVA with Tukey post hoc test. n = 5 animals per condition. b Western blots showing levels of HSP-6, HSP-60 and GFP in proteins isolated from phsp-6::gfp transgenic strain, in N2 wild-type and isp-1(qm150);ctb-1(qm189) genetic background at D1 and D5 of adulthood. Tubulin was used as a loading control. c Targets of the HIF-1 transcription factor, nhr-57 and egl-9 were assayed for their mRNA expression on D1 and D5 of adulthood in N2 wild-type and isp-1(qm150);ctb-1(qm189) mutant. Data are presented as mean ± SEM. p < 0.05, p < 0.01, p < 0.001, one- way ANOVA with Tukey post hoc test. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w n = 4 independent samples per condition 0 5 10 15 N2 D1 isp-1; ctb-1 N2 D5 isp-1; ctb-1 Normalized to N2 D1 phsp-6::gfp EV klf-1 RNAi a N2 wild type isp-1; ctb-1 EV D1 D5 klf-1 RNAi EV phsp-6::gfp a klf-1 RNAi Normalized to N2 D1 b D1 isp-1;ctb-1 N2 isp-1;ctb-1 N2 + + + + + – + – – – – – HSP-6 GFP Tubulin HSP-60 D1 D5 klf-1 RNAi 70 60 27 50 b 2 4 6 8 0 N2 D1 Relative expression (fold change) c nhr-57 isp-1; ctb-1 N2 D5 isp-1; ctb-1 EV klf-1 RNAi c 0 1 2 3 Relative expression (fold change) egl-9 N2 D1 isp-1; ctb-1 N2 D5 isp-1; ctb-1 EV klf-1 RNAi D1 D5 D5 Fig. 2 KLF-1 mildly affects the mitochondrial UPR and does not directly affect the HIF-1 pathway. a Mitochondrial UPR was assayed by activation of gfp expression under the control of hsp-6 promoter. Confocal images (left) were taken at the ﬁrst (D1) and the ﬁfth (D5) day of adulthood in wild type and isp-1 (qm150);ctb-1(qm189) mutant grown on control (EV) or klf-1 RNAi. Scale bar 100 µm. Quantiﬁcation is shown on the right. Data are presented as mean ± SEM. p < 0.05, **p < 0.001, one-way ANOVA with Tukey post hoc test. n = 5 animals per condition. b Western blots showing levels of HSP-6, HSP-60 and GFP in proteins isolated from phsp-6::gfp transgenic strain, in N2 wild-type and isp-1(qm150);ctb-1(qm189) genetic background at D1 and D5 of adulthood. Tubulin was used as a loading control. c Targets of the HIF-1 transcription factor, nhr-57 and egl-9 were assayed for their mRNA expression on D1 and D5 of adulthood in N2 wild-type and isp-1(qm150);ctb-1(qm189) mutant. Data are presented as mean ± SEM. p < 0.05, p < 0.01, p < 0.001, one- way ANOVA with Tukey post hoc test. n = 4 independent samples per condition hormetic response in the isp-1;ctb-1 mutant, we compared the transcriptome of the D5 isp-1;ctb-1 animals with the same ani- mals grown either on klf-1 RNAi during development (long-lived phenotype) or klf-1 RNAi during adulthood (suppressed long- evity). These ﬁndings were then matched to those of WT. In total, we investigated 251 genes, whose expression was altered in isp-1; ctb-1, but normalized to WT levels upon klf-1 knockdown during adulthood (Table 1 and Supplementary Data 1). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w p < 0.01, *p < 0.001, one-way ANOVA with Tukey post hoc test. n = 5 samples per condition. e Fluorescent images of gfp expression under klf-1 promoter in wild type and isp-1(qm150);ctb-1(qm189) at the ﬁrst day of adulthood. Scale bar 100 µm showed increased resistance to paraquat across all different life- stages (Supplementary Fig. 4a). The increased resistance of older isp-1;ctb-1 animals (D5) seemed to be dependent on KLF-1. Likewise, klf-1 expression was speciﬁcally upregulated by the high paraquat treatment, and no other exogenous stressors, such as heat shock or osmotic stress (Fig. 3a). responses were active in the isp-1;ctb-1, one would expect that through adulthood the ROS-induced damage would not rise. Indeed, the amount of carbonylated proteins as a measure of oxidative damage, increased with age in the WT worms, whereas in the isp-1;ctb-1 they were mainly unchanged (Fig. 3b, c). In some cases, we observed the opposing effect where the oxidative damage in isp-1;ctb-1 mutants that was high at the D1, would decrease in the later stages (D5), to match the level of young adult WT controls (Fig. 3b). The increase in oxidative stress at the D1 in isp-1;ctb-1 was accompanied by upregulation of SOD-2/ Increased resistance to oxidative stress has been attributed to a mitohormetic response, where early exposure to stress, in this case ROS, elicits cytoprotective mechanisms that render animals more resistant to the same stress factors later in life17. If hormetic NATURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w n = 3 independent samples per condition a N2 – – + + Oxyblot MnSOD D1 D5 isp-1; ctb-1 N2 – – + + isp-1; ctb-1 b klf-1 RNAi 24.5 0 1 2 Oxyblot * Normalized to WT D1 c D1 isp-1; ctb-1 D5 isp-1; ctb-1 N2 N2 * * * * * * 0 1 2 MnSOD d Normalized to WT D1 D1 isp-1; ctb-1 D5 isp-1; ctb-1 N2 N2 * EV klf-1 RNAi 0 1 2 N2 – – + + Oxyblot MnSOD Oxyblot * * D1 D5 Normalized to WT D1 isp-1; ctb-1 N2 – – + + isp-1; ctb-1 b c D1 isp-1; ctb-1 D5 isp-1; ctb-1 N2 N2 * * * * * * klf-1 RNAi 24.5 b d Normalized to WT D1 D1 D5 D1 D5 D1 Fig. 3 klf-1 expression increases speciﬁcally upon oxidative stress, but not other types of stresses. a Fluorescent images of a strain expressing gfp under the klf-1 promoter. Young adults were exposed to osmotic and heat stress or 16 mM paraquat. For 0.1 mM paraquat, animals were treated with the drug during the larval development and imaged at the ﬁrst day of adulthood. Scale bar 100 µm. b Oxyblot was performed on proteins isolated from the ﬁrst (D1) or the ﬁfth (D5) day old N2 wild-type and isp-1(qm150);ctb-1(qm189) animals. The bottom panel shows the same oxyblot membrane probed against SOD-2/ MnSOD antibody. c Quantiﬁcation of the oxyblot membranes of D1 and D5 old animals. Data are normalized to N2 D1 levels. Ponceau staining was used as loading control. Data are presented as mean ± SEM. p < 0.05, p < 0.01, *p < 0.01, one-way ANOVA with Tukey post hoc test. n = 5 independent samples per condition. d Quantiﬁcation of SOD-2/MnSOD levels of D1 and D5 old animals. Data are normalized to N2 D1 levels. Ponceau staining was used as loading control. Data are presented as mean ± SEM. p < 0.05, *p < 0.01, one-way ANOVA with Tukey post hoc test. n = 3 independent samples per condition To identify direct transcriptional targets of KLF-1 involved in the regulation of detoxiﬁcation response, we performed chroma- tin immunoprecipitation followed by DNA sequencing (ChIP- seq) analyses in WT overexpressing KLF-1-YFP protein or isp-1; ctb-1 mutants (Supplementary Data 3). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w The analysis identiﬁed a number of genomic regions (on average 430 and 550, respectively), out of which 149 were found in both data sets (Supplementary Data 3). We identiﬁed a KLF-1-binding region immediately upstream of the four cyp genes, including cyp-13A11, one of the most upregulated genes in our microarray data set (Supplementary Data 3). Further qPCR analysis on ChIP samples demonstrated that, indeed KLF-1 is present on cyp-13A11 promotor region in WT, but it binds the same region in isp-1; ctb-1 mutants more efﬁciently (Fig. 4c). Remarkably, the ChIP- Seq data not only strongly overlapped with our microarray results (Supplementary Tables 1 and 2 and Supplementary Data 3), but more than 80% of detected KLF-1 targets were in genes previously shown to be responsive to either, treatment with oxidative stress-producing agents (paraquat or rotenone)19, or agents that block mtDNA synthesis and therefore lead to strong mitochondrial OXPHOS dysfunction (EtBr or nucleoside reverse transcriptase inhibitors—NRTIs)20,21. In most cases KLF-1 target genes were found in more than one data set (Supplementary Data 3). Therefore, together with previous microarray and promoter analysis, ChIP-Seq data strongly support our hypoth- esis that KLF-1 directly regulates at least some of the cyps. Table 1 Microarray data available on NCBI Gene Expression Omnibus (GEO) GEO accession number GSE61771 GSM Acc Description GSM1513691 GSM1513692 GSM1513693 D5 isp-1(qm150);ctb-1(qm189) grown on control L4440 RNAi and switched to klf-1 RNAi at L4 stage GSM1513694 GSM1513695 GSM1513696 D5 isp-1(qm150);ctb-1(qm189) grown on klf-1 RNAi and switched to control L4440 RNAi at L4 stage GSM1513697 GSM1513698 GSM1513699 D5 isp-1(qm150);ctb-1(qm189) grown on control L4440 RNAi GSM1513700 GSM1513701 GSM1513702 D5 N2 wild type grown on control L4440 RNAi Table 1 Microarray data available on NCBI Gene Expression Omnibus (GEO) GEO accession number GSE61771 GSM Acc Description GSM1513691 GSM1513692 GSM1513693 D5 isp-1(qm150);ctb-1(qm189) grown on control L4440 RNAi and switched to klf-1 RNAi at L4 stage GSM1513694 GSM1513695 GSM1513696 D5 isp-1(qm150);ctb-1(qm189) grown on klf-1 RNAi and switched to control L4440 RNAi at L4 stage GSM1513697 GSM1513698 GSM1513699 D5 isp-1(qm150);ctb-1(qm189) grown on control L4440 RNAi GSM1513700 GSM1513701 GSM1513702 D5 N2 wild type grown on control L4440 RNAi Table 1 Microarray data available on NCBI Gene Expression Omnibus (GEO) signiﬁcant overrepresentation of genes encoding proteins involved in phase I xenobiotic detoxiﬁcation, particularly cyto- chrome P450 oxidases (CYPs) (Fig. 4a). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w In agreement with the previous results15,16, the list of genes upregulated in the mitochondrial mutant did not show signiﬁcant enrichment for genes involved in the ROS detoxiﬁcation or damage repair. Remarkably, we also did not observe changes in genes encoding mitochondrial proteins, including OXPHOS subunits. The DAVID gene enrichment analysis tool was used to evaluate the statistical representation of gene categories as deﬁned by gene ontology18. This approach identiﬁed “oxidation–reduction” to be the most affected biological process, and “metabolism of xenobiotics by cytochrome P450” and “drug metabolism” to be the two most affected KEGG pathways in isp-1;ctb-1 mutant upon lifespan-shortening klf-1 knockdown (Supplementary Tables 1 and 2). Correspondingly, in the mitochondrial mutant there was a MnSOD, a major mitochondrial antioxidant enzyme that takes care of superoxide (O2−) (Fig. 3b, d). Curiously, KLF-1 depletion prevented the early upregulation of SOD-2/MnSOD, at both transcript and protein level, but had no effect on them later in the adulthood (D5), suggesting that sod-2, is not directly regulated by KLF-1 (Fig. 3b, d and Supplementary Fig. 4b). Likewise, we show that KLF-1 does not regulate transcription of sod-1, a main cytosolic SOD or sod-3, a second mitochondrial MnSOD that is controlled by DAF-16 transcription factor and highly induced in daf-2 long-lived mutants (Supplementary Fig. 4b, c). KLF-1 is also not involved in the transcription regulation of catalases, which are major H2O2 metabolizing enzymes (Supplementary Fig. 4b). Collectively, these data strongly suggest that isp-1;ctb-1 mutants activate pathways, other than bona ﬁde antioxidant response, to combat increased oxidative stress imposed by mitochondrial dysfunction. These responses seem to depend on KLF-1 activity that regulates the hormetic response observed in mitochondrial mutants. KLF-1 regulates phase I detoxiﬁcation response genes. To elucidate the cytoprotective machinery, which mediates the TURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w Control Heat stress Osmotic stress 0.1 mM paraquat 16 mM paraquat pklf-1::gfp a 0 1 2 0 1 2 N2 – – + + Oxyblot MnSOD Oxyblot * D1 D5 Normalized to WT D1 MnSOD isp-1; ctb-1 N2 – – + + isp-1; ctb-1 b c d D1 isp-1; ctb-1 D5 isp-1; ctb-1 Normalized to WT D1 N2 N2 D1 isp-1; ctb-1 D5 isp-1; ctb-1 N2 N2 * * * * * * * klf-1 RNAi EV klf-1 RNAi 24.5 Fig. 3 klf-1 expression increases speciﬁcally upon oxidative stress, but not other types of stresses. a Fluorescent images of a strain expressing gfp under the klf-1 promoter. Young adults were exposed to osmotic and heat stress or 16 mM paraquat. For 0.1 mM paraquat, animals were treated with the drug during the larval development and imaged at the ﬁrst day of adulthood. Scale bar 100 µm. b Oxyblot was performed on proteins isolated from the ﬁrst (D1) or the ﬁfth (D5) day old N2 wild-type and isp-1(qm150);ctb-1(qm189) animals. The bottom panel shows the same oxyblot membrane probed against SOD-2/ MnSOD antibody. c Quantiﬁcation of the oxyblot membranes of D1 and D5 old animals. Data are normalized to N2 D1 levels. Ponceau staining was used as loading control. Data are presented as mean ± SEM. p < 0.05, p < 0.01, *p < 0.01, one-way ANOVA with Tukey post hoc test. n = 5 independent samples per condition. d Quantiﬁcation of SOD-2/MnSOD levels of D1 and D5 old animals. Data are normalized to N2 D1 levels. Ponceau staining was used as loading control. Data are presented as mean ± SEM. p < 0.05, p < 0.01, one-way ANOVA with Tukey post hoc test. NATURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunicatio NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w 0 1 2 3 4 5 0 10 20 30 40 0 2 4 6 0 2 4 0 20 40 60 0 5 10 15 0 100 200 300 0 20 60 100 col-103 cyp-33c2 cdr-1 ugt-62 B0238.18 col-98 cyp-34a8 C05E11.5 col-142 col-119 col-8 gst-1 C52A10.2 K03D3.2 W01C9.1 Y69H2.14.1 col-178 K12D9.1 W01C9.2 C02C2.3 ugt-6 col-19 amt-4 gst-4 C42D4.2 pmt-2 Y45G12C.3 F56A4.4 dct-8 udp-21 dhs-7 dhs-27 C52D10.1 cyp-13a7 F11E6.3.1 col-1 sod-3 B0213.18 abf-2 C17B7.5 F57G8.7 grd-3 Y71HB.1 F59C6.18 Y39B6A.73 asp-8 col-20 cyp-14a11 Y45F10B.13 isp-1; ctb-1 EV isp-1; ctb-1 klf-1 RNAi cyp-13a7 cyp-13a11 Relative expression (fold change) Relative expression (fold change) D1 isp-1; ctb-1 isp-1; ctb-1 D5 D1 isp-1; ctb-1 isp-1; ctb-1 D5 D1 isp-1; ctb-1 isp-1; ctb-1 D5 D1 isp-1; ctb-1 isp-1; ctb-1 D5 D1 isp-1; ctb-1 isp-1; ctb-1 D5 Relative expression (fold change) Relative expression (fold change) Relative expression (fold change) a b CYP3A13 – – + + AA – – + + AA * * * CYP2C70 scr shKLF4 scr shKLF5 Relative expression (fold change) Relative expression (fold change) d N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 cyp-14a1 cyp-14a3 cyp-34a9 * * *** * * * * * * * * * * * *** * *** * *** * * * * * c Relative to WT N2 isp-1;ctb-1 cyp-13a11 EV klf-1 RNAi * 2 4 –2 –4 log2 FC 0 KLF-1 mediates expression of cytochrome P450 oxidases. a log2 fold-change in gene expression in isp-1(qm150);ctb-1(qm189) mutant animals red with N2 wild type, grown either on control (EV) or klf-1 RNAi from L4 larval stage. Microarray analysis was performed at the ﬁfth day of od. b The expression of cytochrome P450 oxidases from (a) was conﬁrmed by qPCR. n = 4 independent samples per condition. Animals were d at the ﬁrst (D1) or the ﬁfth (D5) day of adulthood and grown on control (EV) and klf-1 RNAi whole life. c CHIP-qPCR analysis of WT and isp-1;ctb-1 -13a11 promoter region. n = 2 independent replicates. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w d Levels of Cyp2C70 and Cyp3A13 were assayed with qPCR in Hepa1-6 cells with or without TICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w col-103 cyp-33c2 cdr-1 ugt-62 B0238.18 col-98 cyp-34a8 C05E11.5 col-142 col-119 col-8 gst-1 C52A10.2 K03D3.2 W01C9.1 Y69H2.14.1 col-178 K12D9.1 W01C9.2 C02C2.3 ugt-6 col-19 amt-4 gst-4 C42D4.2 pmt-2 Y45G12C.3 F56A4.4 dct-8 udp-21 dhs-7 dhs-27 C52D10.1 cyp-13a7 F11E6.3.1 col-1 sod-3 B0213.18 abf-2 C17B7.5 F57G8.7 grd-3 Y71HB.1 F59C6.18 Y39B6A.73 asp-8 col-20 cyp-14a11 Y45F10B.13 isp-1; ctb-1 EV isp-1; ctb-1 klf-1 RNAi a 2 4 –2 –4 log2 FC 0 a 0 1 2 3 4 5 0 10 20 30 40 0 2 4 6 0 2 4 0 20 40 60 0 5 10 15 0 100 200 300 0 20 60 100 kl cyp-13a7 cyp-13a11 Relative expression (fold change) Relative expression (fold change) D1 isp-1; ctb-1 isp-1; ctb-1 D5 D1 isp-1; ctb-1 isp-1; ctb-1 D5 D1 isp-1; ctb-1 isp-1; ctb-1 D5 D1 isp-1; ctb-1 isp-1; ctb-1 D5 D1 isp-1; ctb-1 isp-1; ctb-1 D5 Relative expression (fold change) Relative expression (fold change) Relative expression (fold change) b CYP3A13 – – + + AA – – + + AA * * * CYP2C70 scr shKLF4 scr shKLF5 Relative expression (fold change) Relative expression (fold change) d N2 N2 N2 N2 N2 N2 N2 N2 N2 N2 cyp-14a1 cyp-14a3 cyp-34a9 * * *** * * * * * * * * * * * *** * *** * *** * * * * * c Relative to WT N2 isp-1;ctb-1 cyp-13a11 EV klf-1 RNAi *** –4 Fig. 4 KLF-1 mediates expression of cytochrome P450 oxidases. a log2 fold-change in gene expression in isp-1(qm150);ctb-1(qm189) mutant animals compared with N2 wild type, grown either on control (EV) or klf-1 RNAi from L4 larval stage. Microarray analysis was performed at the ﬁfth day of adulthood. b The expression of cytochrome P450 oxidases from (a) was conﬁrmed by qPCR. n = 4 independent samples per condition. Animals were analysed at the ﬁrst (D1) or the ﬁfth (D5) day of adulthood and grown on control (EV) and klf-1 RNAi whole life. c CHIP-qPCR analysis of WT and isp-1;ctb for cyp-13a11 promoter region. n = 2 independent replicates. d Levels of Cyp2C70 and Cyp3A13 were assayed with qPCR in Hepa1-6 cells with or withou antimycin A (AA) treatment as indicated. Cells were exposed to either Klf4, Klf5 or control siRNA. Data are presented as mean ± SEM. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w p < 0.05, p < 0.0 p < 0.001, one-way ANOVA with Tukey post hoc test. n = 4 independent samples per condition 0 100 200 300 cyp-13a7 Relative expression (fold change) D1 isp-1; ctb-1 isp-1; ctb-1 D5 b N2 N2 * *** * * b b 0 20 60 100 cyp-13a11 Relative expression (fold change) D1 isp-1; ctb-1 isp-1; ctb-1 D5 N2 N2 *** * * * * * EV klf-1 RNAi 0 5 10 15 D1 D1 isp-1; ctb-1 isp-1; ctb-1 D5 Relative expression (fold change) N2 N2 cyp-14a1 * * * * 0 20 40 60 D1 isp-1; ctb-1 isp-1; ctb-1 D5 Relative expression (fold change) N2 N2 cyp-14a3 * *** * *** * *** * * 0 2 4 D1 isp-1; ctb-1 isp-1; ctb-1 D5 Relative expression (fold change) N2 N2 cyp-34a9 * * * 0 1 2 3 4 5 CYP3A13 – – + + AA * * scr shKLF4 Relative expression (fold change) 0 10 20 30 40 – – + + AA * CYP2C70 scr shKLF5 Relative expression (fold change) d 0 2 4 6 c Relative to WT N2 isp-1;ctb-1 cyp-13a11 d d c Fig. 4 KLF-1 mediates expression of cytochrome P450 oxidases. a log2 fold-change in gene expression in isp-1(qm150);ctb-1(qm189) mutant animals compared with N2 wild type, grown either on control (EV) or klf-1 RNAi from L4 larval stage. Microarray analysis was performed at the ﬁfth day of adulthood. b The expression of cytochrome P450 oxidases from (a) was conﬁrmed by qPCR. n = 4 independent samples per condition. Animals were analysed at the ﬁrst (D1) or the ﬁfth (D5) day of adulthood and grown on control (EV) and klf-1 RNAi whole life. c CHIP-qPCR analysis of WT and isp-1;ctb-1 for cyp-13a11 promoter region. n = 2 independent replicates. d Levels of Cyp2C70 and Cyp3A13 were assayed with qPCR in Hepa1-6 cells with or without antimycin A (AA) treatment as indicated. Cells were exposed to either Klf4, Klf5 or control siRNA. Data are presented as mean ± SEM. p < 0.05, p < 0.01, *p < 0.001, one-way ANOVA with Tukey post hoc test. n = 4 independent samples per condition A (a complex III inhibitor and a potent ROS inducer) and detected high increase in Cyp2C70 and Cyp3A13, supporting the notion that the response to mitochondrial dysfunction is similar in C. elegans and mammals (Fig. 4d). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w In the isp-1;ctb-1 mutant, most of the identiﬁed cyps showed highly increased, KLF-1- dependent expression early in life, which often declined during aging (Fig. 4b). In agreement, promoter analyses of most sig- niﬁcantly changed genes, including majority of identiﬁed cyps, revealed one or more consensus KLF-binding elements (CA/ GCCC) within 2000 bp upstream and 100 bp downstream of the transcription start site (Supplementary Data 2). Two classes of CYPs whose expression was most highly upregulated in the isp-1;ctb-1 in a KLF-1-dependent manner are homologues of murine CYP2C70 and CYP3A13. To test whether the induction of cyp expression upon mitochondrial dysfunction is conserved, we treated Hepa1-6 cells with low level of antimycin 5 5 TURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w 6b–d), thus mirroring the results obtained from the isp-1;ctb-1 mutants (Fig. 1b, c). Cytochrome P450 oxidases are longevity assurance proteins. The upregulation of CYPs should give advantage to animals upon contact with xenobiotics. To test this, we exposed animals to the known CYP-target vinblastine, a drug that binds tubulin and thus prevents cell proliferation, and assessed the drug-induced devel- opmental delay (Fig. 5a). Remarkably, isp-1;ctb-1 mutants that normally develop slower than WT animals, upon vinblastine treatment reached further stages in development than controls, an effect fully dependent on KLF-1 (Fig. 5a). Moreover, isp-1;ctb-1 showed KLF-1-dependent, increased resistance to levamisole- induced paralysis (Fig. 5b). These data suggest that the high expression of cyps driven by KLF-1 provides higher resistance to xenobiotics in the isp-1;ctb-1 that might be essential for longevity. Furthermore, isp-1;ctb-1 animals showed high resistance to acute, high-dose H2O2 treatment that was fully dependent on KLF-1 and CYPs presence (Fig. 5c). We next measured mitochondrial ROS production at different time points and detected higher levels of H2O2 production in isp- 1;ctb-1 at the fourth larval stage (L4), comparing with WT (Fig. 6e). Interestingly, while the mitochondrial ROS production in WT worms more than trippled on the D1, we observed only a mild increase in isp-1;ctb-1 mutants, in agreement with the mitohormetic theory (Fig. 6e). The knockdown of two highly upregulated cyps (cyp-13a11 and cyp-25a1) strongly suppressed the isp-1;ctb-1 longevity, indicating an important longevity assurance function of these proteins (Supplementary Fig. 6a). Remarkably, the same effect was observed when cyps are depleted only during adulthood, mirroring the results obtained for KLF-1 and further strengthen- ing their interdependence in the longevity pathway mediated by mitochondrial dysfunction (Fig. 5d). y g We have previously shown that mitochondrial biogenesis for all somatic tissues occurs predominantly between late L3 and late L4 stage and ﬁnishes around the time of transitioning into adulthood26. Consistently, increased mitochondrial mass was observed on the D1 in both WT and mutant worms, with higher upregulation in the isp-1;ctb-1 that was not dependent on KLF-1 (Fig. 6f and Supplementary Fig. 7a). Therefore, despite mito- chondrial dysfunction and higher ROS levels during larval development, isp-1;ctb-1 mutants generated less ROS per mitochondria once they reached adulthood (Fig. 6g). This was maintained also later in adulthood and was clearly KLF-1 dependent (Fig. 6h). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w This is in agreement with previous results showing that phase I detoxiﬁcation reactions, catalysed by CYPs, result in the addition of a reactive group onto the toxic compound that is required for the stimulation of phase II metabolism3. transcription factor in the context of mitochondrial dysfunction, outside of regulation of phase I detoxiﬁcation (Supplementary Fig. 5d). Remarkably, combined depletion of klf-1 and klf-3 led to a mild suppression of longevity in isp-1;ctb-1 mutants, again, likely due to dilution of both RNAi (Supplementary Fig. 5d). To address a potential role of KLF-1 in the regulation of autophagy we analysed at the expression of genes encoding proteins involved in the autophagosome formation and did not observe any differences due to mitochondrial dysfunction or depletion of KLF-1 (Supplementary Fig. 5e). Furthermore, the number of GFP-positive puncta in the hypodermal seam cells, commonly used as a marker for autophagosomes, did not change in the absence of klf-1, klf-3 or both factors in the same time (Supplementary Fig. 5f). Remarkably, we detected higher autophagy ﬂux (cleavage of GFP::LGG-1 inside autophagosome) in isp-1;ctb-1 mutants only in conditions where KLF-1 was depleted (Supplementary Fig. 5g). The effect was further exaggerated by addition of vitamin C (Supplementary Fig. 5g), suggesting that KLF-1 and its activation by mitochondrial ROS might play a role in suppression and not activation of autophagy13. Taken together, these results argue against a major role for the autophagy in isp-1;ctb-1 mutants and question proposed role for the KLF-1 in this process. Together our data support the view that KLF-1 directly regulates phase I and not phase II response genes, which instead depend on SKN-1. However, they also signify the necessity of phase I enzymes for the phase II activation and provide an explanation of how KLF-1 mediated activation of cyps regulates the level of oxidative damage and antioxidant response. ROS pulse in development is essential for KLF-1 activation. To further understand the involvement of KLF-1 in the mitohor- metic response and its speciﬁc activation, we treated animals with a low amount of paraquat that has been shown to induce long- evity in WT animals through the same mechanisms described for mitomutants15,25. The observed longevity phenotype was fully suppressed in a klf-1(tm1110) deletion mutant (Fig. 6a). Remarkably, we show that paraquat treatment administered exclusively during development is also able to induce longevity that is suppressed by KLF-1 depletion during adulthood, but not during development (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w The upregulation of these genes was dependent on the closest mammalian homologues of KLF-1, namely KLF4 and KLF5, providing further evidence for the high conservation of this stress response (Fig. 4d). A (a complex III inhibitor and a potent ROS inducer) and detected high increase in Cyp2C70 and Cyp3A13, supporting the notion that the response to mitochondrial dysfunction is similar in C. elegans and mammals (Fig. 4d). The upregulation of these genes was dependent on the closest mammalian homologues of KLF-1, namely KLF4 and KLF5, providing further evidence for the high conservation of this stress response (Fig. 4d). regulation of autophagy genes necessary for caloric restriction and insulin signalling mediated longevity13, we further evaluated possible role of KLF-3 in regulating cyp genes. However, our results argue against the role of KLF-3 in this process, as its depletion did not affect the expression of cyp-25a2, while the combined klf-1 and klf-3 knockdown produced intermittent effect, possibly due to dilution of individual RNAi (Supplemen- tary Fig. 5b). We also show that in contrast to klf-1, the klf-2 and klf-3 expression was not affected in the isp-1;ctb-1 (Supplemen- tary Fig. 5c). However, the loss of KLF-3 strongly suppressed the isp-1; ctb-1 lifespan suggesting an important role for this The activation of cyp genes was exclusively driven by KLF-1 and not the other two C. elegans KLF homologues, KLF-2 and KLF-3 (Supplementary Fig. 5a). As it was recently reported that KLF-3, together with KLF-1, plays an important role in the TURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w compound that notoriously activates a phase II xenobiotic response24. We show that the acrylamide treatment of WT worms strongly activates gst-4 expression independently of KLF- 1, and this upregulation is fully suppressed by SKN-1 depletion (Fig. 5f and Supplementary Fig 6c, d). The gst-4 expression was not upregulated in isp-1;ctb-1 (Fig. 5f and Supplementary Fig 6c, d) and SKN-1 depletion had only a mild effect on isp-1;ctb-1 longevity (Supplementary Fig. 6e). Nevertheless, the expression of phase II genes (gst-4 and F56A4.4, a gst-10 homologue) in isp-1; ctb-1 was strongly dependent on both KLF-1 and CYPs presence (Fig. 5g and Supplementary Fig. 6f). NATURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w D ed as mean ± SEM. p < 0.05, p < 0.01, p < 0.001, one-way ANOVA with Tukey post hoc test. n = 10 animals per condition. f Left pan sentative confocal images of gfp expressed under gst-4 promoter in wild type animals. Animals were treated with acrylamide upon klf-1 a at D1. Scale bar 100 µm. Right panel is the quantiﬁcation of n = 5 animals per condition. Data are presented as mean ± SEM. p < 0.05, **p way ANOVA with Tukey post hoc test. g SKN-1 transcriptional targets gst-4 (left) and F56A4.4 (right) expression levels were quantiﬁed 1(qm150);ctb-1(qm189) animals upon klf-1 RNAi or combined cyp-25a1 and cyp-13a11 RNAi (cyps). Data are presented as mean ± SEM. p < 0. LE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275 0 50 100 L2 L3 L4 D1 EV klf-1 EV klf-1 phb-2 N2 isp-1;ctb-1 Vinblastin toxicity % total worms a 0 20 40 60 0 50 100 Time (min) % paralyzed Levamisole toxicity N2 EV N2 klf-1 isp-1;ctb-1 EV isp-1;ctb-1 klf-1 b b a a 0 20 40 60 c EV klf-1 cyps N2 isp-1;ctb-1 H2O2 stress resistance * *** % survival 0 0.5 1 1.5 2 pcyp-25a2::gfp Control klf-1 RNAi skn-1 RNAi fluorescence intensity (A.U.) N2 isp-1;ctb-1 N2 isp-1;ctb-1 D1 D5 * *** * * *** * * *** * * * e *** c e d isp-1(qm150);ctb-1(qm189) EV EV_cyps RNAi N2 wild type Percent survival Days 0 50 100 0 20 40 d EV EV klf-1 RNAi skn-1 RNAi f pgst-4::gfp Acrylamide 0 2 4 6 8 10 EV EV klf-1 skn-1 Normalized to N2 Acrylamide pgst-4::gfp * * * * f 0 2 4 6 8 10 EV EV klf-1 skn-1 Normalized to N2 Acrylamide pgst-4::gfp 0 0.5 1 1.5 Relative expression (fold change) ** 0.0933 F56A4.4 EV klf-1 cyps * * * * 0 0.5 1 1.5 Relative expression (fold change) EV klf-1 cyps gst-4 ** * g 0 0.5 1 1.5 Relative expression (fold change) 0.0933 F56A4.4 EV klf-1 cyps g Fig. 5 KLF-1 regulates phase I, but not phase II detoxiﬁcation pathway. a L1 larvae of N2 wild type, klf-1(tm1110), isp-1(qm150);ctb-1(qm189), isp-1(qm150);ctb- 1(qm189);klf-1(tm1110) and phb-2(ad2154) were grown in liquid with and without 100 µM vinblastin. When all animals without treatment reached adulthood, the animals in the wells containing vinblastin were assayed for developmental stages. n = 5 wells per condition per experiment. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w 0 0.5 1 1.5 0 2 4 6 8 10 0 0.5 1 1.5 2 0 20 40 60 0 50 100 L2 L3 L4 D1 EV klf-1 EV klf-1 phb-2 N2 isp-1;ctb-1 Vinblastin toxicity % total worms 0 20 40 60 0 50 100 Time (min) % paralyzed Levamisole toxicity N2 EV N2 klf-1 isp-1;ctb-1 EV isp-1;ctb-1 klf-1 a b d EV EV klf-1 RNAi skn-1 RNAi f pgst-4::gfp EV EV klf-1 skn-1 Normalized to N2 Acrylamide pgst-4::gfp Relative expression (fold change) 0 0.5 1 1.5 Relative expression (fold change) EV klf-1 cyps gst-4 * ** 0.0933 F56A4.4 g EV klf-1 cyps c pcyp-25a2::gfp Control klf-1 RNAi skn-1 RNAi fluorescence intensity (A.U.) N2 isp-1;ctb-1 N2 isp-1;ctb-1 D1 D5 * *** * * *** * * *** * * * Acrylamide isp-1(qm150);ctb-1(qm189) EV EV_cyps RNAi N2 wild type Percent survival Days 0 50 100 0 20 40 * * * * e EV klf-1 cyps N2 isp-1;ctb-1 H2O2 stress resistance * * % survival * regulates phase I, but not phase II detoxiﬁcation pathway. a L1 larvae of N2 wild type, klf-1(tm1110), isp-1(qm150);ctb-1(qm189), isp-1(qm150);c -1(tm1110) and phb-2(ad2154) were grown in liquid with and without 100 µM vinblastin. When all animals without treatment reached adultho in the wells containing vinblastin were assayed for developmental stages. n = 5 wells per condition per experiment. Data presented are avera arate experiments. b N2 wild-type and isp-1(qm150);ctb-1(qm189) animals were transferred at the fourth day of adulthood on plates contain isole and assayed every 15 min for movement. Worms that failed to move upon gentle touch with silver wire were considered paralysed. n = 25 worms for each condition. c N2 wild type and isp-1(qm150);ctb-1(qm189) animals on control (EV), klf-1 and combined cyp-25a1 and cyp-13 animals were treated with 20 mM H2O2 at the ﬁrst (D1) day of adulthood and assayed for survival 4 h later. p < 0.01, p < 0.001, one-w h Tukey post hoc test. n = 100 animals per condition. d Survival curve of N2 wild type and isp-1(qm150);ctb-1(qm189) animals on control (E ombined cyp-25a1 and cyp-13a11 RNAi (cyps). Animals were exposed to RNAi from L4 larval stage. e Quantiﬁcation of gfp expression under c ter in N2 wild type and isp-1(qm150);ctb-1(qm189) mutant background upon klf-1 and skn-1 RNAi at D1 and the ﬁfth (D5) day of adulthood. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w Remarkably, KLF-1 depletion at D5 caused much higher ROS burst in WT animals than in isp-1;ctb-1, again supporting the mitohormetic theory (Fig. 6h). To further evaluate transcriptional control of the xenobiotic response in mitomutants, we used reporter strains in which the gfp expression was driven either by cyp-25a2 or cyp-34a8 promoter (Fig. 5e and Supplementary Fig. 6b). Predictably, the gfp levels progressively increased in the isp-1;ctb-1 through adulthood in a KLF-1-dependent manner. In contrast, a skn-1 knockdown, a homologue of mammalian NRF2 (Nuclear factor (erythroid-derived 2)-like 2), a transcription regulator of phase II detoxiﬁcation response22,23, further increased the expression of both reporters (Fig. 5e and Supplementary Fig. 6b). This supports the idea that in the absence of SKN-1, which regulates phase II and the overlapping antioxidant response, endogenous toxic compounds that are produced and not excreted, would further exaggerate phase I response. We next show that the majority of the KLF-1, under normal conditions, is located in the cytoplasm, with nuclear localization observed only in some intestinal cells (Fig. 7a, b). In the isp-1;ctb- 1 mutant, or upon mild oxidative stress induced by treatment with either paraquat or antimycin A, KLF-1 translocates to the nucleus (Fig. 7a, b). As we observed a signiﬁcant increase in ROS production in the isp-1;ctb-1 late developmental stages (L4), we questioned whether redox signalling might be involved in the activation of KLF-1. Indeed, treatment with antioxidants, e.g. N- acetyl cysteine (NAC) and vitamin C, completely abolished KLF-1 Inversely, KLF-1 exclusively regulates a phase I, and not phase II response as shown by the analysis of the phase II/antioxidant gene gst-4 expression upon treatment with acrylamide, a 7 7 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w Data presented are average of three separate experiments. b N2 wild-type and isp-1(qm150);ctb-1(qm189) animals were transferred at the fourth day of adulthood on plates containing 1 mM levamisole and assayed every 15 min for movement. Worms that failed to move upon gentle touch with silver wire were considered paralysed. n = 4 plates with 25 worms for each condition. c N2 wild type and isp-1(qm150);ctb-1(qm189) animals on control (EV), klf-1 and combined cyp-25a1 and cyp-13a11 RNAi (cyps) animals were treated with 20 mM H2O2 at the ﬁrst (D1) day of adulthood and assayed for survival 4 h later. p < 0.01, *p < 0.001, one-way ANOVA with Tukey post hoc test. n = 100 animals per condition. d Survival curve of N2 wild type and isp-1(qm150);ctb-1(qm189) animals on control (EV) plates and combined cyp-25a1 and cyp-13a11 RNAi (cyps). Animals were exposed to RNAi from L4 larval stage. e Quantiﬁcation of gfp expression under cyp- 25a2 promoter in N2 wild type and isp-1(qm150);ctb-1(qm189) mutant background upon klf-1 and skn-1 RNAi at D1 and the ﬁfth (D5) day of adulthood. Data are presented as mean ± SEM. p < 0.05, p < 0.01, p < 0.001, one-way ANOVA with Tukey post hoc test. n = 10 animals per condition. f Left panel shows representative confocal images of gfp expressed under gst-4 promoter in wild type animals. Animals were treated with acrylamide upon klf-1 and skn-1 RNAi at D1. Scale bar 100 µm. Right panel is the quantiﬁcation of n = 5 animals per condition. Data are presented as mean ± SEM. p < 0.05, **p < 0.001, one-way ANOVA with Tukey post hoc test. g SKN-1 transcriptional targets gst-4 (left) and F56A4.4 (right) expression levels were quantiﬁed by qPCR in isp-1(qm150);ctb-1(qm189) animals upon klf-1 RNAi or combined cyp-25a1 and cyp-13a11 RNAi (cyps). Data are presented as mean ± SEM. p < 0.05, p < 0.01, Student’s T-test. n = 4 independent samples per condition Fig. 5 KLF-1 regulates phase I, but not phase II detoxiﬁcation pathway. a L1 larvae of N2 wild type, klf-1(tm1110), isp-1(qm150);ctb-1(qm189), isp-1(qm150);ctb- 1(qm189);klf-1(tm1110) and phb-2(ad2154) were grown in liquid with and without 100 µM vinblastin. When all animals without treatment reached adulthood, the animals in the wells containing vinblastin were assayed for developmental stages. n = 5 wells per condition per experiment. Data presented are average of three separate experiments. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w b N2 wild-type and isp-1(qm150);ctb-1(qm189) animals were transferred at the fourth day of adulthood on plates containing 1 mM levamisole and assayed every 15 min for movement. Worms that failed to move upon gentle touch with silver wire were considered paralysed. n = 4 plates with 25 worms for each condition. c N2 wild type and isp-1(qm150);ctb-1(qm189) animals on control (EV), klf-1 and combined cyp-25a1 and cyp-13a11 RNAi (cyps) animals were treated with 20 mM H2O2 at the ﬁrst (D1) day of adulthood and assayed for survival 4 h later. p < 0.01, **p < 0.001, one-way ANOVA with Tukey post hoc test. n = 100 animals per condition. d Survival curve of N2 wild type and isp-1(qm150);ctb-1(qm189) animals on control (EV) plates and combined cyp-25a1 and cyp-13a11 RNAi (cyps). Animals were exposed to RNAi from L4 larval stage. e Quantiﬁcation of gfp expression under cyp- 25a2 promoter in N2 wild type and isp-1(qm150);ctb-1(qm189) mutant background upon klf-1 and skn-1 RNAi at D1 and the ﬁfth (D5) day of adulthood. Data are presented as mean ± SEM. p < 0.05, p < 0.01, p < 0.001, one-way ANOVA with Tukey post hoc test. n = 10 animals per condition. f Left panel shows representative confocal images of gfp expressed under gst-4 promoter in wild type animals. Animals were treated with acrylamide upon klf-1 and skn-1 RNAi at D1. Scale bar 100 µm. Right panel is the quantiﬁcation of n = 5 animals per condition. Data are presented as mean ± SEM. p < 0.05, **p < 0.001, one-way ANOVA with Tukey post hoc test. g SKN-1 transcriptional targets gst-4 (left) and F56A4.4 (right) expression levels were quantiﬁed by qPCR in isp-1(qm150);ctb-1(qm189) animals upon klf-1 RNAi or combined cyp-25a1 and cyp-13a11 RNAi (cyps). Data are presented as mean ± SEM. p < 0.05, p < 0.01, Student’s T-test. n = 4 independent samples per condition NATURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications 8 8 ocation to the nucleus induced by antimycin A treatment or ctb-1 mutations, suggesting that KLF-1 activation is indeed adulthood (Fig. 7d). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w Percent survival a N2 klf-1(tm1110) klf-1(tm1110) PQ N2 PQ Days 0 0 20 60 100 20 40 Control PQ PQ klf-1 RNAi b Egg L1 L2 L3 L4 Adult Percent survival Days 0 0 20 60 100 20 40 klf-1 RNAi PQ/klf-1 RNAi b a PQ c Egg L1 L2 L3 L4 Adult Control PQ PQ klf-1 RNAi Percent survival Days 0 0 20 60 100 20 40 klf-1 RNAi d Control PQ PQ klf-1 RNAi Egg L1 L2 L3 L4 Adult Percent survival Days 0 0 20 60 100 20 40 PQ/klf-1 RNAi d c Control PQ PQ klf-1 RNAi Percent survival Days 0 0 20 60 100 20 40 y 0 1 2 3 4 Mito mass Normalized to WT L4 L4 stage isp-1; ctb-1 D1 isp-1; ctb-1 f N2 N2 * * * e 0 2 4 6 H2O2 levels Normalized to WT L4 L4 stage isp-1; ctb-1 D1 isp-1; ctb-1 N2 N2 * * * * * f e 0 2 4 6 8 H2O2 levels Fluorescence intensity (A.U.) D1 isp-1; ctb-1 D5 isp-1; ctb-1 h N2 N2 * * * * * * * * EV klf-1 RNAi 0 2 4 6 8 H2O2 levels Fluorescence intensity (A.U.) D1 isp-1; ctb-1 D5 isp-1; ctb-1 h N2 N2 * * * * * * * * g 0 2 4 6 H2O2/mito mass H2O2/mito mass L4 stage isp-1; ctb-1 D1 isp-1; ctb-1 N2 N2 * * * * * h g g D5 D1 D1 Fig. 6 KLF-1 mediates a mitohormetic response in isp-1(qm150);ctb-1(qm189) mutants. a Lifespan curves of N2 wild-type Fig. 6 KLF-1 mediates a mitohormetic response in isp-1(qm150);ctb-1(qm189) mutants. a Lifespan curves of N2 wild-type and klf-1(tm1110) animals grown on 0.1 mM paraquat (PQ) their whole life. b–d Worms were grown on 0.1 mM paraquat (PQ) until L4 larval stage and then transferred to paraquat-free plates. Worms were exposed to klf-1 RNAi either whole life (b), in adulthood (c) or during development (d). e–g N2 wild-type and isp-1(qm150);ctb-1(qm189) animals were stained with Mitotracker Red CM-H2XRos (e) or Deep Red (f) to assay mitochondrial ROS levels and mitochondrial (mito) mass, respectively. Animals were assayed at larval L4 stage or D1 and the ﬂuorescence was measured via Biosorter. A ratio between Mitotracker Red CM- H2XRos and Deep Red ﬂuorescence measurements is shown in (g). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w In the isp-1;ctb-1 animals persistent KLF-1 localization at D1 that is largely Percent survival a N2 klf-1(tm1110) klf-1(tm1110) PQ N2 PQ Days 0 0 20 60 100 20 40 Control PQ PQ klf-1 RNAi b Egg L1 L2 L3 L4 Adult Percent survival Days 0 0 20 60 100 20 40 klf-1 RNAi PQ/klf-1 RNAi PQ c Egg L1 L2 L3 L4 Adult d Control PQ PQ klf-1 RNAi Egg L1 L2 L3 L4 Adult Control PQ PQ klf-1 RNAi Percent survival Days 0 0 20 60 100 20 40 Percent survival Days 0 0 20 60 100 20 40 PQ/klf-1 RNAi klf-1 RNAi 0 1 2 3 4 Mito mass Normalized to WT L4 e L4 stage isp-1; ctb-1 D1 isp-1; ctb-1 f N2 N2 * * *** 0 2 4 6 H2O2 levels Normalized to WT L4 L4 stage isp-1; ctb-1 D1 isp-1; ctb-1 N2 N2 * * * * * 0 2 4 6 8 H2O2 levels Fluorescence intensity (A.U.) D1 isp-1; ctb-1 D5 isp-1; ctb-1 g h N2 N2 * * * * * * * * 0 2 4 6 H2O2/mito mass H2O2/mito mass L4 stage isp-1; ctb-1 D1 isp-1; ctb-1 N2 N2 * * * * * EV klf-1 RNAi KLF-1 mediates a mitohormetic response in isp-1(qm150);ctb-1(qm189) mutants. a Lifespan curves of N2 wild-type and klf-1(tm1110) a M paraquat (PQ) their whole life. b–d Worms were grown on 0.1 mM paraquat (PQ) until L4 larval stage and then transferred to para s were exposed to klf-1 RNAi either whole life (b), in adulthood (c) or during development (d). e–g N2 wild-type and isp-1(qm15 s were stained with Mitotracker Red CM-H2XRos (e) or Deep Red (f) to assay mitochondrial ROS levels and mitochondrial (mit tively. Animals were assayed at larval L4 stage or D1 and the ﬂuorescence was measured via Biosorter. A ratio between Mitotra s and Deep Red ﬂuorescence measurements is shown in (g). h Mitotracker Red CM-H2XRos staining of N2 wild type and isp-1(qm1 s grown on control (EV) or klf-1 RNAi plates and assayed at the D1 or D5 of adulthood. Data are presented as mean ± SEM. *p < A with Tukey post hoc test. n = 60 animals per condition RE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w h Mitotracker Red CM-H2XRos staining of N2 wild type and isp-1(qm150);ctb-1(qm189) animals grown on control (EV) or klf-1 RNAi plates and assayed at the D1 or D5 of adulthood. Data are presented as mean ± SEM. p < 0.001, one-way ANOVA with Tukey post hoc test. n = 60 animals per condition Fig. 6 KLF-1 mediates a mitohormetic response in isp-1(qm150);ctb-1(qm189) mutants. a Lifespan curves of N2 wild-type and klf-1(tm1110) animals grown on 0.1 mM paraquat (PQ) their whole life. b–d Worms were grown on 0.1 mM paraquat (PQ) until L4 larval stage and then transferred to paraquat-free plates. Worms were exposed to klf-1 RNAi either whole life (b), in adulthood (c) or during development (d). e–g N2 wild-type and isp-1(qm150);ctb-1(qm189) animals were stained with Mitotracker Red CM-H2XRos (e) or Deep Red (f) to assay mitochondrial ROS levels and mitochondrial (mito) mass, respectively. Animals were assayed at larval L4 stage or D1 and the ﬂuorescence was measured via Biosorter. A ratio between Mitotracker Red CM- H2XRos and Deep Red ﬂuorescence measurements is shown in (g). h Mitotracker Red CM-H2XRos staining of N2 wild type and isp-1(qm150);ctb-1(qm189) animals grown on control (EV) or klf-1 RNAi plates and assayed at the D1 or D5 of adulthood. Data are presented as mean ± SEM. p < 0.001, one-way ANOVA with Tukey post hoc test. n = 60 animals per condition adulthood (Fig. 7d). In the isp-1;ctb-1 animals we observed persistent KLF-1 localization at D1 that is largely reduced by the treatment with vitamin C, providing further evidence that oxidative stress signalling plays important role in this process (Fig. 7d). A depletion of SKN-1 also induced KLF-1 translocation translocation to the nucleus induced by antimycin A treatment or isp-1;ctb-1 mutations, suggesting that KLF-1 activation is indeed regulated by redox signalling (Fig. 7c). Remarkably, in WT animals KLF-1 is predominantly present in the nucleus at L4 stage, to be quickly excluded upon transition to early TURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications 9 cleus in control animals, but not in isp-1;ctb-1 mutants entary Fig. 7b). As SKN-1 is a major regulator of nt-enzymes22, this effect in WT is likely a consequence ROS levels, while this response is epistatic in isp-1;ctb-1 ons in isp-1 and ctb-1 lead to decreased electron ﬂow this treatment largely prevented the KLF-1 translocation nucleus and therefore the CYPs activation (Fig. 7e, f). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w Remarkably, we could map the initial ROS signal nee the translocation/activation of KLF-1 to the L3–D1 tra which directly coincides with the peak in somatic mitoch biogenesis, as we previously described26. Hence, the 0 20 40 60 0 0.5 1 1.5 2 0 0.5 1 Control isp-1; ctb-1 klf-1-yfp DIC a 0.1 mM PQ S3QEL-2 isp-1;ctb-1 isp-1;ctb-1 c % worms 100 50 0 NAC Vit C N2 ctrl Low Medium High % worms b 100 50 PQ AA isp-1; ctb-1 0 Control Wild type Egg L3 % worms 100 50 0 Control N2 Fluorescence intensity (A.U.) N2 Control S3QEL-2 pcyp-25a2::gfp * *** f g + AA e % worms 100 50 0 ctrl NAC Vit C N2 isp-1;ctb-1 50 100 0 L4 D1 D5 L4 D1 D5 L4 D1 D5 isp-1;ctb-1 isp-1;ctb-1 Vit C N2 % worms d EV isp-1(qm150);ctb-1(qm189) Vit C from L3 Vit C from D1 percent survival Days 0 50 100 0 20 40 H2O2 stress resistance H2O2 stress resistance * Egg L1 L2 L3 L4 AA AA AA No treatment D1 S3QEL S3QEL S3QEL No treatment Egg L1 L2 L3 L4 D1 Vit C Vit C Vit C pcyp-25a1::rfp at D5 Egg L1 L2 L3 L4 D1 D5 h i j % survival % survival Normalized to control (EV) 0 20 40 60 * * CLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w Control isp-1; ctb-1 klf-1-yfp DIC a 0.1 mM PQ c % worms 100 50 0 NAC Vit C N2 ctrl Low Medium High % worms b 100 50 PQ AA isp-1; ctb-1 0 Control Wild type + AA % worms 100 50 0 ctrl NAC Vit C N2 isp-1;ctb-1 50 100 0 L4 D1 D5 L4 D1 D5 L4 D1 D5 isp-1;ctb-1 isp-1;ctb-1 Vit C N2 % worms d Low Medium High % worms b 100 50 PQ AA isp-1; ctb-1 0 Control Wild type Control isp-1; ctb-1 klf-1-yfp DIC a 0.1 mM PQ b a k DIC c % worms 100 50 0 NAC Vit C N2 ctrl % worm 50 PQ AA isp-1; ctb-1 0 Control Wild type + AA % worms 100 50 0 ctrl NAC Vit C N2 isp-1;ctb-1 50 100 0 L4 D1 D5 L4 D1 D5 L4 D1 D5 isp-1;ctb-1 isp-1;ctb-1 Vit C N2 % worms d c % worms 100 50 0 NAC Vit C N2 ctrl + AA % worms 100 50 0 ctrl NAC Vit C N2 isp-1;ctb-1 50 100 0 L4 D1 D5 L4 D1 D5 L4 D1 D5 isp-1;ctb-1 isp-1;ctb-1 Vit C N2 % worms d d 0 0.5 1 isp-1;ctb-1 Fluorescence intensity (A.U.) N2 Control S3QEL-2 pcyp-25a2::gfp * * f S3QEL-2 isp-1;ctb-1 Egg L3 % worms 100 50 0 Control N2 e f e 0 20 40 60 0 0.5 1 1.5 2 H2O2 stress resistance S3QEL S3QEL S3QEL No treatment Egg L1 L2 L3 L4 D1 Vit C Vit C Vit C pcyp-25a1::rfp at D5 Egg L1 L2 L3 L4 D1 D5 h j % survival Normalized to control (EV) * * g H2O2 stress resistance * Egg L1 L2 L3 L4 AA AA AA No treatment D1 % survival 0 20 40 60 0 20 40 60 H2O2 stress resistance S3QEL S3QEL S3QEL No treatment Egg L1 L2 L3 L4 D1 h % survival g h EV isp-1(qm150);ctb-1(qm189) Vit C from L3 Vit C from D1 percent survival Days 0 50 100 0 20 40 i i 0 0.5 1 1.5 2 Vit C Vit C Vit C pcyp-25a1::rfp at D5 Egg L1 L2 L3 L4 D1 D5 j Normalized to control (EV) * * j Normalized to control (EV) to the nucleus in control animals, but not in isp-1;ctb-1 mutants (Supplementary Fig. 7b). ARTICLE ARTICLE Fig. 7 KLF-1 activation is mediated by redox signalling. a Representative images of KLF-1-YFP, expressed under gut speciﬁc vha-6 promoter. Arrows indicate gut nuclei. Scale bar is 200 µm. b–e Animals were assayed based on KLF-1 nuclear localization as follows: “low” as less than 2 nuclei, “medium” 3–10 nuclei, and “high” where all nuclei were stained. Animals were imaged at D1, unless otherwise stated. n = 20 animals per condition. b WT animals were grown on control or plates containing 0.1 mM paraquat (PQ), or 1 µM Antimycin A (AA). c N2 animals were grown on control or plates containing 1 µM antymicin A or antimycin A in combination with 10 mM NAC or 10 mM vitamin C (left). Right, isp-1(qm150);ctb-1(qm189) were grown on 10 mM NAC or 10 mM vitamin C. As control, KLF-1-YFP nuclear localization in WT was used. d KLF-1-YFP expressing animals in N2 or isp-1(qm150);ctb-1(qm189) were assayed at L4 stage, D1 or D5, with or without vitamin C treatment. e isp-1(qm150);ctb-1(qm189) were grown on 100 µM S3QEL-2 either from egg stage or L3 stage and assayed for KLF-1 nuclear localization at D1. f Quantiﬁcation of rfp expression under the cyp-25a2 promoter in N2 and isp-1(qm150);ctb-1(qm189) upon treatment with 100 µM S3QEL-2 during whole development at the second day of adulthood. Data are presented as mean ± SEM. **p < 0.001, one-way ANOVA with Tukey post hoc test. n = 10 animals per condition. g N2 animals were treated with 20 mM H2O2 at D1 and survival was assayed 4 h later. Animals were treated with 2 µM antimycin at designated developmental stages. Data are presented as mean ± SEM. p < 0.05, Student’s T-test. n = 100 animals per condition. h isp-1(qm150);ctb-1(qm189) were treated with S3QEL-2 at designated developmental stages and the animals were assayed for H2O2 resistance at D1. Data are presented as mean ± SEM. *p < 0.01, Student’s T-test. n = 100 animals per condition. i Lifespan curve of isp-1(qm150);ctb-1(qm189) grown on control (EV) or treated with 10 mM vitamin C from either L3 stage or D1. j Fluorescence quantiﬁcation from pcyp-25a2::rfp reporter strain. isp-1(qm150); ctb-1(qm189) were assayed at D5, upon the designated treatments with 10 mM vitamin C. Data are normalized to control animals (presented as dashed line). p < 0.05, Student’s T-test. n = 10 animals per condition mitochondrial function. ARTICLE The longevity inducing signal, arising from mitochondrial dysfunction, coincides with a time when majority of somatic mitochondrial biogenesis occurs, between the L3 and D1 stages in worms26,35. We show that the abrupt increase in the number of dysfunctional mitochondria in mutant worms produces a signalling pulse of mtROS, which facilitates nuclear translocation, and therefore, activation of the KLF-1- mediated response. Remarkably, this is also the time when the aerobic respiration peaks36 and it was previously reported that downregulation of mitochondrial function only during this per- iod is sufﬁcient to prolong the lifespan37. Therefore, we propose that this speciﬁc period has been set as an important checkpoint for mitochondrial function that is carefully monitored as vital for the adult worm. A safety mechanism in the form of ROS- regulated stress response mediated by KLF-1 was also put in place to ensure a damage control, upregulation of detoxiﬁcation response to safeguard proper functioning during the adulthood. We further show that the KLF-1 activation has to be timed to D1, in order to support reproduction and provoke the life-extending mitohormetic response observed in mitomutants. antimycin A, a potent inducer of ROS production at complex III, during this speciﬁc period, paradoxically, provides strong resistance to oxidative stress (Fig. 7g). Conversely, treatment with S3QEL-2 during the same period largely decreased H2O2 resistance in isp-1;ctb-1 mutants (Fig. 7h). Consistently, block of ROS production from L3 fully suppresses the longevity in isp-1; ctb-1, unlike suppression from D1 that had only a partial effect (Fig. 7i). This is further in agreement with ﬁnding that vitamin C treatment also decreases expression of cyp-25a2 reporter when administered during the L3–D1 period (Fig. 7j). antimycin A, a potent inducer of ROS production at complex III, during this speciﬁc period, paradoxically, provides strong resistance to oxidative stress (Fig. 7g). Conversely, treatment with S3QEL-2 during the same period largely decreased H2O2 resistance in isp-1;ctb-1 mutants (Fig. 7h). Consistently, block of ROS production from L3 fully suppresses the longevity in isp-1; ctb-1, unlike suppression from D1 that had only a partial effect (Fig. 7i). This is further in agreement with ﬁnding that vitamin C treatment also decreases expression of cyp-25a2 reporter when administered during the L3–D1 period (Fig. 7j). Therefore, our data strongly imply that ROS directly or indirectly modiﬁes KLF-1 resulting in a subsequent nuclear translocation and activation of target genes. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w As SKN-1 is a major regulator of antioxidant-enzymes22, this effect in WT is likely a consequence of higher ROS levels, while this response is epistatic in isp-1;ctb-1 mutants. Mutations in isp-1 and ctb-1 lead to decreased electron ﬂow through respiratory complex III and increased ROS production15. To show that the initial redox signal needed for KLF-1 activation originates from mitochondrial ROS, we treated the isp-1;ctb-1 animals with S3QEL-2, a compound that speciﬁcally blocks ROS production from complex III, although not complex I27. Indeed, to the nucleus in control animals, but not in isp-1;ctb-1 mutants (Supplementary Fig. 7b). As SKN-1 is a major regulator of antioxidant-enzymes22, this effect in WT is likely a consequence of higher ROS levels, while this response is epistatic in isp-1;ctb-1 mutants. this treatment largely prevented the KLF-1 translocation to the nucleus and therefore the CYPs activation (Fig. 7e, f). Remarkably, we could map the initial ROS signal needed for the translocation/activation of KLF-1 to the L3–D1 transition, which directly coincides with the peak in somatic mitochondrial biogenesis, as we previously described26. Hence, the KLF-1 translocation could be prevented by the administration of S3QEL- 2 from the egg, but not if the treatment started at late L3 stage (Fig. 7e). The essential role of the L3 to young adulthood period for the activation of protective stress responses in isp-1;ctb-1 mutant is further supported by our ﬁnding that treatment with Mutations in isp-1 and ctb-1 lead to decreased electron ﬂow through respiratory complex III and increased ROS production15. To show that the initial redox signal needed for KLF-1 activation originates from mitochondrial ROS, we treated the isp-1;ctb-1 animals with S3QEL-2, a compound that speciﬁcally blocks ROS production from complex III, although not complex I27. Indeed, NATURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications 10 ARTICLE In line with this, we show that KLF-1 overexpression leads to only a moderate increase in lifespan, with little effect on analysed target genes (Supplementary Fig 7c, d). However, the increase was fully dependent on the cyps expression (Supplementary Fig. 7e). We could further show that the klf-1 overexpressors are more resistant to treatment with high H2O2 levels (Supplementary Fig. 7f). Collectively, these results suggest that longevity assurance adaptations that are regulated by KLF-1, although initiated by ROS, activate more elaborate stress responses rather than directly combating oxidative stress. This complex adaptation then contributes to mitohormesis that renders animals more resistant to toxic endogenous and xenobiotic compounds leading to an increased longevity. KLFs moderate many different fundamental processes in the cell38. Intriguingly, many of the mammalian 17 KLF homologues are involved in different kind of stress responses or are essential for switching developmental to adult gene programmes, as in the case of beta-globin genes38. Our results argue that both of these functions are preserved in KLF-1. The closest homologues (KLF2, KLF4 and KLF5) of nematode KLF-1 are also highly responsive to oxidative stress and seem to have complementary, very precisely timed functions in mammalian cardiomyocytes and vascular endothelium13,39. The dynamic regulation of expression of mul- tiple KLF family members suggests that they are actively involved in regulating phenotypic responses to extracellular stimuli39. Furthermore, some members of mammalian KLF family are shown to be important for mitochondrial function in different tissues, including KLF6, that is essential for mitochondrial gene expression and morphology in kidney or KLF4 and KLF15 that seem to regulate mitochondrial biogenesis and function in heart40–42. NATURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications Discussion h Our study now sug- gests that increased resistance to oxidative stress in long-lived animals stems from the activation of a complex xenobiotic detoxiﬁcation pathway shown to be upregulated in by all major longevity models. In view of this study, future research efforts should focus on establishing whether our ﬁndings extend to mammals as a prelude to developing novel interventions and new therapeutic strategies to combat aging in general and age- associated diseases in particular. Lifespan analysis. For lifespan analysis, worms were grown at 20 °C, unless otherwise stated. For the experiments with paraquat, the drug was added to the plates at a concentration of 0.1 mM and worms were exposed to the drug either their whole life or from hatching until L4 larval stage. First day of adulthood was deﬁned as day 1 of lifespan. Unless otherwise stated, 25 worms were transferred to each plate at day 0, for a total of 100–120 worms per experiment. Worms were examined every second day by prodding with a silver wire. The worms that escaped the plate, or died due to internal hatching or protrusions, were censored. For the “switch” experiments, worms were grown either on empty L4440, klf-1, cyc-1, atp-5 or cco-1 RNAi until the L4 larval stage or the third or ﬁfth day of adulthood, as stated. Worms were then transferred to either L4440 or klf-1 RNAi plates. Compilation of lifespan assays is listed in Supplementary Data 4. Lifespan analysis. For lifespan analysis, worms were grown at 20 °C, unless otherwise stated. For the experiments with paraquat, the drug was added to the plates at a concentration of 0.1 mM and worms were exposed to the drug either their whole life or from hatching until L4 larval stage. First day of adulthood was deﬁned as day 1 of lifespan. Unless otherwise stated, 25 worms were transferred to each plate at day 0, for a total of 100–120 worms per experiment. Worms were examined every second day by prodding with a silver wire. The worms that escaped the plate, or died due to internal hatching or protrusions, were censored. For the “switch” experiments, worms were grown either on empty L4440, klf-1, cyc-1, atp-5 or cco-1 RNAi until the L4 larval stage or the third or ﬁfth day of adulthood, as stated. Worms were then transferred to either L4440 or klf-1 RNAi plates. Compilation of lifespan assays is listed in Supplementary Data 4. Discussion h However, other longevity promoting pathways, such as decreased insulin/IGF1 signalling or dietary restriction, also activate the expression of DMEs in a wide array of organisms ranging from worms29,54 to mammals5. Therefore, it is possible that coupling of detoxiﬁcation pathways to longevity has arisen across different mechanisms that are regulated by similar or even overlapping pathways. Suitably, KLF-1 seems to be essential for the longevity in the mitomutants, upon dietary restriction and suppression of insulin signalling pathway, although different set of genes have been proposed to be essential for this effect12,13. RNAi Screen. For the isp-1(qm150);ctb-1(qm189) longevity suppression screen, RNAi clones from chromosome III of the Ahringer library8,57 were inoculated in 384-deep well plates and grown overnight. Bacteria were then seeded on 24-well NGM plates containing 100 μg/ml ampicillin and 2 mM IPTG. Each clone was seeded on four wells and induced overnight at room temperature. Afterwards, 15–20 eggs were placed into each well, and the worms were grown at 25 °C. To avoid separating the parental generation from the progeny, the isp-1(qm150);ctb-1 (qm189) strain was crossed into the eri-1(mg366) mutant background, since this mutation leads to embryonic lethality at 25 °C. As a control, eri-1(mg366) and isp-1 (qm150);ctb-1(qm189);eri-1(mg366) strains were grown on a separate 24-well plate, on L4440. Nine to ten days after reaching adulthood, 50% of the eri-1(mg366) population was scored as dead. On that day, survival of isp-1(qm150);ctb-1(qm189); eri-1(mg366) was assayed and the RNAi clones that gave a similar phenotype as the eri-1(mg366) single mutant, were identiﬁed. Among all the candidates identiﬁed, transcription factors were retested using eri-1(mg366) and isp-1(qm150);ctb-1 (qm189);eri-1(mg366) strains, at 25 °C. In summary, our results provide clear evidence that KLF-1 regulates longevity assurance pathway through its role in activa- tion of genes involved in xenobiotic detoxiﬁcation. This pathway seems to interconnect with many mechanisms of lifespan exten- sion, including mitochondrial dysfunction, caloric restriction and insulin/IGF-1 signalling. We show that the initial signal in mitochondrial mutants is precisely timed with a mild increase in mtROS production. Importantly, our results also provide an answer to a long-standing puzzle in the ﬁeld: Why have studies of antioxidant genes failed to reveal a major causal role in the reg- ulation of lifespan, despite the fact that most long-lived animals show higher resistance to oxidative stress? Discussion h (qm189);zcIs13, klf-1(tm1110), GR1373 eri-1(mg366), ATR2640 isp-1(qm150);ctb-1 (qm189);eri-1(mg366), TK22 mev-1(kn1), CW152 gas-1(fc21), CL2166 N2;dvIs19 [pgst-4::gfp-nls], ATR1040 isp-1(qm150);ctb-1(qm189);dvIs19, SD1444 gaIs237 [cyp- 25a2p::his-24::mCherry;unc-119(+)], ATR4029 isp-1(qm150); ctb-1(qm189); gaIs237, CF1553 muIs84 [(pAD76)sod-3p::gfp;rol-6(su1006)], ATR4051 isp-1 (qm150);ctb-1(qm189);muIs84 ATR4052 daf-2(e1370);muIs84, DA2154 phb-2 (ad2154), DA2123 N2;adIs2122 [plgg-1::gfp-lgg-1] and ATR1043 isp-1(qm150);ctb-1 (qm189);adIs2122. q ATR1022 N2; atEx100 [pklf-1::gfp;rol-6(su1006)] and ATR4030 N2; atEx4030 [pcyp-34a8::gfp;prab-3::mCherry], were generated by injecting the pklf-1::gfp (50 ng/ μl) and pcyp-34a8::gfp (50 ng/μl), respectively, with pRF4 (50 ng/μl) plasmids into the N2 wild-type animals. ATR1022 and ATR4030 were crossed into isp-1(qm150); ctb-1(qm189) to create ATR4050 isp-1(qm150);ctb-1(qm189);atEx100 and ATR4026 isp-1(qm150);ctb-1(qm189);atEx4030. ATR4086 N2;atEx4086 [pvha-6::klf-1-yfp; prab-3::mCherry;rol-6(su1006)] was created by using pvha-6::klf-1-yfp (40 ng/μl), pGH8 (20 ng/μl) and pRF4 (40 ng/μl) plasmids. Plasmid mixtures were then injected using standard procedures56. ATR4086 was subsequently integrated and outcrossed ﬁve times (ATR4081 strain) before being crossed into isp-1(qm150);ctb- 1(qm189) to create ATR4082 strain. RNAi treatment. RNAi knockdown was performed as described previously57. All genes for RNAi were obtained from the Ahringer RNAi library57 and conﬁrmed by sequencing. As a control, empty L4440 vector was used. All clones were trans- formed into the E. coli HT115 (DE3) strain. The overnight culture grown in Luria broth media was grown to OD595 = 0.5, and then IPTG was added to a con- centration of 1 mM. The bacteria were then induced for 3 h at 37 °C, shaking and seeded on NGM plates containing 100 μg/ml ampicillin and 1 mM IPTG. Worms were treated with RNAi from hatching and phenotype was observed as indicated. In order to obtain 5-day-old worms, beginning from the ﬁrst day of adulthood, worms were washed every day with M9 and allowed to settle by gravity, in order to remove eggs and lighter larvae. y Xenobiotic/detoxiﬁcation pathways are increased in isp-1;ctb-1, resulting in increased resistance toward different drugs, in agreement with reports on other mitomutants that exhibit resis- tance to multiple drugs53. Even a simultaneous knockdown of both mitochondrial SODs (SOD-2 and SOD-3) boosted the resistance to multiple drugs, suggesting that an increase in mitochondrial ROS is needed for this adaptation53. Previous studies have detected increased expression of genes encoding proteins involved in xenobiotic detoxiﬁcation, including cyp, ugt and gst genes, in long-lived mitochondrial mutants6,7,28. Since many bacterial species from C. elegans habitats target mito- chondria, the evolutionary advantage of coupling detection of mitochondrial dysfunction to antibacterial gene expression is clear7. Discussion h Oxidative stress sensitivity assay. In order to avoid bagging or egl phenotypes in worms, upon exposure to high oxidative stress, experiments were performed on strains with an eri-1(mg366) background at 25 °C, since this mutation leads to embryonic lethality at this temperature. At the L4 larval stage, the ﬁrst and the ﬁfth day of adulthood, worms were transferred onto NGM plates containing 16 mM paraquat. Survival of worms was checked every 8–12 h. H2O2 resistance assay. One-day-old adult worms were picked into 96-well plates ﬁlled with M9 buffer. H2O2 was then added to ﬁnal concentration of 20 mM. Animals were scored for survival every hour. Presented are the data 4 h after treatment. For each condition, 12 wells of 8 worms each were assayed. Discussion h The upregulation of genes involved in xenobiotic detoxiﬁcation has been increasingly recognized as a transcriptional signature of many long-lived mutants of different species1,7,28,29. Mice har- bouring various life-promoting mutations in growth hormone pathway, including Ames dwarf mice, Snell dwarf mice, GHRKO mice and Little mice, all show a transcriptomic signature of ele- vated xenobiotic detoxiﬁcation genes5,30. Different pharmacolo- gical and dietary interventions that promote longevity in mice also increase expression of DME encoding genes, including mice subjected to dietary restriction, reduced access to the mother during the breastfeeding period (‘crowded litter’), or treated with rapamycin5,31. In agreement, many of them also showed increased resistance to different xenobiotics, including hepato- toxins32–34. These ﬁndings suggest that increased metabolism of endo- and xenobiotics might be a downstream mechanism mediating the effects of multiple life-prolonging interventions. Here, we identify KLF-1 as the ﬁrst transcription factor regulating this response that is essential for a longevity mediated by reduced Our study also identiﬁed CYPs as direct effectors of the KLF-1 mediated response that promotes longevity. In mammals, the majority of cyp expression is governed by a very complex reg- ulation process that includes four nuclear transcription factors, two different cofactors, and an elaborate signalling cascade43. Intriguingly, some of mammalian KLFs were also shown to bind the basic transcription element (BTE) in different cyp promoters, and therefore, potentially regulate their expression44,45. In fact, some KLFs were initially identiﬁed as BTEB (BTE-binding) 11 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w proteins and only later renamed (BTEB/KLF9, BTEB2/KLF5, BTEB3/KLF13, BTEB4/KLF16)46–48. Recently, mammalian KLF6 was identiﬁed as a novel DNA binding partner of the aryl hydrocarbon receptor (AhR), a bona ﬁde transcription factor that activates expression of many cyps and ugts through binding to xenobiotic response elements (XRE)49, while KLF9 was shown to regulate hepatic CYP2D6 expression during pregnancy50. How- ever, very little is known about the physiological relevance of this regulation. Similarly, potential effect of KLFs on mammalian ageing has not been explored although they are involved in the regulation of many molecular and cellular hallmarks of aging as well as age-associated diseases51. We also demonstrated that both KLF-1 and CYPs are needed for the activation of phase II genes, mainly enzymes of glutathione metabolism22. However, KLF-1 does not directly regulate phase II genes and evidently SKN-1 does not regulate the expression of most cyp genes, as shown here and by others52. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w Brood size. Single worms were transferred on individual plates at the L4 larval stage and allowed to lay eggs. The worms were transferred to a fresh plate every day until they stopped laying eggs. The total amount of hatched progeny was scored and plotted as total brood size. The DNA fragments were sequenced using Hi-Seq Illumina platform. ChIP-Seq data was analysed by using QuickNGS (Next-Generation Sequencing) pipeline. The ChIP-Seq workﬂow takes advantage of BWA for genomic alignment of the reads. Reads was mapped to the Caenorhabditis_elegans (Ensembl database version 93). Quality check of the sequencing data was performed with FastQC version 0.10.1. For peak calling used MACS2 version 2.0.10. QuickNGS pipeline identiﬁes all the genes which are 2000 bp up- or downstream from the MACS2 peaks. The results comprise lists of signiﬁcant peaks. Result was uploaded into MySQL database. Alternatively, ChiP-Seq data were analysed with qPCR using primers listed in Supplementary Data 5. CHIP-seq data are available at NCBI Gene Expression Omnibus (GEO) under GSE130035. Movement. Worms were grown on indicated RNAi plates. On the ﬁrst and the ﬁfth day of adulthood, worms were transferred to non-seeded NGM plates, and allowed to settle for an hour. Afterwards, movement was scored as the number of full sinusoidal curves, body bends that a worm made moving forward or back- wards, over a period of 3 min. The data were presented as the number of body bends per minute. RNA isolation and qPCR. Worms were collected from a 9 cm plate and total RNA was isolated with Trizol (Invitrogen). DNAse treatment was performed using DNA-freeTM, DNAse and removal (Ambion, Life Technologies), according to the manufacturer’s protocol. RNA was quantiﬁed by spectrophotometry and 0.8 μg of total RNA was reversely transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For each condition, ﬁve independent samples were prepared. qPCR was performed using the Step One Plus Real-Time PCR System (Applied Biosystems), with the following PCR conditions: 3 min at 95 °C, followed by 40 cycles of 5 s at 95 °C and 15 s at 60 °C. Ampliﬁed products were detected with SYBR Green (Brilliant III Ultra Fast SYBR Green qPCR Master Mix, Agillent Technologies). Relative quantiﬁcation was performed against either act-1 or Y45F10D.4. Primers used in this study are presented in Supplementary Data 5. Data were analysed using ΔΔCt analysis. For each condition, ﬁve inde- pendent replicates were used. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w Western blotting was performed using antibodies against MnSOD (1:1000, Upstate #06-984), Grp75 (1:1000, Abcam #82591), HSP-60 (1:2000, BD Transduction Laboratories #611562), GFP (1:2000, Kindly provided by Jan Riemer), HSC70 (1:2000, Santa Cruz #sc-7298) and Tubulin (1:1000, Calbiochem #CP06). Oxyblot analysis was performed using the manufacturer’s protocol (Millipore) and the data were normalized to Ponceau S staining of the membrane. All uncropped blots are supplied in the Source Data File. Microarray analysis. For the microarray experiment, RNA was isolated from worms collected from a 9-cm plate, using the Qiagen RNeasy kit. Conditions and strains used are as follows: N2 and isp-1(qm150);ctb-1(qm189) grown on L4440 plates, isp-1(qm150);ctb-1(qm189) grown on klf-1 RNAi plates from hatching to the L4 larval stage and then switched to L4440 plates and isp-1(qm150);ctb-1(qm189) grown on L4440 from hatching to the L4 larval stage and then switched to klf-1 RNAi. All worms were collected on the ﬁfth day of adulthood. For each condition, three independent isolates were used. Reversely transcribed RNAs were hybridized to Affymetrix EleGene 1.0 ST microarrays, according to the manufacturer’s instructions. Affymetrix CEL ﬁles were processed with the Affymetrix Power Tools, version 1.15.2 and the Robust Multiarray Average normalization algorithm58. Analysis of differentially regulated genes was carried out using the R language for statistical computing, version 3.0.2. For functional annotation and GO term enrichment analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID), version 6.7 was used18. For the data presented in Supple- mentary Tables 1 and 2 and Supplementary Data 1, a cutoff of p < 0.05 and fold change >1.2 was used. For the data in the Fig. 4, a cutoff of p < 0.01 and fold change > 1.5 was used. Data availability is stated in Table 1. Microscopy. Animals were immobilized on 2% agarose pads in 5 mM levamisole buffer and imaged using an AxioImager Z.1 epiﬂuorescence microscope, equipped with a Hammamatsu camera (OrcaR2) and AxioVision software 4.8. Images were analysed using ImageJ (National Institutes of Health), as previously described60. Oxygen consumption. Oxygen consumption rates were measured using an Oro- boros Oxygraph 2k (Oroboros Instruments GmbH). Three hundred animals, on the ﬁrst or the ﬁfth day of adulthood, were used for each measurement. Each measurement was performed at 20 °C and repeated at least three times. Data were analysed using DatLab4 software (Version 4.3). Hepa1-6 cell experiments. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w ATR1022 strain was grown until adulthood on 20 °C degrees on normal NGM plates and then treated as follows: (i) for osmotic stress animals were transferred to NGM plates containing 500 mM NaCl and kept for 20 h, after which, the animals were washed off the plates using M9 buffer containing 300 mM NaCl and transferred to normal NGM plates; (ii) for heat stress, animals were incubated on 35 °C for a period of 9 h; (iii) for oxidative stress, young adults were either transferred to 16 mM paraquat plates for 24 h, or worms were grown from eggs until adulthood on 0.1 mM paraquat throughout the development. At the end of treatment, animals were imaged as described bellow. Xenobiotic resistance assays. For vinblastin treatment, ~20–30 synchronized L1 larvae were incubated in S-Complete media with 6 mg/ml HT115 bacteria. Wells were divided into controls without vinblastin and with 100 μM vinblastin (Sigma Aldrich). Worms were checked every day. When animals in the control wells reached adulthood, the animals treated with vinblastin were scored for develop- mental stages. At least ﬁve wells were used per condition and experiment was repeated three times. Western blotting. For protein sample preparations, worms were collected from three full 9-cm plates and wash extensively with M9 buffer, in order to remove the bacteria. The pellets were then frozen in liquid nitrogen. Two hundred microlitres of lysis buffer (25 mM Tris-HCl pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, 0.5% SDS, 10 mM DTT and proteinase inhibitor cocktail) was added to the pellet prior to thawing. Three freeze-thaw cycles were performed in liquid nitrogen, followed by sonication. The debris was spun down for 10 min at 16,000 × g at 4 °C and the supernatant was transferred to a fresh tube. Protein concentration was measured with Bradford assay. p For levamisole assay, levamisole was added directly to the NGM plates to ﬁnal concentration of 1 mM. Animals were transferred to these plates on the second day of adulthood and scored every 15 min for paralysis by prodding gently with the silver wire. If no movement was observed, animals were scored as paralysed. A minimum of ﬁve plates were used per condition with 25 worms each. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w Each gene is tested in two independent experiments. Data presented are average of one experiment. KLF-1 localization assay. To assess KLF-1 intracellular localization, ATR4081 and ATR4082 strains were used. For antioxidant treatments, animals were grown from eggs or as otherwise stated on 10 mM NAC (Sigma Aldrich), 10 mM vitamin C (Sigma Aldrich) and 100 μM S3QEL-2 (Caymann Chemicals). The chemicals were added directly to the NGM plates prior to seeding with HT115 bacterial strain. For antimycin A treatment, antimycin A was added directly to the NGM medium to ﬁnal concentration of 1 μM. These plates were than seeded with HT115 bacteria and allowed to dry from couple of days. Animals were grown until L4 larval stage and then transferred to the fresh NGM plates containing antimycin A. All animals were imaged as young adults unless otherwise stated. Activation was quantiﬁed as “low” when less than 2 nuclei per animal were stained, “medium” when 2–6 nuclei were stained, and “high” when more than 6 nuclei were observed. Each experiment was repeated at least three times on different days. ROS measurements. For ROS measurements and mitochondrial staining Mito- tracker Red CM-H2XRos and Mitotracker Deep Red were used, respectively. Animals were grown until indicated stages. Day before the experiments, NGM plates were seeded with heat-inactivated bacteria and let dry overnight on room temperature. On the day of the experiments, 200 μl of 10 μM dye solutions were added on top of the bacteria and let dry. Animals were then added to the plates and incubated for 1 h in the dark. Afterwards, animals were washed few times with M9 buffer and transferred to fresh plates without the dye for 2 h. Subsequently, worms were washed and analysed with Biosorter Instrument (Union Biometrica). klf-1 expression under stress conditions. Methods St i St Strains. Strains were cultured on OP50 E. coli-seeded NGM plates, according to standard protocols unless otherwise stated55. The following strains were used in this study: Bristol N2, MQ887 isp-1(qm150), MQ989 isp-1(qm150);ctb-1(qm189), CB1370 daf-2(e1370), SJ4100 N2;zcIs13 [hsp-6::gfp], ATR1010 isp-1(qm150);ctb-1 Post embryonic development. Worms were exposed to RNAi from hatching and scored every 8 h for vulva formation. The total time from hatching until the vulva formation was taken as developmental time. NATURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications 12 NATURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunicatio References S. & Ahringer, J. Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30, 313–321 (2003). 37. Rea, S. L., Ventura, N. & Johnson, T. E. Relationship between mitochondrial electron transport chain dysfunction, development, and life extension in Caenorhabditis elegans. PLoS Biol. 5, e259 (2007). 9. Feng, J., Bussiere, F. & Hekimi, S. 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NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w incubated for additional 24 h. After the incubation, the cells were collected and used for RNA isolation using Trizol Reagent (Thermo Fisher Scientiﬁc) according the manufacturer’s instructions. After DNase I treatment (New England Biolabs, UK), the total RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientiﬁc) with random primer. Quantitative polymerase chain reaction (qPCR) was performed with a QuantStudio 12 K Flex Real-Time PCR System (Thermo Fisher Scientiﬁc) in 12 μl aliquots of reaction mixtures containing cDNA, appropriate pairs of primers and Brilliant III Ultra-Fast SYBR®Green QPCR Master Mix (Agilent). Expression levels of the genes were calculated by the comparative CT method using HPRT as endogenous housekeeping genes. Primers used are enlisted in Supplementary Data 5. 19. 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Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development. Nucleic Acids Res. 37, 1817–1828 (2009). 27. Orr, A. L. et al. Suppressors of superoxide production from mitochondrial complex III. Nat. Chem. Biol. 11, 834–836 (2015). Received: 18 June 2018 Accepted: 28 June 2019 28. Shore, D. E., Carr, C. E. & Ruvkun, G. Induction of cytoprotective pathways is central to the extension of lifespan conferred by multiple longevity pathways. PLoS Genet. 8, e1002792 (2012). 29. McElwee, J. J., Schuster, E., Blanc, E., Thomas, J. H. & Gems, D. Shared transcriptional signature in Caenorhabditis elegans Dauer larvae and long- lived daf-2 mutants implicates detoxiﬁcation system in longevity assurance. J. Biol. Chem. 279, 44533–44543 (2004). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11275-w Hepa1-6 cells (ATCC®-CRL-1830™) were cultured in DMEM medium containing 4 mM L-Glutamine, 1 mM sodium pyruvate, 100 units/mL penicillin-streptomycin and 10% foetal bovine serum (FBS). For the gene silencing of Klf4, Klf5 and negative control 1.2 × 105 Hepa1-6 cells were transfected in a reverse transfection protocol with Lipofectamine RNAiMAX (Thermo Fisher Scientiﬁc) and each siRNA (purchased from Eurogentec) at a dose of 1 nM in 12 wells following the manufacturer’s instructions. siRNAs used are as follows: Klf4— GGAACUCUCUCACAUGAAG and Klf5—GACCAUGCGUAAC ACAGAU. ChiP-Seq assay. ChiP-Seq assay was performed as described previously59 using ATR4081 and ATR4082 strains at the ﬁrst day of adulthood. Crosslinking was performed in 1% formaldehyde for 20 min at room temperature. After lysis and sonication to obtain 300–1000 bp fragments, protein concentration was measured and proteins were normalized to 2 mg in 500 µl volume. Fifty microlitres of the volume was kept as input and the rest of the lysate was incubated with GFP-Trap (Chromotek) for 2 h at 4 °C. After washing, the crosslinks were reversed by incubation at 65 °C overnight. DNA was then isolated using phenol–chloroform–isoamylalcohol extraction and ethanol precipitation protocol. After 24-h cultivation at 37 °C in culture medium, antimycin A (purchased from Sigma Aldrich, 5 mM stock solution in 100% Ethanol, diluted to 2.5 µM working solution in H2O) was added to a ﬁnal concentration of 50 nM and 13 Competing interests: The authors declare no competing interes 53. Zubovych, I. O., Straud, S. & Roth, M. G. Mitochondrial dysfunction confers resistance to multiple drugs in Caenorhabditis elegans. Mol. Biol. Cell 21, 956–969 (2010). 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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/. 59. Mukhopadhyay, A., Deplancke, B., Walhout, A. J. & Tissenbaum, H. A. Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans. Nat. Protoc. 3, 698–709 (2008). 60. Sumakovic, M. et al. Competing interests: The authors declare no competing interes UNC-108/RAB-2 and its effector RIC-19 are involved in dense core vesicle maturation in Caenorhabditis elegans. J. Cell Biol. 186, 897–914 (2009). Author contributions A.T. and M.H. conceived the project, designed the experiments, analysed the data and wrote the paper. 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Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf. Aging Cell 8, 524–541 (2009). Caenorhabditis elegans SKN-1/Nrf. Aging Cell 8, 524–541 (20 Acknowledgements g The authors wish to thank the Caenorhabditis Genetics Center (which is funded by National Institutes of Health Ofﬁce of Research Infrastructure Programs, P40 The authors wish to thank the Caenorhabditis Genetics Center (which is funded by National Institutes of Health Ofﬁce of Research Infrastructure Programs, P40 © The Author(s) 2019 15 15 NATURE COMMUNICATIONS \| (2019) 10:3323 \| https://doi.org/10.1038/s41467-019-11275-w \| www.nature.com/naturecommunications
https://openalex.org/W4386908783	https://zenodo.org/records/8366253/files/V18I09A30_8366253.pdf	English	null	THIRD PARTY FUNDS, OTHER BANK LOANS, AND CAPITAL ON FEE-BASED INCOME AT ISLAMIC BANKS IN INDONESIA	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	4,940	DOI: 10.5281/zenodo.8366253 DOI: 10.5281/zenodo.8366253 Abstract The main income of the bank including Islamic banks derived from financing. In Islamic banking is the main income can be derived from the margin and profit sharing. In one side financing give benefit to Islamic banking, but on the other hand face with credit risk, so the quality of financing is influential on Islamic banking income. Which in turn affects existing of the Islamic banking itself. It is necessary to diversify income sources of Islamic banking. Another source of revenue that can be obtained Islamic banking comes from fees derived from Islamic banking services that can be provided to the public in the form of wakalah, kafalah, hawalah and rahn. Islamic banking revenues derived from these services known as fee-based income (FBI). This research was conducted to analysis any factors that may affect the fee-based income of Islamic Banking. The independent variables used in this study a Third-Party Fund (DPK), Other Bank Loan (PBL) and Capital (MDL). Methods of this study using is quantitative where data used in the form of secondary data. The results of this study indicate that the Third-Party Fund (DPK) and Other Bank Loan (PBL) positive and significant impact on the achievement of Islamic banking fee-based income, while Capital (MDL) significant with negative effect. DPK, PBL and MDL influence significantly on the achievement of Islamic Banking Fee Based Income. Keywords: Fee Based Income, Islamic Banking, TSR Model, Circular Causations, eywords: Fee Based Income, Islamic Banking, TSR Model, Circular Causations, RADEN BAMBANG BUDHIJANA Department of Management, Indonesia Banking School, Jl. Kemang Raya no. 35, Jakarta Selatan. Email: r.bambang.budhijana@ibs.ac.id DIKDIK SALEH SADIKIN Department of Accounting, Indonesia Banking School, Jl. Kemang Raya no. 35, Jakarta Selatan. Email: dikdik.sadikin@ibs.ac.id @ibs.ac.id THIRD PARTY FUNDS, OTHER BANK LOANS, AND CAPITAL ON FEE-BASED INCOME AT ISLAMIC BANKS IN INDONESIA DIKDIK SALEH SADIKIN DOI: 10.5281/zenodo.8366253 DOI: 10.5281/zenodo.8366253 the DPK, Sharia Banking Financing has increased significantly from year to year. Sharia banking financing growth in turn has a direct effect on Sharia Banking. Operating Income generated from channeling financing. In Sharia Banking Operating Income in 3 years is estimated to grow by 1.95% (Aisy, 2016; Rahman, 2016; Lubis, 2016; Budhijana, 2022). the DPK, Sharia Banking Financing has increased significantly from year to year. Sharia banking financing growth in turn has a direct effect on Sharia Banking. Operating Income generated from channeling financing. In Sharia Banking Operating Income in 3 years is estimated to grow by 1.95% (Aisy, 2016; Rahman, 2016; Lubis, 2016; Budhijana, 2022). Services that can be provided by Islamic banks in the form of debt transfer, carry out a debit card business, provide a place for storing goods and securities, move money, perform trustee functions and provide facilities for issuing letters of credit and bank guarantees. Where the covenant used is the covenant used is the Akad Hawalah bil Ujroh, Akad Wakalah bil Ujroh, Akad Kafalah bil Ujroh and other contracts in accordance with Sharia Principles. In these services the bank will get a fee or ujrah as wages for the services provided. Sharia Banking Fee sourced from these services is also known as Fee Based Income (FBI). Fee Based Income is the profit gained from transactions provided in other bank services or spread based. The Sharia Bank FBI is recorded in the Sharia Bank Financial Statement Balance Sheet and recorded in other bank's income. Islamic Banking FBI within ten years grew quite high (Kasmir, 2001; Benamraoui, 2008; Choudhury, 2013; Budhijana, 2023: Lubis, 2016). Third-Party Funds are recorded in the Sharia Bank Report Balance Sheet as a Liability. Included in the category of liability because DPK must be returned at any time to the owner. In addition, Islamic Banking must provide compensation for deposits placed in the form of profit sharing or in other forms in accordance with the agreement and Sharia Principles. Furthermore, Financing is recorded in the Sharia Bank Report Balance as Bank Assets or Assets. Categorized in Assets because Financing is a productive Bank and generates income in the form of Profit Sharing or Margin (Lubis, 2016; Aisy, 2016). Sharia Bank service activities can generate income. Services also increase Sharia Bank assets such as debt takeover services. 1. INTRODUCTION Sharia banking in Indonesia in 2019 has 14 Sharia Commercial Banks (BUS) and 19 Commercial Banks that have 156 Sharia Business Units (UUS). Sharia Commercial Bank business activities include collecting funds in the form of Savings and Investment, Distributing Financing based on contracts that are not in conflict with Sharia Principles, Providing Services, among others, taking over debt, conducting debit card business, providing a place to store goods and securities, move money, perform trustee functions, provide letter of credit and bank guarantee facilities (OJK, 2015a; 2015b; 2019) The Bank business activity in its function as a business entity that collects funds from the public in the form of Demand Deposits, Savings and Deposits. By managing this, from year to year Sharia Banking showed a significant increase. In the last 3 years Third-Party Funds (DPK) successfully collected by Islamic banking have grown by 1.45%. The second Sharia Banking business activity is the distribution of financing to the public. In terms of distribution of financing, Islamic Banking is able to implement it optimally. This can be seen from the value of Financing to Deposit Ratio (FDR), which ranges from 89% - 103% in the last 3 years. As in 401 \| V 1 8 . I 0 9 DOI: 10.5281/zenodo.8366253 DOI: 10.5281/zenodo.8366253 that the source of bank funds comes from bank capital, community deposits and loans from other institutions where each source of funds has its own characteristics. that the source of bank funds comes from bank capital, community deposits and loans from other institutions where each source of funds has its own characteristics. Hadinoto further explained that the acquisition of funds was adjusted to the purpose of using the funds. Funding of the main activities of banks, namely the distribution of financing obtained from the DPK, funding for investment is obtained from own capital, while special funding needs such as liquidity needs are sourced from interbank loans. On the other hand, the Financing to Deposit Ratio (FDR) is quite high. Riyanto (2014) argues that the ideal FDR of Islamic banks is currently in the range of 90% to 95%. If FDR is too high, there is a risk of liquidity, while if it is too low, the profit sharing for the community is not attractive. With the current high FDR, Sharia Banking must be in the form of lowering the value of FDR, which can be pursued by increasing depositor funds or by reducing financing. This is where the important role of the FBI is as an effort to maintain the profitability of Islamic banking while reducing the risk of financing. The average amount of Third-Party Funds (DPK), Financing to deposit Ratio (FDR), Capital (MDL) and Other Bank Loans (PBL) need to be considered in increasing Fee Based Income (FBI). DPK has increased movements from 2009- 2018 in the direction of the FBI movement (positive indication). MDL has increased movement from 2009-2018 in the direction of the FBI movement (positive indication) except for 2015. In 2015, the FBI experienced a decline while the MDL increased. Then in 2017 the opposite happened. The FBI has increased but MDL has weakened and has a worse economic impact (negative indication). Considering the importance of the FBI for the continuity of bank growth, research is needed to find out what factors influence the FBI of Islamic Banking in Indonesia. By knowing these factors, the Islamic Bank can arrange appropriate steps in dealing with changes that occur in the variables that become these influential factors so that Islamic Banking can maintain or even increase the FBI. The purpose of this study is to Analyse the influence of Third-Party Funds (DPK); Financing Given (PYD); Capital (MDL); Other Bank Loans (PBL) towards Achievement of Sharia Banking Fee Based Income. Revenues in the form of FBI will be recorded as other operating income. Sharia Bank Revenues can be grouped into two major parts, namely Main Operational Revenues sourced from financing and other Operational Revenues sourced from the services provided. Other sources of income of Islamic banks can come from the distribution of financing and activities in the form of services (Wiroso, 2005; Purboastuti, 2015). Until now, income derived from financing is still dominant. Therefore, a method of obtaining income from banking services is needed in accordance with the provisions of Bank Indonesia through Bank Indonesia Circular Letter Number 9/24 / DPbS 2007. Based on this provision, Diversification of Revenue is the proportion between Fee Based Income and main operating income to one of the criteria in the assessment of Islamic bank health. If measured by these criteria for the past six years, Islamic Banking is ranked 1 (one) or in very good condition. This is due to the past 3 years the ratio of Islamic banking DP is always above 12% (OJK, 2019; Rahman, 2016; Lubis, 2016). Fee Based Income based on some previous researchers as stated by Aprillya (2013) and Adiyanti (2013) has a positive effect on increasing profitability and has a relatively smaller level of risk compared to the main operating income. Funding that can be channelled is highly dependent on the DPK successfully compiled by Islamic Banking. Hadinoto (2008) explains 402 \| V 1 8 . I 0 9 Y1 = f {(X1), (X2), (X3)} The financing functions provided by Shariah banks can be formulated using the Tauhidi String Relation (TSR) Model as follows: Simulation FBI[θ] = f {(DPK), (MDL), (PBL)} [θ] Where: FBI = Fee Based Income DPK = Third-party funds PBL = Other Bank Loans MDL = Owned Capital [θ] = Tauhidi Knowledge Subject to the functions of causation (Circular Causation / CC) are as follows: FBI [θ] = f {(DPK), (PBL), (MDL)} [θ] …. (1) DPK [θ] = f {(FBI), (PBL), (MDL)} [θ] …. (2) PBL [θ] = f {(FBI), (DPK), (MDL)} [θ] …. (3) MDL [θ] = f {(FBI), (DPK), (PBL)} [θ] …. (4) Subject to the functions of causation (Circular Causation / CC) are as follows: Subject to the functions of causation (Circular Causation / CC) are as follows: FBI [θ] = f {(DPK), (PBL), (MDL)} [θ] …. (1) According to Choudhury (2014), this research model approach known as an Interaction, Integration and Evolution Process (IIE) process. The IIE process does not recognize mutations as stated by Darwin, so cell mutations can be avoided. In the discussion of this analysis, Correlation Matrix which in conventional research is used to determine multicollinearity, in this study is used to determine the level of correlation between variables. With this Correlation Matrix, it can be seen the extent of the Interaction, Integration and Evolution Process (IIE) process between its variables. By using this Model, Sharia Banking Fee Based Income in Indonesia in the long run is theorized to experience the process of interaction, integration and evolution of a learning process as in Al-Ma'arij: 19-35. Fee Based Income is one of the important factors in the development of Islamic Banking. Fee Based Income has systemic interdependent variables. Each of these variables, need each other, fully support each other support and influence each other. Code of conduct needs one another in the system while reflecting patterns of interacting and pairing with each other Y1 = f {(X1), (X2), (X3)} Y1 = f {(X1), (X2), (X3)} DOI: 10.5281/zenodo.8366253 2. RESEARCH METHODS The Qur'an and the Hadith are references and form the basis of this research. Fee Based Income Shariah Banking is an important factor in the development of Shariah Banking. The Shariah Bank's Fee Based Income (Y1) is thought to be influenced by the following factors: Third Party Funds (X1), Other Bank Loans (X2) and Owned Capital (X3), in this regard the Shariah Banking Fee Based Income can be formulated as: 403 \| V 1 8 . I 0 9 DOI: 10.5281/zenodo.8366253 DOI: 10.5281/zenodo.8366253 Table 3.1: Research Regression Results From the regression results that can be seen in Table 3.1 above, the equation can be arranged as follows: Table 3.1: Research Regression Results Table 3.1: Research Regression Results From the regression results that can be seen in Table 3.1 above, the equation can be arranged as follows: 3. RESULTS AND DISCUSSION The estimation of multiple regression performed on panel data for Fee Based Income (FBI) as an independent variable with Third Party Funds (DPK), Other Bank Loans (PBL) and Owned Capital (MDL) as independent variables using the Fixed Effects Model obtained results as listed in the following table 3.1: 404 \| V 1 8 . I 0 9 404 \| V 1 8 . I 0 9 Circular Causation Circular Causation (Shuratic Process) is a causal relationship of interactions between variables leading to integration through evaluation and discussion that results in the evolution of learning. Also called Interaction, Integration & Evolution (IIE). Circular Causation is a process of inherent interaction in order to provide problems faced dynamically so that new knowledge or provisions are obtained in order to obtain the benefit of the people. Fee Based Income Sharia Banking is a long process which in its implementation will include the process of interaction, integration and evolution as a learning process. Fee Based Income as an important factor in the development of Islamic Banking has a special character. Fee Based Income has systemic interrelated variables. Each of these variables, need each other, full of mutual support and mutual influence. The mutual need for behavior in the system reflects between these variables interacting and pairing with each other. Circular Causation is applied in this study to find out whether each variable, both the independent variable and the independent variable influences and complement each other. DOI: 10.5281/zenodo.8366253 DOI: 10.5281/zenodo.8366253 1 billion rupiah increase in other bank loans, it will affect an increase in Fee Based Income of 191 million rupiah. Unlike the case with Third Party Funds and Other Bank Loans, Capital has a negative relationship with the acquisition of Fee Based Income. With the same assumption, each additional bank capital will have an impact on the reduction of Fee Based Income of 629 million rupiah. The regression model used uses the Fixed Effect Model which means that each cross section has a different intercept. From the linear regression equation, it can be seen that Bank Muamalat has the smallest constant, namely - 7.612197. While Bank Syariah Mandiri was recorded at - 7.172867 and Bank Syariah Mega Indonesia at -6,936309. FBI=C+1.676172DPK+0.190546PBL-0.628243*MDL Based on the regression using the Fixed Effects Model, it is known that in general Sharia Banking Fee Base Income (FBI) acquisition is influenced by the amount of the collection of Third-Party Funds (DPK), Loans to Other Banks (PBL) and Owned Capital (MDL). Variable deposits, PBL, and MDL both individually and together have a significant influence on the FBI. All independent variable t-values are greater than t-table values. The coefficient of determination (R2) for this model is very good at 0.980863 which means about 98 percent, meaning that 98% of the variation in the FBI dependent variable can be explained by the independent variable. And only the remaining 2% is explained by other variables outside this model. Based on the compound linear regression equation above it can also be seen that Third-Party Funds and Other Bank Loans have a positive relationship with the acquisition of Fee Based Income. Assuming there are no additions to other independent variables, for every increase of 1 billion rupiah from Third Party Funds, it will also affect the Fee Based Income increase of 1.68 billion rupiah. The same is true for other bank loans. With the same assumption for every 405 \| V 1 8 . I 0 9 DOI: 10.5281/zenodo.8366253 DOI: 10.5281/zenodo.8366253 positive value implies a good and mutually reinforcing interaction between Islamic banks. Likewise, Fee Based Income can encourage cooperation (complementary) between Islamic banking institutions. In optimizing Sharia Banking funding sources, it can be in the form of collecting Third Party Funds and Other Bank Loans. That is because these funding sources are more suitable to be used to increase the achievement of Fee Based Income compared to funding sources that come from capital (Kasmir, 2001; Rahman, 2016; Lubis, 2016) positive value implies a good and mutually reinforcing interaction between Islamic banks. Likewise, Fee Based Income can encourage cooperation (complementary) between Islamic banking institutions. In optimizing Sharia Banking funding sources, it can be in the form of collecting Third Party Funds and Other Bank Loans. That is because these funding sources are more suitable to be used to increase the achievement of Fee Based Income compared to funding sources that come from capital (Kasmir, 2001; Rahman, 2016; Lubis, 2016) PBL's interaction with the FBI shows that loans from other banks also have risks that if not managed properly will have an impact on a 6 percent reduction in FBI achievement. These risks include: (1) Other bank loans cannot be disbursed or idle, (2) Fees obtained are smaller than the cost of funds and bank costs, (3) Mismatch between the maturity of other bank loans and the maturity of services to customers. While PBL Interaction with PBL shows negative value. This shows the occurrence of unfavorable competition between Islamic banks to get loans from other banks. Where one of the factors is the lack of instruments used by Islamic banks to get loans from other banks. The interaction between MDL and the FBI is positive. This shows that the additional capital of an Islamic bank will increase the ability of the Islamic bank in providing excellent service to its customers. Likewise, the acquisition of Fee Based Income for an Islamic bank will increase the capital of an Islamic bank, both directly in the form of retained earnings or indirectly in the form of trust from shareholders to increase its participation. Based on these interactions form an integrative relationship between the FBI, PBL and MDL towards the FBI positively. This means that overall all variables are integrated well. Variables Interaction, Integration and Evolution Matrix From the results of the measurement of correlation levels in Circular Causation (CC) shown by each of the Matrix tables above, the discussion continues the process of measuring the level of interaction, integration and evolution (IIE). In sharia principles, the parties that establish cooperation in economic transactions are expected to develop together in a balanced manner. A balanced development will occur when each institution interacts properly. Over time, integration between banks, shareholders (capital owners), financing banks (lenders), shohibul mal (funding customers) & service customers are firmly formed through the role of government, compliance (compliance) in sharia, culture community and economic factors. 406 \| V 1 8 . I 0 9 DOI: 10.5281/zenodo.8366253 DOI: 10.5281/zenodo.8366253 Figure 3.1: Learning Process (Interaction, Integration and Evolution / IIE) Figure 3.1: Learning Process (Interaction, Integration and Evolution / IIE) In this case the learning process includes Interaction, Integration and Evolution. Learning Process is formed from pairs of each variable (called, namely: FBI, DPK, PBL and MDL) in Circular Causation equations, both in CC1, CC2, and CC3. The value formed from this process shows the level of interaction between variables. The stages of the learning process are described in detail as follows: Table 3.2: Variables Interaction, Integration and Evolution Matrix on CC1 DPK [θ] = f {(FBI), (PBL), (MDL)} [θ] Based on the results of the analysis of the interaction process in CC1 shows that interactions that occur between Islamic Banks in getting Fee Based Income / FBI occur positively. This Based on the results of the analysis of the interaction process in CC1 shows that interactions that occur between Islamic Banks in getting Fee Based Income / FBI occur positively. This Based on the results of the analysis of the interaction process in CC1 shows that interactions that occur between Islamic Banks in getting Fee Based Income / FBI occur positively. This 407 \| V 1 8 . I 0 9 407 \| V 1 8 . I 0 9 DOI: 10.5281/zenodo.8366253 DOI: 10.5281/zenodo.8366253 This competition can be in the form of: (1) Providing high Ratio, (2) Providing direct prizes and prize draws, (3) freeing certain service fees such as withdrawal fees at ATMs and so on. This condition certainly has an unfavorable impact which in turn will have an impact on the decline in the ability of Islamic banks to generate profits. This condition occurs not only in Islamic banking but also in conventional banking. The Financial Services Authority / OJK (2015) explains that due to the upward trend in deposit rates that are above the reference of Bank Indonesia and the Deposit Insurance Corporation, the OJK sets limits on bank funds interest rates. Sharia banking supervision is a must that must be carried out strictly (Aisy, 2016; Rahman, 2016; Lubis, 2016). Table 3.4: Variables Interaction, Integration and Evolution Matrix on CC3 MDL [θ] = f {(FBI), (DPK), (PBL)} [θ] Based on the analysis of the interaction process in CC3, it shows that the interaction that occurs between Fee Base Income and Third-Party Funds will weaken by 0.27 percent on the capital growth of Islamic banking. .4: Variables Interaction, Integration and Evolution Matrix on CC3 MDL [θ] = f {(FBI), (DPK), (PBL)} [θ] Based on the analysis of the interaction process in CC3, it shows that the interaction that occurs between Fee Base Income and Third-Party Funds will weaken by 0.27 percent on the capital growth of Islamic banking. Based on the analysis of the interaction process in CC3, it shows that the interaction that occurs between Fee Base Income and Third-Party Funds will weaken by 0.27 percent on the capital growth of Islamic banking. Therefore, innovative efforts are needed to encourage the emergence of banking products that have an easily controlled risk factor. If this can be developed, Sharia Banking products, especially those based on services that produce fees, will automatically reduce the dependence of Sharia Banking on income from financing sources and at the same time avoid the risk of problematic financing so that banking capital will be well accumulated (Lubis, 2016; Aisy, 2016) Bundling products specifically between financing and Sharia Banking services must be monitored in their implementation so that in the future the distribution of financing will affect the achievement of Fee Based Income. Opportunities that can be achieved in the future in this effort can reach 87.3 percent. Furthermore, in developing service products in order to encourage an increase in third party funds, banks still have a 64 percent chance to turn it into a service appeal. Table 3.3: Variables Interaction, Integration and Evolution Matrix on CC2 PBL [θ] = f {(FBI), (DPK), (MDL)}[θ] Based on the results of the analysis of the interaction process in CC2 shows that the interaction that occurred between Fee Base Income and Third Party Funds weakened by 3.74 percent. This indicates the occurrence of unfavorable competition between Islamic banks in collecting public funds. Table 3.3: Variables Interaction, Integration and Evolution Matrix on CC2 PBL [θ] = f {(FBI), (DPK), (MDL)}[θ] Based on the results of the analysis of the interaction process in CC2 shows that the interaction that occurred between Fee Base Income and Third Party Funds weakened by 3.74 percent. This indicates the occurrence of unfavorable competition between Islamic banks in collecting public funds. Based on the results of the analysis of the interaction process in CC2 shows that the interaction that occurred between Fee Base Income and Third Party Funds weakened by 3.74 percent. This indicates the occurrence of unfavorable competition between Islamic banks in collecting public funds. Based on the results of the analysis of the interaction process in CC2 shows that the interaction that occurred between Fee Base Income and Third Party Funds weakened by 3.74 percent. This indicates the occurrence of unfavorable competition between Islamic banks in collecting public funds. 408 \| V 1 8 . I 0 9 408 \| V 1 8 . I 0 9 DOI: 10.5281/zenodo.8366253 DOI: 10.5281/zenodo.8366253 banking has a significant influence in its role in contributing to providing funds community and has a useful business aspect. banking has a significant influence in its role in contributing to providing funds community and has a useful business aspect. 4. CONCLUSIONS From the results of the data analysis process and the discussion that has been presented, it can be concluded that in simulation the variables of third party funds, PBL (other bank loans) and MDL (owned capital) included in the TSR model have a significant effect on the achievement of the FBI (Fee Based Income) Islamic Banking in Indonesia. Circular Causation analysis shows that Islamic banking in Indonesia has great development opportunities. Increasing the achievement of Fee Based Income is expected to further enhance the confidence of Customers, Counterparties and Shareholders in Sharia Banking in strengthening their existence in the national economy. Developing Islamic Banking products needs to be focused on services that generate fees, by avoiding the risk of problematic financing. When bundling products, optimizing Islamic banking financing and services, it is necessary to specifically monitor their implementation When bundling products, optimizing Islamic banking financing and services, it is necessary to specifically monitor their implementation Avoid unfair competition for third party funds and prioritize the acquisition of alternative funds. Other bank loans are utilized effectively and efficiently so that the funds are not idle and detrimental. This needs to be a concern given that idle and misallocation of beneficiaries has the potential to weaken by 0.22 percent. Opportunity value in all processes of business change (evolution), interaction and integration that occurs both in CC1, CC2 and CC3 for each accumulated value variable which tends to strengthen and the whole process shows the development of Sharia 409 \| V 1 8 . I 0 9 References 1) Adiyanti, Sulis Khutijah. (2013). 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https://openalex.org/W3107012313	http://intellekt-izdanie.osu.ru/arch/2020_5_72.pdf	Russian	null	VALUE-BASED MANAGEMENT IN THE AGE OF DIGITIZATION	Intellekt. Innovacii. Investicii	2,020	cc-by	7,575	А. Г. Мнацаканян, А. Г. Харин А. Г. Мнацаканян, А. Г. Харин УДК 336.6 DOI: 10.25198/2077-7175-2020-5-72 СТОИМОСТНЫЙ ПОДХОД К УПРАВЛЕНИЮ БИЗНЕСОМ В ЭПОХУ ЦИФРОВИЗАЦИИ А. Г. Мнацаканян1, А. Г. Харин2 Калининградский государственный технический университет, Калининград, Россия 1e-mail: mag@klgtu.ru 2e-mail: aleksandr.harin@klgtu.ru А. Г. Мнацаканян1, А. Г. Харин2 Калининградский государственный технический университет, Калининград, Россия 1e-mail: mag@klgtu.ru 2e-mail: aleksandr.harin@klgtu.ru Аннотация. Цифровая экономика играет все более значимую роль в процессах социально-экономиче ского развития. Поддержка этой тенденции и повышение вклада цифровизации для роста частного и об щественного благосостояния возможны путем совершенствования методов и практик корпоративного управления. Одним из распространенных способов управления бизнесом является управление, основанное на стоимости. Однако в случае цифровой экономики применение стоимостных моделей управления ком паниями сопряжено с определенными трудностями. Существует два типа проблем при реализации сто имостного управления цифровым бизнесом. Они обусловлены несовершенством методологии измерения, а также труднодоступностью и несопоставимостью информации. Целью статьи является выработка подходов к управлению компаниями в условиях цифровизации эко номики на основе их стоимости. Для этого выявляются и анализируются ключевые проблемы, возника ющие в процессе применения в бизнесе цифровых технологий, рассматриваются особенности различных методов измерения стоимости и предлагаются пути совершенствования методологии оценки и управле ния стоимостью компаний, отражающие сущность цифрового бизнеса. Научную основу работы составляют эмпирические и теоретические методы экономических иссле дований. В статье используются результаты работ аналогичной тематики и собственные разработки авторов, анализ и обобщение которых позволило получить новый взгляд на решение проблемы. Результатом анализа стал вывод, что в эпоху цифровизации подходы к оценке и управлению стоимо стью бизнеса претерпевают значительные изменения. Роль детерминант стоимости переходит к фак торам, имеющим неосязаемую форму. Поэтому ключевым источником создания стоимости, роста богатства и благосостояния является способность компаний использовать возможности цифровых технологий, не забывая при этом об устойчивости бизнеса. Наилучшим решением является разработка такой системы управления, которая, с одной стороны, охватывала бы все основные операционные, финансовые и социальные аспекты деятельности компании, а с другой, учитывала особенности цифро вого бизнеса. Сконструированная с учетом данных требований система предоставит в распоряжение менеджеров эффективный инструмент управления, обеспечивающий устойчивое развитие компании. Ключевые слова: цифровизация, цифровая экономика, стоимость, методы управления, модели оценки стоимости. Для цитирования: Мнацаканян А. Г., Харин А. Г. Стоимостный подход к управлению бизнесом в эпоху цифровизации // Интеллект. Инновации. Инвестиции. – 2020. – № 5. – С. 72–82. DOI: 10.25198/2077-7175- 2020-5-72. Введение фундаментальных проблем. К числу таких проблем, прежде всего, относится концентрация цифровых новаций и выгод от них в рамках узкого круга стран, компаний и частных лиц, усиливающая экономиче ское неравенство и социальную сегрегацию. В силу разных причин существует значительная неоднород ность протекания процесса цифровизации на меж государственном, межотраслевом и внутриотрасле вом уровнях. Результатом этого являются серьезные диспропорции, создающие новые угрозы как для стран, так и для компаний. Устранению указанного недостатка – сглаживанию неравномерности цифро вого развития в различных сегментах общественной жизни и расширению круга его выгодоприобретате лей служит поиск эффективных направлений и спо собов реализации потенциала цифровизации. Такой поиск требует не только творческого мышления, но и ясного понимания основ и движущих сил форми рования ценности в условиях цифровой экономики. Понятие стоимости является одним из ключевых в современной экономической теории и практике. Оно служит объяснению многих экономических процессов и явлений. Стоимость также широко ис пользуется для решения задач управления на всех его уровнях, выступая эффективным инструментом пла нирования и контроля экономической деятельности. Не является исключением и цифровая экономика, в понимании результатов развития которой стоимость играет столь же важную роль, как и в традиционной экономике. Вместе с тем, оценка стоимости, как и в целом любые измерения цифровой экономики, со пряжена с большими трудностями. Причины этих трудностей разнообразны, они носят фундаменталь ный и технический характер. Приведем лишь неко торые из них. Во-первых, до сих пор не сформулиро вано общепринятое определение цифровой экономи ки, нет также ясного понимания ее состава и границ. Во-вторых, не выработана общепринятая методоло гия оценки стоимости в цифровой экономике, охва тывающая все значимые аспекты ее деятельности (в том числе, бесплатные или условно бесплатные услуги), удобная для практического применения. В-третьих, отсутствуют надежные статистические данные о ключевых сторонах деятельности и резуль татах цифровой экономики, особенно в развиваю щихся странах1. В результате существующие спосо бы оценки стоимости в цифровой экономике могут давать различные результаты. Одной из основополагающих категорий совре менной экономики является понятие стоимости, обычно рассматриваемой как частный случай, де нежный эквивалент экономической ценности. Оли цетворяя богатство, стоимость может выступать одновременно и целью и инструментом управления. В том случае, когда показатель стоимости служит индикатором оценки эффективности экономическо го развития [2], речь идет о т. н. «стоимостном управ лении» или «управлении, базирующемся на стоимо сти» (Value-based management). Хотя многие прин ципы и технологии данной концепции управления универсальны и не зависят от степени цифровизации экономики, тем не менее, имеется ряд особенностей, определяющих различия в стоимостном управлении в рамках традиционного и цифрового бизнеса. VALUE-BASED MANAGEMENT IN THE AGE OF DIGITIZATION A. G. Mnatsakanyan1, A. G. Kharin2 Kaliningrad State Technical University, Kaliningrad, Russia 1e-mail: mag@klgtu.ru 2e-mail: aleksandr.harin@klgtu.ru A. G. Mnatsakanyan1, A. G. Kharin2 Kaliningrad State Technical University, Kaliningrad, Russia 1e-mail: mag@klgtu.ru 2e-mail: aleksandr.harin@klgtu.ru 2e-mail: aleksandr.harin@klgtu.ru Abstract. The digital economy has a growing role in the processes of socio-economic development. To support this trend and the growing contribution of the digital economy to the growth of private and public welfare, it is nec essary to improve corporate governance methods and practices. Among modern business management methods, an important place belongs to value-based management. However, in the case of companies in the digital economy, the application of this management model is difficult. There are two types of digital business value-based manage ment issues. They are due to the imperfection of the measurement methodology, as well as to the inaccessibility and incompatibility of information. The purpose of this paper is to formulate approaches to value-based management of digital economy 72 Стоимостный подход к управлению бизнесом в эпоху цифровизации nies. We analyze the key problems that arise in the process of managing a digital business, consider various meth ods for measuring the value of digital companies, and suggest ways to improve their value-based management.i nies. We analyze the key problems that arise in the process of managing a digital business, consider various meth ods for measuring the value of digital companies, and suggest ways to improve their value-based management. h fi b f k l d h l h h d h h l f nies. We analyze the key problems that arise in the process of managing a digital business, consider various meth ods for measuring the value of digital companies, and suggest ways to improve their value-based management. The scientific basis of our work is empirical and theoretical research methods. This paper uses the results of similar topics and the authors’ own developments. Analysis and generalization of this material made it possible to The scientific basis of our work is empirical and theoretical research methods. This paper uses the results of similar topics and the authors’ own developments. Analysis and generalization of this material made it possible to synthesize new approaches to solving the problem of digital business management.i We conclude that value-based management changes significantly at the digitalization stage. The role of the determinants of value goes to factors that have an intangible form. VALUE-BASED MANAGEMENT IN THE AGE OF DIGITIZATION Therefore, the key source of value creation, wealth growth is the ability of companies to use the capabilities of digital technology, while not forgetting the sus tainability of the business. We think that the best solution to this problem is to create a management system that, on the one hand, covers the main operational, financial and social aspects of the company, and on the other, takes into account the features of digital business. A system that is designed to meet these requirements will provide manag ers with an effective tool for managing the company, which will ensure its sustainable development. Cite as: Mnatsakanyan, A. G., Kharin, A. G. (2020) [Value-based management in the age of digitization]. Intellekt. Innovatsii. Investitsii [Intellect. Innovations. Investments]. Vol. 5, рр. 72–82. DOI: 10.25198/2077-7175-2020-5-72. International Monetary Fund (2018). Measuring the Digital Economy. Washington, D.C. International Monetary Fund, 48 р Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 Введение Но прежде чем приступить к рассмотрению этих разли чий, следует определить смысл, вкладываемый в по нятие ценность/стоимость в цифровой экономике. Проблемы управления связанные с цифровизацией бизнеса В настоящее время почти не подвергается сом нению тезис, что цифровизация становится одним из ключевых факторов экономического развития и роста благосостояния. Между тем, процесс циф ровизации в его современном виде порождает ряд 73 А. Г. Мнацаканян, А. Г. Харин Как отмечалось выше, расширение сферы циф ровой экономики создает новые экономические возможности. Использование цифровых инноваций и технологий становится дополнительным драйве ром, обеспечивающим рост производительности, способствует выпуску новых или более качест венных старых товаров и услуг, снижению затрат на их производство. Благодаря достижениям циф ровой экономики, например, таким как цифровые платформы, облегчаются транзакции и расширя ются возможности для обмена информацией меж ду участниками рынка. Все это, в конечном счете, улучшает результаты экономической деятельности и ведет к росту создаваемой стоимости. можно найти обзорном отчете ЮНКТАД, опубли кованном в 2019 г.2 Авторами этого отчета выделя ется ряд возможных последствий роста цифровой экономики, как позитивного, так и негативного характера. В частности, к ключевым позитивным результатам цифровизации они относят: ускорение экономического роста, модификацию или создание новых цепочек ценности, приводящие к росту эф фективности и добавленной стоимости, создание рабочих мест в цифровом секторе. Негативные же последствия, в основном сосредоточены в слабо оцифрованных отраслях и в целом сводятся к утра те конкурентоспособности и вытеснению с рынка «нецифровых» фирм, вплоть до разрушения от дельных отраслей в экономиках отставших стран. Вызнанные цифровизацией, глубокие структур ные изменения трансформируют цепочки создания стоимости и открывают новые способы создания добавленной стоимости. Однако положительный эффект от цифровизации не гарантирован всем участникам рынка и не возникает автоматически. Хотя цифровизация и обладает высоким потенци алом, способным поддерживать экономическое развитие, существует пока не до конца осознанная проблема вольного или невольного участия в со здании и потреблении создаваемых ею благ. Даже если отдельные лица, фирмы и страны ограничи вают свое участие в цифровой экономике, они кос венно, помимо своей воли, являются объектами ее влияния. Так, например, работники, не владеющие или плохо владеющие цифровыми навыками, могут оказаться в невыгодном положении по сравнению с теми, кто лучше подготовлен к требованиям цифро вой экономики, слабо цифровизированные фирмы могут оказаться не готовыми к конкуренции с более продвинутыми компаниями. Аналогичные пробле мы также возможны на региональном и межстра новом уровнях. Все это нивелирует возможности, открывающиеся в связи с цифровизацией общест венной и деловой среды, вплоть до отрицательного влияния цифровизации на процессы создания стои мости на всех уровнях экономики. 2 UNCTAD (2019). Digital Economy Report 2019. Value Creation and Capture: Implications for Developing Countries. Geneva. United Nations. UNCTAD, 22 р Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 Проблемы управления связанные с цифровизацией бизнеса в столь динамичной среде, какой является рынок цифровых товаров и услуг, оказывается невозмож ным для большинства компаний. 2. Особенности спроса. Более трудным, чем в случае предприятия, использующего цифровые технологии для модернизации производства явля ется определение реальной стоимости компаний, весь бизнес которых строится на предоставлении коллективного доступа к различного рода сетевым цифровым продуктам (онлайн-платформы, соци альные сети, справочно-информационные базы данных, электронные почтовые сервисы и т. д.). По скольку на цифровых рынках полезность товара или услуги часто зависит от количества и от активности потребителей, имеют место т.н. «прямые» и «кос венные сетевые эффекты»3, приводящие к тому, что на стоимость цифрового бизнеса сильное вли яние оказывают клиенты – потребители его услуг. С одной стороны, возникает своего рода эффект от масштаба рынка, когда полезность услуги непо средственно зависит от размеров рынка (например, от количества участников сети). С другой стороны, в условиях многосторонних рынков посредством центрального элемента сетевой инфраструктуры – онлайн-платформы – между клиентами формиру ются сложные связи, помогающие им извлекать выгоду из такого взаимодействия. Таким образом, цифровизация меняет психологию пользователей, предоставляя им возможность прямо или косвенно участвовать в процессе создания стоимости. В том случае, когда речь идет об управлении бизнесом на основе его стоимости, почти безаль тернативной остается концепция экономической ценности. И хотя в последнее время эта концепция часто критикуется за утрату способности выявлять реальную стоимость современного бизнеса и неко торых его активов, особенно элементов неосязае мого капитала, благодаря универсальности и гибко сти показателя стоимости потенциал концепции не исчерпан. Наиболее простым (хотя и не всегда оп тимальным) решением является модернизация су ществующих методов измерения стоимости с тем, чтобы вернуть этому показателю способность адек ватно оценивать все блага, создаваемые цифровой экономикой. При этом для того, чтобы модифици рованный показатель стоимости лучше отражал специфику управленческих процессов в цифровом бизнесе, его методология должна учитывать ряд но вых обстоятельств и факторов. К числу таких фак торов, влияющих на стоимость бизнеса в условиях цифровизации, относятся: 3. Быстрое устаревание производственных и нематериальных активов. Известно, что акти вы и продукты ИКТ, олицетворяющие технологи ческие знания, со временем устаревают и теряют свою стоимость быстрее, чем многие другие това ры. Темпы амортизации столь эфемерного объекта, каким являются знания, довольно трудно точно из мерить, поэтому часто сложно установить досто верную величину стоимости того или иного актива или продукта ИКТ в конкретный момент време ни. 3 OECD (2018). Tax Challenges Arising from Digitalisation – Interim Report 2018. OECD/G20 Base Erosion and Profit Shifting Proj ect. Available at: https://www.oecd-ilibrary.org/sites/9789264293083-en/index.html?itemId=/content/publication/9789264293083-en (дата обращения: 12.04.2020.) Проблемы управления связанные с цифровизацией бизнеса Поскольку ранее успешно применявшиеся биз нес-модели зачастую оказываются несостоятельны ми в условиях массового внедрения принципиально новых продуктов и услуг, кардинального измене ния структуры затрат, снижения входных барьеров и прочих последствий цифровизации, влекущих за собой изменения в цепочках создания стоимо сти, требуется переосмысление того, как создается, распространяется и фиксируется стоимость в этих новых реалиях. Данное требование означает необ ходимость использования методов управления биз несом, базирующихся на идеях, принципах и техно логиях цифровой экономики. Основой для принятия любых управленческих решений, особенно если речь идет об управлении бизнесом на основе его стоимости, является изме рение величины стоимости. Однако обозначенные выше особенности нынешнего этапа развития циф ровой экономики обусловливают ряд проблем, не избежно возникающих при попытке как-либо коли чественно оценить цифровой бизнес. Укрупненно выделяют два типа проблем. Они связаны, во-пер вых, с несовершенством методологии измерения и, во-вторых, с недоступностью и несопоставимостью информации [11]. Несмотря на различия в природе возникновения этих проблем, их общей фундамен тальной причиной можно считать незавершенный характер теоретической концепции цифровой эко номики, обусловливающий отсутствие ясного по нимания того, что должны измерять показатели стоимости, чтобы адекватно отражать специфику цифрового бизнеса и обеспечивать эффективное управление им. Для устранения этой причины, по мимо ликвидации теоретических пробелов в опи сании механизмов функционирования цифровой экономики, также необходимо улучшение сущест вующих и разработка новых методов оценки стои мости, адаптированных к особенностям цифрового Воздействие цифровизации на создание и рас пределение стоимости можно рассматривать в раз ных экономических аспектах (например, анализи ровать ее влияние на добавленную стоимость, на доход, производительность, занятость и пр.), с точ ки зрения различных субъектов (влияние на работ ников, на фирмы, правительство), а также с пози ций различных трактовок понятия цифровой эконо мики (влияние на ядро цифрового сектора, узко или широко трактуемую сферу цифровой экономики). Пример такого разностороннего анализа влияния цифровой экономики на создаваемую стоимость 2 UNCTAD (2019). Digital Economy Report 2019. Value Creation and Capture: Implications for Developing Countries. Geneva. United Nations. UNCTAD, 22 р Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 74 Стоимостный подход к управлению бизнесом в эпоху цифровизации бизнеса. Очевидно, что фундаментальность ука занной задачи обусловливает сложность ее реше ния. Избегая методологических сложностей можно предложить ряд направлений улучшения качества управления цифровым бизнесом в рамках сущест вующих подходов к управлению стоимости. Одним из первых шагов на этом пути должна стать систе матизация инструментария, способного служить измерению стоимости бизнеса в условиях перехода экономики к цифровому этапу развития. Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 Проблемы управления связанные с цифровизацией бизнеса Одной из особенностей цифровой экономики является денационализация бизнеса, обусловливающая тенденцию снижения трансграничных барьеров, размещения отдельных частей единого производственного процесса в раз ных странах и регионах. Благодаря использованию технологий удаленного доступа для многих пред приятий открываются возможности для экономиче ского присутствия и наращивания масштабов своего бизнеса одновременно на рынках многих стран без сколько-либо существенного физического присутст вия в этих странах. Таким образом, цифровизация нивелирует зависимость бизнеса от местоположе ния производства и клиентов и даже от расстояния между ними. В силу этого географическое располо жение предприятия не может рассматриваться в ка честве значимого фактора, влияющего на стоимость бизнеса, основанного на цифровых технологиях. Вышеназванные обстоятельства довольно убе дительно указывают на то, что в эпоху цифровых технологий подходы к оценке и управлению стои мостью бизнеса претерпевают значительные изме нения. Если в традиционной экономике основными детерминантами стоимости были внутренние свой ства товаров и их дефицитность, а относительная важность знаний в основном ограничивалась их вкладом в процесс производства, то в цифровой экономике детерминантами стоимости выступают трудноуловимые обстоятельства и факторы, многие из которых имеют неосязаемую форму. Стоимость все большего числа товаров и услуг определяется главным образом используемыми для их производ ства или содержащимися в них идеями и знаниями. Поэтому в новой экономике ключевым источником создания стоимости, роста богатства и благососто яния является способность бизнеса к инновациям. 5. Дифференциация продукта и цены. Цифровые технологии обеспечивают широкие возможности для дифференциации и адаптации к потребностям клиентов, выпускаемых фирмами товаров и услуг. Более того, некоторыми исследователями делается вывод, что расширенные возможности сбора дан ных о поведении потребителей позволяют компани ям индивидуализировать продукты и услуги в соот ветствии со специфическими потребностям каждого клиента4. В результате фирмы могут дополнительно получать часть излишка потребителей и, тем самым, создавать дополнительную стоимость. Отметим, однако, что пока нет эмпирических исследований, однозначно подтверждающих данное утверждение, поэтому остается открытым вопрос о конкурентных преимуществах индивидуализированного бизнеса по сравнению с массовым производством [15]. Тем не менее, в ряде случаев учет данного фактора необ ходим для адекватной оценки бизнеса. Изменение детерминант формирования стои мости, а также обусловленные этими изменениями сложность и неопределенность измерения стоимо сти цифрового бизнеса и многих его активов имеют не только экономические, но и социально-полити ческие последствия. Во-первых, бурный рост компаний цифрового сектора в сочетании с перманентной нестабильно стью современной мировой экономики обусловли вают дефицит безопасных с точки зрения инвести рования активов. В результате инвесторы испыты вают значительные трудности в поиске надежных инструментов и объектов для вложений капитала. Отсутствие безопасности само по себе усиливает 6. Проблемы управления связанные с цифровизацией бизнеса Как правило, на скорость амортизации объек тов ИКТ их внутренние характеристики (например, такие как, интенсивность использования или воз раст), оказывают меньшее влияние по сравнению с изменениями во внешних условиях (например, по явление новых технологий, изменение условий кон куренции или спроса). Данная особенность имеет существенное значение как при оценке стоимости активов предприятия, так и при определении вели чины создаваемой ими добавленной стоимости. 1. Изменения в структуре производства. До стижения в области цифровых технологий ради кально меняют структуру себестоимости конеч ной продукции, для которой становится типичной относительно низкая доля компонентов издержек, обычно относимых к категории постоянных. Как следствие, для такого бизнеса по мере роста объ емов производства характерно быстрое снижение предельных издержек. Подобный эффект от мас штаба проявляется, в первую очередь, в отраслях, активно использующих цифровые технологии и иные технологические новации. Фирмы, пользу ющиеся эффектом экономии от масштаба, как пра вило, обладают какими-либо активами с уникаль ными свойствами, позволяющими доминировать на рынке, получать монопольную прибыль и, сле довательно, создавать более высокую относитель но конкурентов новую стоимость. В тоже время, хотя новые технологии и приводят к консолидации бизнеса, компаниям цифровой экономики редко удается сохранять свою монопольную власть на долго [15]. Удержание доминирующих позиций 4. Независимость бизнеса от его местополо жения. В течение долгого времени географическое расположение предприятия рассматривалось как 75 нтеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 А. Г. Мнацаканян, А. Г. Харин тивы. Исследования показывают устойчиво расту щую долю нематериальных активов в финансовых отчетах фирм в течение, как минимум, последних полутора десятков лет [9]. В этот период общее ко личество заявок на права интеллектуальной собст венности в мире ежегодно увеличивалось в среднем на 7%5. Важность нематериальных активов в созда нии стоимости подтверждается непосредственной связью между величиной нематериальных активов и экономическим ростом в ведущих промышленно развитых странах [10]. Таким образом, сильная зави симость современного бизнеса от неосязаемых акти вов определяет необходимость рассмотрения этого рода активов в качестве одного из ключевых драйве ров стоимости. Хотя пока нерешенной остается про блема адекватной стоимостной оценки данного типа активов [4], их использование в моделях управления бизнесом в настоящее время не оспаривается [3]. один из ключевых факторов успеха деятельности и величины его стоимости. В основе принятия ре шений в традиционной экономике лежал принцип выбора выгодного местоположения, которое обес печивает компании относительно ее конкурентов такие преимущества, как возможность получения дохода, низкие удельные затраты, высокий уровень прибыли, доступ к ресурсам и к рабочей силе. Од нако в цифровой экономике этот фактор быстро утрачивает свое значение. 4 OECD (2015). OECD Science. Technology and Industry Scoreboard 2015: Innovation for growth and society. OECD Publishing. Avail able at: https://www.oecd-ilibrary.org/docserver/sti_scoreboard-2015-en.pdf?expires=1588687937&id=id&accname=guest&checksum=0E45 20594F0FF92C43BF39FAB182D627 (дата обращения: 12.04.2020.). 4 OECD (2015). OECD Science. Technology and Industry Scoreboard 2015: Innovation for growth and society. OECD Publishing. Avail able at: https://www.oecd-ilibrary.org/docserver/sti_scoreboard-2015-en.pdf?expires=1588687937&id=id&accname=guest&checksum=0E45 20594F0FF92C43BF39FAB182D627 (дата обращения: 12.04.2020.). 5 WIPO (2016). WIPO IP Facts and Figures. 2016. Available at: http://www.wipo.int/edocs/pubdocs/en/wipo_pub_943_2016.pdf (дата б 12 04 2020 ) 5 WIPO (2016). WIPO IP Facts and Figures. 2016. Available at: http://www.wipo.int/edocs/pubdocs/en/wipo_pub_943_2016.pdf (дата обращения: 12.04.2020.) Проблемы управления связанные с цифровизацией бизнеса Особая роль интеллектуальной собствен ности и прочих неосязаемых активов. Значимым фактором, влияние которого на стоимость бизнеса постоянно возрастает, являются неосязаемые ак 76 Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 Стоимостный подход к управлению бизнесом в эпоху цифровизации к измерению этой величины. Во-первых, оценка, основанная на внешних по отношению к бизнесу источниках, того, насколько высоко ценят бизнес рынок и общество. Во-вторых, оценка внутренних свойств бизнеса, главным образом, сводящихся к его способности генерировать достаточный по величине и устойчивый денежный поток. При этом следует учитывать, что объективная экономическая ценность бизнеса не может быть установлена толь ко одним способом, каким бы убедительным он не выглядел. Выбирая метод измерения стоимости, следует также помнить о предназначении данного показателя – обеспечивать принятие управленче ских решений. Исходя из этого, важным качеством используемого показателя стоимости, как части системы управления бизнесом является его способ ность удовлетворять требованиям оперативности и гибкости. Это означает несложность расчетов и простоту интерпретации данного показателя. неопределенность и риски. Более того, опыт пока зывает, что дефицит безопасных активов способен быть одной из причин финансовых кризисов. к измерению этой величины. Во-первых, оценка, основанная на внешних по отношению к бизнесу источниках, того, насколько высоко ценят бизнес рынок и общество. Во-вторых, оценка внутренних свойств бизнеса, главным образом, сводящихся к его способности генерировать достаточный по величине и устойчивый денежный поток. При этом следует учитывать, что объективная экономическая ценность бизнеса не может быть установлена толь ко одним способом, каким бы убедительным он не выглядел. Выбирая метод измерения стоимости, следует также помнить о предназначении данного показателя – обеспечивать принятие управленче ских решений. Исходя из этого, важным качеством используемого показателя стоимости, как части системы управления бизнесом является его способ ность удовлетворять требованиям оперативности и гибкости. Это означает несложность расчетов и простоту интерпретации данного показателя. Во-вторых, цифровые технологии кардинально меняют принципы функционирования экономики, что определяет приоритетность задачи совершен ствования методов управления как на корпоратив ном, так и на национальном уровнях. Межсекто ральный характер цифровизации обусловливает важность разработки и реализации общегосудар ственной политики в области цифровых техноло гий. Кроме того, поскольку цифровые технологии легко пересекают национальные границы, сущест вует необходимость поиска новых форм междуна родного сотрудничества в области регулирования деятельности фирм, активно использующих эти технологии. В-третьих, поскольку практически все основные теоретические положения концепции управления на основе стоимости были разработаны в доциф ровую эпоху, по мере развития цифровой экономи ки они утрачивают способность выступать в роли научного базиса эффективного управлении бизне сом. 1. Классические модели оценки. Наш обзор следует начать с универсального ин струмента оценки бизнеса – DCF-модели, которая, несмотря на солидный «возраст», по-прежнему спо собна давать хорошие результаты в самых сложных случаях, в том числе, при оценке бизнеса, использу ющего цифровые технологии. Но в отличие от тра диционного бизнеса, измерение стоимости компа ний цифровой экономики с помощью классической DCF-модели может оказаться довольно трудной за дачей. Современный бизнес тесно связан с быстро развивающимися технологиями, имеющими весьма неопределенное и трудно прогнозируемое будущее. В частности, исследование, MIT Sloan Management Review, показало, что более 78% руководителей компаний цифрового сектора считают невозможным точное экономическое обоснование планов разви тия своего бизнеса, таким образом, ставя под сомне ние целесообразность использования DCF-модели6. Другой существенный недостаток модели – слож ность определения многих ее компонентов при опи сании цифрового бизнеса, особенно, ведущегося в социальных сетях. В результате оценка цифровых Отмеченные аспекты, как представляется, долж ны определять магистральные направления разви тия теории и методологии управления стоимостью бизнеса в условиях цифровизации всех сторон об щественной и деловой жизни. С другой стороны, анализ и систематизация существующих инстру ментов и методов управления стоимостью позволя ет выделить среди них потенциально эффективные и легко адаптируемые к специфике и потребностям цифровой экономики. 6 Fitzgerald М., Kruschwitz N., Bonnet D., Welch M. (2013). Embracing Digital Technology: A New Strategic Imperative. Capgemini Consulting and MIT Sloan Management Review. Available at: https://www.capgemini.com/wpcontent/uploads/2017/07/embracing_digital_ technology_a_new_strategic_imperative.pdf (дата обращения: 12.04.2020.) Проблемы управления связанные с цифровизацией бизнеса Немаловажным недостатком существующих подходов к управлению на основе стоимости также является доминирование интересов одной группы стейкхолдеров – собственников и акционеров, что не отражает характер цифрового бизнеса и затруд няет принятие оптимальных решений о распре делении произведенной стоимости в интересах обеспечения социальной справедливости и общест венного благосостояния. В силу этого, существует необходимость в прикладных и фундаментальных исследованиях, нацеленных на формирование но вых научных взглядов, в полной мере отражающих реалии и перспективы цифровой экономики. Учитывая переходной характер этапа цифрови зации экономики, обеспечить одновременное вы полнение названных выше требований к оценке, с одной стороны, объективности и всесторонности, а с другой, простоты и оперативности, довольно сложно. Имеется ограниченное количество мето дов, в разной мере удовлетворяющих этим требова ниям. Перечислим некоторые из них, имеющие, на наш взгляд, перспективы для измерения стоимости цифрового бизнеса в целях управления. 1. Классические модели оценки. Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 7 Equidam (2016). Available at: https://www.equidam.com/resources/Equidam-Valuation-Methodology.pdf (дата обращения: 12.04.2020.) Методы оценки стоимости цифрового бизнеса: анализ и выводы Пожалуй, одной из центральных проблем управ ления стоимостью бизнеса является оценка величи ны стоимости. Существуют два основных подхода 77 Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 А. Г. Мнацаканян, А. Г. Харин в рамках традиционных технологий экономического анализа. Основу подхода составляет попытка выя вить стоимость отказа от использования продукта на определенный период времени. В частности, в одном из исследований, выполненных с помощью этого ме тода, были проведены крупномасштабные опросы, в которых потребителям предлагалось назвать цену, которая, по их мнению, компенсирует потерю досту па к ряду популярных бесплатных онлайн-сервисов. Авторы метода полагают, что анализ потребитель ского излишка – разницы между суммой, которую потребители готовы платить за товар или услугу, и тем, сколько они фактически платят, приблизитель но, но, тем не менее, адекватно измеряет стоимость многих цифровых товаров. Поскольку «лучший спо соб оценить цифровые товары – обратиться к людям напрямую и спросить их» [8]. предприятий с помощью классической DCF-модели часто оказывается малопродуктивной. Для решения проблемы ограниченной функци ональности DCF-модели в случае оценки бизнеса, основанного на цифровых технологиях, предлага ются различные подходы. Один из них состоит в по пытке нивелировать неопределенность, неизбежную при описании быстрорастущих компаний цифровой экономики посредством разработки и анализа взве шенных по вероятности сценариев. Авторы данного решения полагают, что даже разработка всего не скольких сценариев значительно повышает качест во DCF-моделей по сравнению с другими методами моделирования, позволяет ясно понять, что опреде ляет ценность современного бизнеса, основанного на цифровых технологиях [12]. Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 2. Альтернативные подходы. 2. Альтернативные подходы. Однако метод оценки потребительского излиш ка, основанный на опросах людей, имеет ряд из вестных недостатков. Во-первых, люди склонны скрывать истинную ценность тех благ, которыми они пользуются. Во-вторых, опросы, как правило, не оперативны и дорогостоящи. В-третьих, опросы потребителей позволяют выявить их субъективную готовность платить за какое-то благо, однако непо средственно не раскрывают стоимость этого блага [14]. Учитывая последний недостаток, развитием метода является использование моделей дискрет ного выбора, сочетающих данные о готовности платить с атрибутами (социально-экономическими характеристиками) респондентов [13]. Такая мето дология позволяет точнее измерить потребитель ский излишек, что, в конечном счете, дает более объективную оценку трудно поддающегося стои мостному измерению цифровому бизнесу. Поскольку имеются обоснованные сомнения в способности стандартных моделей стоимостной оценки обеспечивать надежность и гибкость про цесса управления, предлагаются альтернативные решения. Одним из таких подходов, призванных нивелировать неопределенность и учесть риски, связанные с цифровизацией бизнеса является ис пользование для принятия управленческих реше ний теории реальных опционов. Имеется обширная литература, посвященная применению теории реальных опционов для эко номической оценки проектов, связанных с исполь зованием объектов интеллектуальной собственно сти и ИКТ [7, 18], а также для разработки, оценки и принятия стратегических инвестиционных реше ний в области цифровых технологий [6, 17]. При менение методов реальных опционов позволяет ми нимизировать последствия ситуации неопределен ности при реализации сложных проектов цифровой трансформации бизнеса. В частности, разработана модификация метода NPV, дополняемого опцией оценки высокорисковых проектов с высокой вола тильностью ожидаемых будущих денежных потоков [16]. Подобного рода решения, улучшающие качест во традиционных методов оценки бизнеса, отчасти устраняют проблему неопределенности стратегиче ского выбора в случае цифрового бизнеса. Нетипичный характер цифрового бизнеса, в котором возрастающую роль играют различные проявления и формы человеческого капитала, об условливает поиск решений, способных соединить в рамках одной многофункциональной модели различные подходы к управлению. В результате появляются комплексные решения, построенные на сочетании методологических подходов и техно логий стратегического и финансового менеджмен та. Примером такого рода является прикладная методика, разработанная компанией Equidam – поставщиком продуктов для онлайн-оценки биз неса7, которая позволяет оценивать бизнес с раз ных точек зрения, дающих всестороннее и точное представление о его ценности. Полученные таким образом ключевые показатели эффективности (KPI) делают возможным оперативно оценивать влияние тех или иных цифровых новаций, при не Еще одним простым и, вместе с тем, эффек тивным способом оценки стоимости, создаваемой цифровым бизнесом, является непосредственное измерение благосостояния потребителей с помощью метода массовых экспериментов. dam (2016). Available at: https://www.equidam.com/resources/Equidam-Valuation-Methodology.pdf (дата обращения: ) 2. Альтернативные подходы. Данный подход призван решать проблему денежной оценки создава емых бизнесом, использующим цифровые техноло гии, немонетизируемых продуктов (например, таких как бесплатные цифровые сервисы), непреодолимую 7 Equidam (2016). Available at: https://www.equidam.com/resources/Equidam-Valuation-Methodology.pdf (дата обращения: 12.04.2020.) Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 78 Стоимостный подход к управлению бизнесом в эпоху цифровизации обходимости корректируя процесс их внедрения и обеспечивать движение к желаемой цели. Ме тодика допускает использование стандартных ме трик, разработанных в «доцифровую» эпоху с тем лишь условием, что эти метрики обеспечивают сбалансированность качественной и количествен ной оценки. Они также должны соответствовать отраслевой направленности компании и учиты вать приоритеты ее стратегии. В качестве таких метрик могут использоваться, в частности, такие известные и хорошо себя зарекомендовавшие ин струменты стратегического менеджмента как си стема сбалансированных показателей (Scorecard Method) и метод контрольного списка (Checklist Method), а также многие методы денежной оценки, например, модифицированные DCF-модели. обходимости корректируя процесс их внедрения и обеспечивать движение к желаемой цели. Ме тодика допускает использование стандартных ме трик, разработанных в «доцифровую» эпоху с тем лишь условием, что эти метрики обеспечивают сбалансированность качественной и количествен ной оценки. Они также должны соответствовать отраслевой направленности компании и учиты вать приоритеты ее стратегии. В качестве таких метрик могут использоваться, в частности, такие известные и хорошо себя зарекомендовавшие ин струменты стратегического менеджмента как си стема сбалансированных показателей (Scorecard Method) и метод контрольного списка (Checklist Method), а также многие методы денежной оценки, например, модифицированные DCF-модели. в распоряжение менеджеров эффективный инстру мент управления компанией, обеспечивающий ее устойчивое развитие. Пожалуй, наиболее сложным на пути создания такой методики является учет социальных аспектов. Цифровая трансформация влияет на многие сторо ны жизни и деятельности людей. Соответственно, для оценки этого влияния необходимы инструменты и методы, позволяющие измерить то, в какой сте пени цифровые технологии и новые бизнес-модели помогают решению социальных проблем. Решение задачи интеграции социальных аспектов в модели управления стоимостью цифровым бизнесом также осложняется неполнотой или отсутствием статисти ческих данных о том, как цифровизация влияет на благосостояние9. Это затрудняет выявление причин но-следственных связей между экономическими ре зультатами цифрового бизнеса и создаваемыми им социальными эффектами в различных областях. р р ф р Несмотря на заметный прогресс, достигнутый за счет разносторонней оценки, комплексному подходу присущ главный недостаток большинства существующих методов и моделей измерения стои мости – он опирается на старые метрики, изначаль но сконструированные для традиционного бизнеса. 8 Overby S. (2020). 3 Digital Transformation Metrics That Work For Everyone. CMO by Adobe. Analytics. Available at: https://cmo. adobe.com/articles/2017/9/3-digital-transformation-metrics-that-work-for-everyone.html#gs.50kv3l (дата обращения: 12.04.2020.) 9 OECD (2019). A measurement roadmap for the future, in Measuring the Digital Transformation: A Roadmap for the Future. Paris. OECD Publishing. Available at: www.oecd.org/going-digital/measurement-roadmap.pdf (дата обращения: 12.04.2020.) 2. Альтернативные подходы. Это дает основания некоторым авторам высказы вать мнение, что использование доцифровых KPI для комплексной оценки стоимости бизнеса, в ос нове которого лежат цифровые технологии, может оказаться «хуже, чем бесполезным»8. Использо вание организацией такой несовершенной кросс- функциональной метрики может приводить к тому, что у каждого структурного подразделения форми руется свой набор целей, которые могут противоре чить друг другу, что затрудняет измерение или даже снижает стоимость бизнеса. Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 rby S. (2020). 3 Digital Transformation Metrics That Work For Everyone. CMO by Adobe. Analytics. Available at: https://cmo. articles/2017/9/3-digital-transformation-metrics-that-work-for-everyone.html#gs.50kv3l (дата обращения: 12.04.2020.) D (2019). A measurement roadmap for the future, in Measuring the Digital Transformation: A Roadmap for the Future. Paris. i hi A il bl d / i di i l/ d df ( б 12 04 2020 ) Литература 1. Мнацаканян А. Г., Харин А. Г. Принципы устойчивого развития в управлении компанией // Соци ально-экономические явления и процессы. – 2016. – Т. 11. – № 10. – С. 41–50. 1. Мнацаканян А. Г., Харин А. Г. Принципы устойчивого развития в управлении компанией // Соци ально-экономические явления и процессы. – 2016. – Т. 11. – № 10. – С. 41–50. Сергеев Л. И. Развитие теоретических положений оценки экономической эффективности в конте атегории стоимости // Балтийский экономический журнал. – 2018. – № 3 (23). – С. 90–100. 3. Харин А. Г. 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Angelou G., Economides A. (2008). A real options approach for prioritizing ICT business alternatives: A case study from broadband technology business field. Journal of the Operational Research Society. Vol. 59. Issue 10, pp. 1340–1251. 6. Angelou G., Economides A. (2008). A real options approach for prioritizing ICT business alternatives: A case study from broadband technology business field. Journal of the Operational Research Society. Vol. 59. Issue 10, pp. 1340–1251. 7. Benaroch M., Kauffman R.J. (1999). A case for using real options analysis to evaluate information tech nology investments. Information Systems Research. Vol. 10. No. 1, pp. 70–86. 8. Brynjolfsson E., Collis A. (2020). How Should We Measure the Digital Economy? Hutchins Center on Fiscal & Monetary Policy at Brookings. Working Paper 57. Available at: https://www.brookings.edu/research/ how-should-we-measure-the-digital-economy (дата обращения: 12.04.2020.) 9. Clausen S., Hirth S. (2016). Measuring the value of intangibles // Journal of Corporate Finance. Vol. 40, pp. 110–127. 10. Corrado C. et al. (2012). Заключение Российская экономика находится на начальном этапе цифровой трансформации, в ней набирает силу процесс формирования комплексной инфор мационной инфраструктуры и массового внедрения в практику бизнеса информационных технологий. Цифровизация оказывает возрастающее влияние на воспроизводственные процессы, ведет к трансфор мации основ осуществления многих видов деятель ности как в традиционных, так и в новых отраслях [5]. Все это становится мощным стимулом для вне дрения в практику цифровизируемого бизнеса но вых методов управления, в том числе, основанных на стоимости. Кардинальным решением проблемы является разработка такой метрики, которая, с одной сторо ны, охватывала бы все основные аспекты деятель ности компании, а с другой, учитывала особенности бизнеса, активно использующего цифровые техно логии. При этом показатели, предназначенные для отслеживания текущего воздействия цифровых пре образований на бизнес должны находиться в тес ной взаимосвязи с его окружением и охватывать все ключевые стратегические направления, такие как, улучшение работы фирмы, качество обслужива ния клиентов и финансовые результаты. Важными также являются требования непрерывности, пре емственности и сбалансированности операцион ных, социальных и финансовых показателей такой метрики [1]. Сконструированная с учетом данных требований метрика, позволит не только адекват но оценивать цифровой бизнес, но и предоставит В настоящее время есть ряд предпосылок, позво ляющих реализовать стоимостной подход к управле нию бизнесом, базирующимся на цифровых техно логиях, используя для этого имеющиеся инструмен ты и модели, либо модифицируя их. Выбор того или иного способа управления во многом определяется особенностями самого бизнеса, целями и задачами его развития, а также и внешними условиями. Вместе с тем, перспективы стоимостного подхода в управ лении бизнесом, преимущественно основанном на использовании цифровых технологий, связаны не с адаптацией и совершенствованием существующих методов, а с разработкой новой методологии управ ления, в полной мере учитывающей специфику циф ровой экономики, как с позиций организации и про текания внутрифирменных процессов, так и с точки зрения интересов общества. нтеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 79 А. Г. Мнацаканян, А. Г. Харин Несмотря на многочисленные проблемы, име ющиеся на этом пути, перспективы дальнейшего социально-экономического развития нашей стра ны, как и будущее управления производственны ми и бизнес-процессами, несомненно, связаны с цифровизацией. Одним из ее аспектов является рассмотренный нами стоимостной подход – способ управления бизнесом, наиболее точно отражающий специфику цифровой экономики. References 70–86. (In Engl.). 8. Brynjolfsson, E., Collis, A. (2020) How Should We Measure the Digital Economy? Hutchins Center on Fiscal & Monetary Policy at Brookings. Working Paper 57. Available at: https://www.brookings.edu/research/ how-should-we-measure-the-digital-economy (accessed 12.04.2020). 9. Clausen, S., Hirth, S. (2016) Measuring the value of intangibles. Journal of Corporate Finance. Vol. 40, pp. 110–127. (In Engl.). 10. Corrado, C. et al. (2012) Intangible Capital and Growth in Advanced Economies: Measurement and Compar ative Results. CEPR Discussion Paper. Available at: https://papers.ssrn.com/sol3/papers.cfm?abstract_id=2153512 (accessed 12.04.2020). 11. G20 DETF (2018) Toolkit for Measuring the Digital Economy. G20 DETF. Argentina. Available at: https://www.itu.int/en/ITU-D/Statistics/Documents/g20-detf-toolkit_FINAL.pdf (accessed 12.04.2020). 12. Goedhart, M., Koller, Т., Wessels, D. (2016) Valuing high-tech companies. McKinsey. 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Available at: https://www.itu.int/en/ITU-D/Statistics/Documents/g20-detf-toolkit_FINAL.pdf (дата обращения: 12.04.2020.) 12. Goedhart M., Koller Т., Wessels D. (2016). Valuing high-tech companies. McKinsey. Available at: https:// www.mckinsey.com/ ~/media/McKinsey/Business%20Functions/Strategy%20and%20Corporate%20Finance/ Our%20Insights/Valuing%20high%20tech%20companies/Valuing%20high-tech%20companies.ashx (дата обращения: 12.04.2020.) 13. Herzog B. (2018). Valuation of Digital Platforms: Experimental Evidence for Google and Facebook. International Journal of Financial Studies. MDPI. Open Access Journal. Vol. 6(4), pр. 1-13. Available at: https:// www.mdpi.com/2227-7072/6/4/87/pdf (дата обращения: 12.04.2020.) p p ( р ) 14. Miller K., Hofstetter R., Krohmer H., Zhang J. (2011). How should consumers’ willingness to pay be measured? An empirical comparison of state-of-the-art approaches. Journal of Marketing Research. No. 48, pр. 172–184. 15. Papadimitriou P. 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Continuous strategic alignment: Exploiting informa tion technology capabilities for competitive success. European Management Journal, Vol. 11. No. 2, pр. 139–149. Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 80 Стоимостный подход к управлению бизнесом в эпоху цифровизации References 1. Mnatsakanyan, A. G., Kharin, A. G. (2016) [Principles of sustainable development in company manage ment]. Sotsial’no-ekonomicheskiye yavleniya i protsessy [Socio-economic phenomena and processes]. Vol. 11. No. 10, рр. 41–50. (In Russ.).i 1. Mnatsakanyan, A. G., Kharin, A. G. (2016) [Principles of sustainable development in company manage ment]. Sotsial’no-ekonomicheskiye yavleniya i protsessy [Socio-economic phenomena and processes]. Vol. 11. No. 10, рр. 41–50. (In Russ.).i 2. Sergeyev, L. I. (2018) [Development of the theoretical provisions for evaluating economic efficiency in the context of the category of value]. Baltiyskiy ekonomicheskiy zhurnal [Baltic Economic Journal]. Vol. 3 (23), pp. 90–100. (In Russ.). 2. Sergeyev, L. I. (2018) [Development of the theoretical provisions for evaluating economic efficiency in the context of the category of value]. Baltiyskiy ekonomicheskiy zhurnal [Baltic Economic Journal]. Vol. 3 (23), pp. 90–100. (In Russ.). 3. Kharin, A. G. (2012) [Study of the prerequisites and opportunities for the formation of a single value-ori ented approach to business management]. Ekonomicheskiy analiz: teoriya i praktika [Economic analysis: theory and practice]. Vol. 42(297), pp. 26–33. (In Russ.). 3. Kharin, A. G. (2012) [Study of the prerequisites and opportunities for the formation of a single value-ori ented approach to business management]. Ekonomicheskiy analiz: teoriya i praktika [Economic analysis: theory and practice]. Vol. 42(297), pp. 26–33. (In Russ.). p ] ( ) pp ( ) 4. Kharin, A.G. (2017) [Theoretical and practical aspects of the valuation of intangible assets of a company]. Baltiyskiy ekonomicheskiy zhurnal [Baltic Economic Journal]. Vol. 1(17), pp. 23–37. (In Russ.).i p ( ) pp ( ) 4. Kharin, A.G. (2017) [Theoretical and practical aspects of the valuation of intangible assets of a company]. Baltiyskiy ekonomicheskiy zhurnal [Baltic Economic Journal]. Vol. 1(17), pp. 23–37. (In Russ.).i 5. Kharin, A.G. (2019) [Digital economy and its prospects in the fishing industry]. Baltiyskiy ekonomiches kiy zhurnal [Baltic Economic Journal]. Vol. 3(27), pp. 75–86. (In Russ.). 5. Kharin, A.G. (2019) [Digital economy and its prospects in the fishing industry]. Baltiyskiy ekonomiches kiy zhurnal [Baltic Economic Journal]. Vol. 3(27), pp. 75–86. (In Russ.). 6. Angelou, G., Economides, A. (2008) A real options approach for prioritizing ICT business alternatives: A case study from broadband technology business field. Journal of the Operational Research Society. Vol. 59. Issue 10, pp. 1340–1251. (In Engl.). 7. Benaroch, M., Kauffman, R. J. (1999) A case for using real options analysis to evaluate information tech nology investments. Information Systems Research. Vol. 10. No. 1, pp. Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 Статья поступила в редакцию:12.05.2020; принята в печать: 19.08.2020. Статья поступила в редакцию:12.05.2020; принята в печать: 19.08.2020. прочитали и одобрили окончательный вариант рукописи. Авторы прочитали и одобрили окончательный вариант рукописи. Авторы прочитали и одобрили окончательный вариант рукописи. The paper was submitted: 12.05.2020 Accepted for publication: 19.08.2020. Информация об авторах: Альберт Гургенович Мнацаканян, доктор экономических наук, профессор, заведующий кафедрой отраслевых и корпоративных финансов, Калининградский государственный технический университет, Ка лининград, Россия 81 Интеллект. Инновации. Инвестиции / Intellect. Innovations. Investments • № 5, 2020 А. Г. Мнацаканян, А. Г. Харин ORCID ID: 0000-0001-8437-9852, Web of Science Researcher ID: AAB-7623-2020, Scopus Author ID: 0000-0001-8437-9852 e-mail: mag@klgtu.ru Александр Геннадьевич Харин, кандидат экономических наук, доцент кафедры отраслевых и кор поративных финансов, Калининградский государственный технический университет, Калининград, Россия ORCID ID: 0000-0002-4375-7666, Web of Science Researcher ID: AAF-1199-2019 e-mail: aleksandr.harin@klgtu.ru ORCID ID: 0000-0002-4375-7666, Web of Science Researcher ID: AAF-1199-2019 e-mail: aleksandr.harin@klgtu.ru оступила в редакцию:12.05.2020; принята в печать: 19.08.2020. Information about the authors: Albert Gurgenovich Mnatsakanyan, Doctor of Economics, Professor, Head of the Department of Industrial and Corporate Finance, Kaliningrad State Technical University, Kaliningrad, Russia ORCID ID 0000 0001 8437 9852 W b f S i R h ID AAB 7623 2020 S A th ID ORCID ID: 0000-0001-8437-9852, Web of Science Researcher ID: AAB-7623-2020, Scopus Author ID: 0000-0001-8437-9852 e-mail: mag@klgtu.ru Alexander Gennadievich Kharin, PhD in Economics, Associate Professor, Department of Industrial and Corporate Finance, Kaliningrad State Technical University, Kaliningrad, Russia ORCID ID: 0000-0002-4375-7666, Web of Science Researcher ID: AAF-1199-2019 e-mail: aleksandr.harin@klgtu.ru Alexander Gennadievich Kharin, PhD in Economics, Associate Professor, Department of Industrial and Corporate Finance, Kaliningrad State Technical University, Kaliningrad, Russia ORCID ID: 0000-0002-4375-7666, Web of Science Researcher ID: AAF-1199-2019 e-mail: aleksandr.harin@klgtu.ru The paper was submitted: 12.05.2020 Accepted for publication: 19.08.2020. The authors have read and approved the final manuscript. he authors have read and approved the final manuscr 82
https://openalex.org/W4294267021	https://www.researchsquare.com/article/rs-2006323/latest.pdf	English	null	Primary intestinal-type adenocarcinoma of the vulva with high tumor mutational burden and cancer-associated mutations: a case report	Research Square (Research Square)	2,022	cc-by	4,623	Primary intestinal-type adenocarcinoma of the vulva with high tumor mutational burden and cancer- associated mutations: a case report Hanako Sato Kindai University Faculty of Medicine Kosuke Murakami (  kmurakami@med.kindai.ac.jp ) Kindai University Faculty of Medicine Conclusions In cases of intestinal-type adenocarcinoma of the vulva, it may be helpful to check tumor mutational burden and gene mutations for treatment selection. Case presentation A 63-year-old Japanese woman came to the hospital because she was aware of a vulvar mass. There was a 1 cm mass on the dorsal side of the vulva, just outside the remains of the hymen. Biopsy revealed suspected adenocarcinoma, and wide local excision was performed. Based on histopathology and immunohistochemistry, the mass was diagnosed as intestinal-type adenocarcinoma. No other primary lesions were found, and the vulva was considered the primary site. The Gene panel test (FoundationOneCDx assay) showed a high tumor mutational burden and gene mutations in TP53, KEL, RB1, RNF43, PTEN, GNAS, and PIK3CA. Background Vulvar cancer is a rare disease, accounting for about 5% of gynecological malignancies. Intestinal-type adenocarcinoma of the vulva is extremely rare, and details regarding its origin and related genetic mutations are unknown. Treatment options for this cancer have not been defined. Case Report License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/14 Page 1/14 Background Vulvar cancer accounts for only approximately 3–4% of gynecologic malignancies [1]. Approximately 80% of vulvar cancers are squamous cell carcinoma, followed by basal cell carcinoma, Paget's disease, and melanoma. Adenocarcinoma is very rare [1]. Intestinal-type adenocarcinoma of the vulva was first reported in 1964 [2] and is diagnosed based on characteristic pathologic findings and immunohistochemistry (IHC). Several theories have been reported regarding the origin of intestinal-type adenocarcinoma of the vulva: the urethra, lower vaginal area, and rectum are derived from the cloaca, so the lower vaginal area contains remnants of bowel tissue [3]; ectopic bowel epithelium or intestinal metaplasia within the tissue derived from Müllerian duct [4]; and Bartholin’s glands in the vulva [5]. The precise origin of intestinal-type adenocarcinoma of the vulva remains unknown and there is no established treatment. In addition, the genetic mutation patterns of intestinal-type adenocarcinoma of the vulva have not been investigated. Here, we report a case of intestinal-type adenocarcinoma of the vulva with multiple cancer-associated mutations and high tumor mutational burden (TMB). Case Presentation Page 2/14 Page 2/14 A 63-year-old Japanese woman, gravida 4, para 3, was aware of a mass in the vulva and visited the clinic. She has no medical or family history and no smoking history. There was a 1 cm mass on the vulva that had self-destructed. The histopathological diagnosis of biopsy was adenocarcinoma (Fig. 1A). The pathological findings were not typical for vulvar primary cancer and metastatic carcinoma of gastrointestinal origin was suspected. The patient was referred to our hospital. Gross findings at the time of the visit to our hospital were redness with ulceration of approximately 5 mm just outside the 7 o'clock remains of the hymen, but the mass had already disappeared (Fig. 1B). On magnetic resonance imaging, no obvious mass was detected on both T1-weighted and T2-weighted images, but there was a high-signal area of approximately 5 mm on the diffusion-weighted image that was presumed to be a tumor lesion (Fig. 1C, D). Contrast-enhanced computed tomography and positron emission tomography showed no enlarged lymph nodes or distant metastases. Tumor markers were as follows: CEA, 1.8 ng/mL; CA125, 12 U/mL; and CA19-9, 40 U/mL. Only CA19-9 was slightly elevated. Upper gastrointestinal endoscopy revealed erosions in the gastric angulus, from which one biopsy was performed, but no malignant findings were observed. Lower gastrointestinal endoscopy revealed no abnormal findings. Wide local excision of the vulvar tumor was performed under spinal anesthesia. The resected specimen size was 2 × 1 × 1 cm with a margin at the site of the probable tumor (Fig. 1E). Grossly, there was an approximately 5 mm depression in the skin. The operation took 23 min and there was minimal blood loss. The postoperative course was good, and the patient was discharged the day after surgery. On histologic examination, the tumor was situated in the mucosa covered by squamous epithelium, some portion of which had sebaceous glands. The tumor was a cystic lesion invaginating from the surface and lined by a papillary and tubular proliferation of dysplastic columnar epithelium (Fig. 2A). Some dysplastic glands were on the mucosal surface and continuous with the non-neoplastic squamous epithelium (Fig. 2A, B). The tumor was well-circumscribed, with no infiltrative growth or lymphovascular infiltration. The histological picture was not unlike that of a colorectal adenoma, but especially in the tissue obtained by the biopsy, high-grade dysplasia was obvious with fused glands and enlarged nuclei with prominent nucleoli (Fig. 1A). Discussion And Conclusions Thirty cases of intestinal-type adenocarcinoma of the vulva or vagina have been reported. The countries and races are diverse, with a mean age of 54.8 (31–80) years (Table 2) [3–30]. This is the first report of primary intestinal-type adenocarcinoma of the vulva with high TMB accompanied by many cancer- associated mutations identified by gene panel testing. Symptoms of vulvar intestinal-type adenocarcinoma often include pain, itching, and bleeding. In the early stage, as in the present case, the patient may present only with a mass (Table 2). Histopathologically, some tumors are known to resemble colorectal villous adenoma [31]. It is also necessary to exclude metastatic gastrointestinal cancers, such as metastatic colorectal cancer. For diagnosis, IHC is important. Typically, the normal intestinal epithelium is CK7 negative, CK20 positive, and CDX2 positive. Most colorectal cancers are CK7 negative, CK20 positive, and CDX2 positive, while rectal cancers are somewhat more CK7 positive [32]. The present case was also a CK7-positive intestinal-type adenocarcinoma. In previous reports, among 20 cases in which IHC for CK20 and CK7 were performed, 19 cases (95%) were positive for CK20, and 13 cases (65%) were positive for CK7 were positive (Table 2). CDX2 is expressed in the mucosal epithelium from the duodenum to the rectum and is also positive in intestinal-type adenocarcinoma. In previous reports, IHC for CDX2 was positive in all 7 cases in which it was performed (Table 2). ER and PgR were mostly negative, including in the present case (Table 2). Interestingly, p16 was positive in the present case. In addition to this case, there are 5 reports of IHC for p16, 4 cases of which were positive (Table 2). In another report, reverse transcription polymerase chain reaction for HPV in p16- positive intestinal-type adenocarcinoma of the vulva did not detect HPV type 16, but only low-risk HPV [30]. HPV was negative in the present case. The significance of HPV is unclear and further study is needed. In the present case, p53 is a null pattern, which may reflect the TP53 mutation described below. Although there is one report of a patient who received neoadjuvant chemotherapy followed by surgery [22], most cases are preceded by surgery (Table 2). The most common surgical techniques are wide local excision and radical vulvectomy, with lymph node dissection in some cases (Table 2). Case Presentation The difference in the degree of atypia seen in the two specimens was not so pronounced as to allow for different diagnoses assigned separately to the biopsy and the resected specimen and both samples showed a cytologically similar neoplasm with brisk mitotic activity. Sebaceous glands were the only non-dysplastic glandular elements present in the vicinity of the tumor: Skene glands, minor vestibular glands, endometriosis, ectopic intestinal mucosa, or intestinal metaplasia were not identified; a portion of Bartholin gland was included in the periphery of the resected specimen, apart from the tumor. The IHC results were as follows: cytokeratin (CK) 20: positive, CK7: focal positive, CDX2: positive, estrogen receptor (ER): negative, progesterone receptor (PgR): negative, and PAX8: negative (Fig. 3A-F). p16 expression was strong and diffusely positive (Fig. 3G), but human papillomavirus (HPV) was negative (Fig. 3H). p53 was null pattern (Fig. 3I). PMS2 and MSH6 expressions were detected (Fig. 3J, K). Based on these results, the patient was diagnosed with primary intestinal-type adenocarcinoma of the vulva. A gene panel test (FoundationOneCDx assay, Foundation Medicine, Inc. Cambridge, MA, USA) was performed on the tumor portion of the explanted specimen. The TMB was 13 ds were the only non-dysplastic glandular elements present in the vicinity of the tumor: minor vestibular glands, endometriosis, ectopic intestinal mucosa, or intestinal metaplasia ed; a portion of Bartholin gland was included in the periphery of the resected specimen, umor. The IHC results were as follows: cytokeratin (CK) 20: positive, CK7: focal positive, estrogen receptor (ER): negative, progesterone receptor (PgR): negative, and PAX8: Page 3/14 mutations/megabase (Mut/Mb), which was high. The tumor was microsatellite stable. Gene mutations in TP53, KEL, RB1, RNF43, PTEN, GNAS, and PIK3CA were detected (Table 1). mutations/megabase (Mut/Mb), which was high. The tumor was microsatellite stable. Gene mutations in TP53, KEL, RB1, RNF43, PTEN, GNAS, and PIK3CA were detected (Table 1). The patient did not receive any adjuvant therapy. At 30 months postoperatively, there has been no recurrence. Discussion And Conclusions In the present case, the lesion was less than 2 cm grossly and there were no suspicious findings of lymph node or distant metastasis on imaging. Since the depth of invasion could not be accurately determined by biopsy, wide local excision was performed. Whether this case was an in situ or invasive lesion was a moot point: well- defined tumor border and overall cystic appearance argued for a possibility that this was an in situ carcinoma that arose within a pre-existing duct or cystic structure; on the other hand, background non- dysplastic epithelium to prove the existence of preceding benign structure was lacking and the tumor was not situated at the site of known major glands such as Bartholin glands or Skene glands; moreover, the Page 4/14 Page 4/14 underlying stroma of the dysplastic epithelium showed prominent inflammation, which was worrisome for tissue destruction. We considered it a minimally invasive carcinoma for the staging purpose and assigned it FIGO stage IA. There was a sufficient margin, and no adjuvant therapy was performed. Similarly, adjuvant therapy has not been used in most cases that could have been adequately removed by surgery (Table 2). While there is a report that intestinal-type adenocarcinoma of the vulva has a poor prognosis and that endoscopic follow-up of the colon is mandatory given the high propensity for associated gastrointestinal tumors [19], the course is often gradual, and the prognosis is good (Table 2). In the present case, several cancer-associated mutations were detected. No other reports have performed gene panel testing for intestinal-type adenocarcinoma of the vulva. TP53, KEL, RB1, RNF43, and PTEN showed high variant allele frequency and were considered clonal mutations. The variant allele frequency for PIK3CA and GNAS were very low and were considered to be subclonal mutations. KRAS mutation was not detected. In the gastrointestinal tract, except the colorectum, cancer that histopathology may show the pattern of intestinal-type adenocarcinoma is common in the esophagus [32], stomach [33], and duodenal papilla [34]. Outside of the gastrointestinal tract, it is most common in the sinuses and is the second most common histologic type of primary sinus cancer [35]. Rare cases have been reported of intestinal-type adenocarcinoma of the tongue [36], gallbladder [37], lung [38], bladder [39], and ureter [40] as primary sites. Regarding genetic mutations in cancers with histopathology showing intestinal-type adenocarcinoma, TP53 and KRAS mutations are often detected. Abbreviations IHC: immunohistochemistry TMB: tumor mutational burden HE: hematoxylin-eosin CK: cytokeratin ER: estrogen receptor PgR: progesterone receptor HPV: human papillomavirus Mut/Mb: mutations/megabase MSI: microsatellite instability Ethics approval and consent to participate Not applicable. Discussion And Conclusions TP53 mutations are particularly frequent in intestinal-type adenocarcinoma of the stomach and primary sinus [35, 41]. In intestinal-type adenocarcinomas of the duodenal papilla, TP53 and KRAS mutations were reported in approximately 40% of cases and RNF43 mutations in approximately 15% of cases (32), Another study reported mutations in KRAS, PIK3CA, and SMAD4 [42]. In intraductal papillary mucinous neoplasms of the pancreas of the intestinal type, the frequency of KRAS mutations is approximately 50% [43]. There is also a report of an intestinal-type adenocarcinoma with KRAS mutation arising from a mature cystic teratoma of the ovary [44]. On the other hand, KRAS and BRAF mutations are rare in intestinal-type adenocarcinomas of the sinus [35]. In the present case, TMB was high, at 13 Mut/Mb. TMB is an indicator of response to immunotherapy [45]. The KEYNOTE-158 study showed that advanced solid tumors with TMB ≥ 10 were more likely to respond to pembrolizumab, an anti-PD-1 antibody [46]. The present case was early stage and not treated with adjuvant therapy, but if the patient were in an advanced stage or had a recurrence, an immune checkpoint inhibitor like pembrolizumab could be a treatment option. Therefore, in intestinal-type adenocarcinoma of the vulva, a gene panel test may be helpful to select treatment options. In addition, although the present case was microsatellite stable, there are reports that microsatellite instability (MSI) - high and mismatch repair deficiency is more frequent in gastric intestinal-type adenocarcinoma [47, 48], and microsatellite status and IHC for mismatch repair protein may also be checked in intestinal-type adenocarcinoma of the vulva. In conclusion, here we report a case of intestinal-type adenocarcinoma of the vulva with multiple cancer- associated mutations and high TMB. In cases of intestinal-type adenocarcinoma of the vulva, it may be Page 5/14 helpful to check tumor mutational burden and gene mutations for treatment selection. helpful to check tumor mutational burden and gene mutations for treatment selection. Acknowledgments We thank Gabrielle White Wolf, Ph.D., from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. We thank Gabrielle White Wolf, Ph.D., from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. Competing interests The authors declare that they have no competing interests. The authors declare that they have no financial support. Authors' contributions HS and KM managed the patient and wrote the original draft. TO participated in the pathological evaluation. NM reviewed and edited the manuscript. All authors read and approved the final manuscript. HS and KM managed the patient and wrote the original draft. TO participated in the pathological evaluation. NM reviewed and edited the manuscript. All authors read and approved the final manuscript. Acknowledgments HS and KM managed the patient and wrote the original draft. TO participated in the pathological evaluation. NM reviewed and edited the manuscript. All authors read and approved the final manuscript. References 1. Wohlmuth C, Wohlmuth-Wieser I. Vulvar malignancies: an interdisciplinary perspective. J Dtsch Dermatol Ges. 2019;17:1257-76. 1. Wohlmuth C, Wohlmuth-Wieser I. Vulvar malignancies: an interdisciplinary perspective. J Dtsch Dermatol Ges. 2019;17:1257-76. 2. Lulenski CR, Naji AF. Mucin-secreting adenocarcinoma of bartholin gland. report of a case. Obstet Gynecol. 1964;24:542-4. 2. Lulenski CR, Naji AF. Mucin-secreting adenocarcinoma of bartholin gland. report of a case. Obstet Gynecol. 1964;24:542-4. 3. Tiltman AJ, Knutzen VK. Primary adenocarcinoma of the vulva originating in misplaced cloacal tissue. Obstet Gynecol. 1978;51:30s-3s. 3. Tiltman AJ, Knutzen VK. 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Mucinous Bladder Adenocarcinoma: Case Report and Literature Review. Case Rep Urol. 2015;2015:783109. 39. Kato H, Hayama M, Kobayashi M, Ota H, Nishizawa O. Large intestinal type-urachal adenocarcinoma with focal expression of prostatic specific antigen. Int J Urol. 2004;11:1033-5. 39. Kato H, Hayama M, Kobayashi M, Ota H, Nishizawa O. Large intestinal type-urachal adenocarcinoma with focal expression of prostatic specific antigen. Int J Urol. 2004;11:1033-5. 40. Network CGAR. Comprehensive molecular characterization of gastric adenocarcinoma. Nature. 2014;513:202-9. 40. Network CGAR. References Comprehensive molecular characterization of gastric adenocarcinoma. Nature. 2014;513:202-9. 41. Mikhitarian K, Pollen M, Zhao Z, Shyr Y, Merchant NB, Parikh A, et al. Epidermal growth factor receptor signaling pathway is frequently altered in ampullary carcinoma at protein and genetic levels. Mod Pathol. 2014;27:665-74. 41. Mikhitarian K, Pollen M, Zhao Z, Shyr Y, Merchant NB, Parikh A, et al. 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Cristescu R, Lee J, Nebozhyn M, Kim KM, Ting JC, Wong SS, et al. Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes. Nat Med. 2015;21:449-56. 46. Cristescu R, Lee J, Nebozhyn M, Kim KM, Ting JC, Wong SS, et al. Tables Table 1-2 is available in the Supplemental Files section. Table 1-2 is available in the Supplemental Files section. References Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes. Nat Med. 2015;21:449-56. 46. Cristescu R, Lee J, Nebozhyn M, Kim KM, Ting JC, Wong SS, et al. Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes. Nat Med. 2015;21:449-56. Page 9/14 47. Zhang L, Wang Y, Li Z, Lin D, Liu Y, Zhou L, et al. Clinicopathological features of tumor mutation burden, Epstein-Barr virus infection, microsatellite instability and PD-L1 status in Chinese patients with gastric cancer. Diagn Pathol. 2021;16:38. 47. Zhang L, Wang Y, Li Z, Lin D, Liu Y, Zhou L, et al. Clinicopathological features of tumor mutation burden, Epstein-Barr virus infection, microsatellite instability and PD-L1 status in Chinese patients with gastric cancer. Diagn Pathol. 2021;16:38. 47. Zhang L, Wang Y, Li Z, Lin D, Liu Y, Zhou L, et al. Clinicopathological features of tumor mutation burden, Epstein-Barr virus infection, microsatellite instability and PD-L1 status in Chinese patients with gastric cancer. Diagn Pathol. 2021;16:38. Figures Page 10/14 Figure 1 Page 11/14 pecimen. Magnification: 100×, scale bar: 250 µm he vaginal entry at 7 o'clock (black arrowhead). Figure 1 MRI and visual findings (after biopsy) A. Hematoxylin-eosin staining of the biopsy specimen. Magnification: 100×, scale bar: 250 µm Figure 1 MRI and visual findings (after biopsy) A. Hematoxylin-eosin staining of the biopsy specimen. Magnification: 100×, scale bar: 250 µm B. Visual findings. There was a red lesion at the vaginal entry at 7 o'clock (black arrowhead). Page 11/14 Page 11/14 Page 11/14 C. MRI T2-weighted image (axial). The lesion showed a high signal (white arrowhead). C. MRI T2-weighted image (axial). The lesion showed a high signal (white arrowhead). D. MRI Diffusion-weighted image (axial). The lesion showed restricted diffusion (white arrowhead). E. Surgical specimen. The thread indicates the ventral direction. D. MRI Diffusion-weighted image (axial). The lesion showed restricted diffusion (white arrowhead). E. Surgical specimen. The thread indicates the ventral direction. E. Surgical specimen. The thread indicates the ventral direction. Figure 2 Figure 2 Page 12/14 Pathological findings of hematoxylin-eosin staining Pathological findings of hematoxylin-eosin staining A. Hematoxylin-eosin staining. A magnified view of the arrowhead is shown in Fig.2B. An asterisk shows contamination. Magnification: 12.5×, scale bar: 1000 µm Page 13/14 contamination. Magnification: 12.5×, scale bar: 1000 µm B. Magnified view of the arrowhead in Fig.2A. Magnification: 100×, scale bar: 100 µm Figure 3 Pathological findings of immunohistochemistry A. Cytokeratin 20. Magnification: 100×, scale bar: 200 µm B. Cytokeratin 7. Magnification: 100×, scale bar: 200 µm C. CDX2. Magnification: 100×, scale bar: 200 µm D. Estrogen receptor. Magnification: 100×, scale bar: 200 µm E. Progesterone receptor. Magnification: 100×, scale bar: 200 µm F. PAX8. Magnification: 100×, scale bar: 200 µm G. p16. Magnification: 100×, scale bar: 200 µm B. Magnified view of the arrowhead in Fig.2A. Magnification: 100×, scale bar: 100 µm B. Magnified view of the arrowhead in Fig.2A. Magnification: 100×, scale bar: 100 µm Figure 3 Figure 3 Figure 3 Figure 3 Pathological findings of immunohistochemistry Pathological findings of immunohistochemistry Page 13/14 Pathological findings of immunohistochemistry A. Cytokeratin 20. Magnification: 100×, scale bar: 200 µm B. Cytokeratin 7. Magnification: 100×, scale bar: 200 µm C. CDX2. Magnification: 100×, scale bar: 200 µm D. Estrogen receptor. Magnification: 100×, scale bar: 200 µm E. Progesterone receptor. Magnification: 100×, scale bar: 200 µm F. PAX8. Magnification: 100×, scale bar: 200 µm G. p16. Magnification: 100×, scale bar: 200 µm H. HPV. Magnification: 100×, scale bar: 200 µm I. p53. Magnification: 100×, scale bar: 200 µm J. PMS2. Magnification: 100×, scale bar: 200 µm K. MSH6. Magnification: 100×, scale bar: 200 µm Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Table120220825.xlsx Table220220825.xlsx Page 14/14
https://openalex.org/W4393357677	https://parasitesandvectors.biomedcentral.com/counter/pdf/10.1186/s13071-024-06243-3	English	null	Spatiotemporal distribution and bionomics of Anopheles stephensi in different eco-epidemiological settings in Ethiopia	Parasites & vectors	2,024	cc-by	12,537	Abstract Background Malaria is a major public health concern in Ethiopia, and its incidence could worsen with the spread of the invasive mosquito species Anopheles stephensi in the country. This study aimed to provide updates on the distri- bution of An. stephensi and likely household exposure in Ethiopia. Methods Entomological surveillance was performed in 26 urban settings in Ethiopia from 2021 to 2023. A kilometer- by-kilometer quadrant was established per town, and approximately 20 structures per quadrant were surveyed every 3 months. Additional extensive sampling was conducted in 50 randomly selected structures in four urban centers in 2022 and 2023 to assess households’ exposure to An. stephensi. Prokopack aspirators and CDC light traps were used to collect adult mosquitoes, and standard dippers were used to collect immature stages. The collected mosquitoes were identified to species level by morphological keys and molecular methods. PCR assays were used to assess Plas- modium infection and mosquito blood meal source. Results Catches of adult An. stephensi were generally low (mean: 0.15 per trap), with eight positive sites among the 26 surveyed. This mosquito species was reported for the first time in Assosa, western Ethiopia. Anopheles stephensi was the predominant species in four of the eight positive sites, accounting for 75–100% relative abundance of the adult Anopheles catches. Household-level exposure, defined as the percentage of households with a perido- mestic presence of An. stephensi, ranged from 18% in Metehara to 30% in Danan. Anopheles arabiensis was the pre- dominant species in 20 of the 26 sites, accounting for 42.9–100% of the Anopheles catches. Bovine blood index, ovine blood index and human blood index values were 69.2%, 32.3% and 24.6%, respectively, for An. stephensi, and 65.4%, Spatiotemporal distribution and bionomics of Anopheles stephensi in different eco‑epidemiological settings in Ethiopia Temesgen Ashine1,2†, Adane Eyasu3†, Yehenew Asmamaw2, Eba Simma4, Endalew Zemene5, Adrienne Epstein6, Rebecca Brown6, Nigatu Negash2, Abena Kochora2, Alison M. Reynolds6, Mikiyas Gebremichael Bulto2, Temesgen Tafesse2, Alemayehu Dagne3, Biniyam Lukus3, Endashaw Esayas2, Sinknesh Wolde Behaksra2, Kidist Woldekidan2, Fikregabrail Aberra Kassa2, Jimma Dinsa Deressa2, Muluken Assefa2, Dereje Dillu7, Gudissa Assefa7, Hiwot Solomon7, Ahmed Zeynudin5, Fekadu Massebo1, Luigi Sedda8, Martin James Donnelly6, Anne L. Wilson6, David Weetman6, Endalamaw Gadisa2† and Delenasaw Yewhalaw3,5† © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. †Temesgen Ashine and Adane Eyasu contributed equally to this work. †Temesgen Ashine and Adane Eyasu contributed equally to this work. †Endalamaw Gadisa and Delenasaw Yewhalaw contributed equally to this work. †Endalamaw Gadisa and Delenasaw Yewhalaw contributed equally to this work. Correspondence: Temesgen Ashine ashine.temesgen@gmail.com Full list of author information is available at the end of the article Correspondence: Temesgen Ashine ashine.temesgen@gmail.com Full list of author information is available at the end of the article †Temesgen Ashine and Adane Eyasu contributed equally to this work. Ashine et al. Parasites & Vectors (2024) 17:166 https://doi.org/10.1186/s13071-024-06243-3 Ashine et al. Parasites & Vectors (2024) 17:166 https://doi.org/10.1186/s13071-024-06243-3 Parasites & Vectors Ashine et al. Parasites & Vectors (2024) 17:166 https://doi.org/10.1186/s13071-024-06243-3 RESEARCH Open Access Parasites & Vectors Spatiotemporal distribution and bionomics of Anopheles stephensi in different eco‑epidemiological settings in Ethiopia Temesgen Ashine1,2†, Adane Eyasu3†, Yehenew Asmamaw2, Eba Simma4, Endalew Zemene5, Adrienne Epstein6, Rebecca Brown6, Nigatu Negash2, Abena Kochora2, Alison M. Reynolds6, Mikiyas Gebremichael Bulto2, Temesgen Tafesse2, Alemayehu Dagne3, Biniyam Lukus3, Endashaw Esayas2, Sinknesh Wolde Behaksra2, Kidist Woldekidan2, Fikregabrail Aberra Kassa2, Jimma Dinsa Deressa2, Muluken Assefa2, Dereje Dillu7, Gudissa Assefa7, Hiwot Solomon7, Ahmed Zeynudin5, Fekadu Massebo1, Luigi Sedda8, Martin James Donnelly6, Anne L. Wilson6, David Weetman6, Endalamaw Gadisa2† and Delenasaw Yewhalaw3,5† Open Access Background pharoensis, An. funestus and An. nili, were incriminated as vectors of malaria [20]. Anopheles arabiensis is the primary malaria vector in most malaria-endemic areas [21–23]. However, its abundance, host preference and Plasmodium sporozoite rate vary across ecological gra- dients and epidemiological settings [22–25]. Anopheles pharoensis is of secondary importance [20, 23], with An. funestus and An. nili playing lesser roles in malaria trans- mission [22, 23].fi Malaria remains a threat to global public health, with 249 million cases and 608,000 deaths in 2022 [1]. The WHO African region is disproportionately affected, and approx- imately 78% of malaria-related deaths in the region were in children aged < 5 years [1–4]. More than half of all Ethiopians, mainly those in rural areas, are at risk of con- tracting malaria [5–7]. Unlike most African countries, clinical malaria of public health importance in Ethiopia is caused by both Plasmodium falciparum (P. falciparum) and P. vivax, which co-occur in all malarious areas, with the prevalence attributable to each parasite dependent on ecological settings and seasons [7, 8]. The transmission of malaria is highly variable due to the diverse eco-topogra- phy and climate conditions and, in general, transmission is bimodal, mainly occurring following the rainy seasons, “Kiremt” and “Belg,” which are associated with major (long) and minor (short) transmission periods, respec- tively [8]. Since the 2000s, comprehensive preventative and case management interventions, including improved coverage of long-lasting insecticide-treated bed-nets and indoor residual spraying, the rollout and scale-up of artemisinin-based combination therapy, the deployment of a more sensitive and specific rapid diagnostic test (his- tidine-rich protein-2/3 [HRP2/3]-based test) and treat- ment at the grassroots level through health extension programs have achieved successive reductions in malaria burden [7, 9–11]. As a consequence, the aim of Ethio- pia is to achieve zero indigenous malaria cases by 2030 [8, 12]. However, the country has experienced a nation- wide resurgence in recent years and an unprecedented increase in case burden [1, 13]. Possible contributing fac- tors include insecticide resistance in the primary malaria vector, the COVID-19 pandemic, the emergence of the HRP2/3 deletion, deterioration of the healthcare system, internal conflicts and invasion by the exotic malaria vec- tor, Anopheles stephensi [14–17]. © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ashine et al. Parasites & Vectors (2024) 17:166 Page 2 of 18 46.7% and 35.8%, respectively, for An. arabiensis. None of the 197 An. stephensi mosquitoes assayed tested positive for Plasmodium sporozoite, while of the 1434 An. arabiensis mosquitoes assayed, 62 were positive for Plasmodium (10 for P. falciparum and 52 for P. vivax). Conclusions This study shows that the geographical range of An. stephensi has expanded to western Ethiopia. Strongly zoophagic behavior coupled with low adult catches might explain the absence of Plasmodium infection. The level of household exposure to An. stephensi in this study varied across positive sites. Further research is needed to better understand the bionomics and contribution of An. stephensi to malaria transmission. Keywords Anopheles stephensi, Spatiotemporal distribution, Blood meal source, Sporozoite rate, Household’s exposure, Ethiopia Methods Study area dwellings/households. Based on the availability of aquatic habitats, we delineated a 1- × 1-km quadrant for entomological sampling (Fig. 2). In each quadrant, 20 households were selected for adult mosquito collection, of which four households were purposively selected based on their proximity to a major aquatic habitat and four were randomly selected in different directions from each of the four purposively chosen dwellings. Immature-stage mosquitoes were collected from all potential aquatic habitats within the compounds/prop- erty limits of the households selected for adult collec- tion as well as from the purposively identified aquatic habitats within the quadrant beyond the compounds/ property limits of the selected dwellings. Catches from the immature-stage collections were pooled by habitat type (either artificial or natural) and reared to adults for morphological species identification as described below. Written informed consent was obtained from the head of each household prior to mosquito col- lection. To increase the probability of detecting An. stephensi, adaptive sampling was employed [44, 45]. Thus, 50% of the households for adult collection were replaced randomly in subsequent collection rounds. y Twenty-six urban centers were selected for this study (Fig. 1). A range of variables, including ecological set- ting, presence of dry ports and major transportation corridors, An. stephensi habitat suitability modeling and malaria endemicity, were considered when selecting the sites [36, 42]. The study sites (Additional file 1: Table S1) are located at altitudes between 339 and 2355 m a.s.l. and range from hot-desert lowland to humid highland envi- ronments. The mean annual temperature ranges from 30.9 °C in Afambo, the northeastern tip of the country, to 15.6 °C in Akaki, central Ethiopia, and the mean annual rainfall ranges from 224 to 1883 mm2 [43]. Background Anopheles stephensi is an efficient urban malaria vec- tor in southeast Asia and the Gulf Region [26] but is currently expanding its geographical range in Africa, where there have been reports of its presence in Djibouti [27], Ethiopia [17], Sudan [28], Somalia [29] and, more recently, Nigeria, Eritrea, Ghana and Kenya [30]. Anoph- eles stephensi is known to readily invade urban environ- ments, and immature stages thrive in artificial aquatic habitats, with the consequent potential to increase malaria incidence in cities [27, 31] or reintroduce the disease into regions where it has been successfully elimi- nated [32–35]. In Ethiopia, since the first detection of An. stephensi in Kebri Dehar, Somali region [17], surveillance has con- firmed its presence in the central, northeast, northwest and southwest parts of the country [36, 37]. Recent stud- ies have shown that An. stephensi is a permissive host to P. falciparum and P. vivax infection [16, 37]. The results of a study from Dire Dawa City, eastern Ethiopia, suggest that An. stephensi was responsible, at least in part, for a malaria outbreak [38]. Similarly, in Djibouti, an upsurge in malaria incidence was observed following the detec- tion of An. stephensi, providing further evidence for the potential for increased risk [39, 40]. In line with the WHO’s call for strengthened entomological surveillance of An. stephensi [41], this study aims to update current data on the distribution of An. stephensi across Ethiopia and to increase understanding of the patterns of house- hold exposure to An. stephensi across the country. Until recently, about 46 Anopheles species and subspe- cies were recorded in Ethiopia [18, 19]. However, only a few Anopheles species, including An. arabiensis, An. Ashine et al. Parasites & Vectors (2024) 17:166 Page 3 of 18 Spatiotemporal distribution of An. stephensi Four rounds of adult and immature stage collections were conducted at approximately 3-month intervals from November 2021 to January 2023. At each of the 26 study sites, a preliminary survey was first conducted to locate potential aquatic habitats of Anopheles mos- quitoes outside compound/property limit of human Fig. 1 Map of study urban centers, with the colored dots indicating Anopheles stephensi-positive (red) and -negative sites (green) ig. 1 Map of study urban centers, with the colored dots indicating Anopheles stephensi-positive (red) and -negative sites (gre Ashine et al. Parasites & Vectors (2024) 17:166 Page 4 of 18 Random households Purposive households 100 – 200 meter 500 meter 1 kilometer Major aquatic habitat Fig. 2 Schematic showing the approach used to select habitats and households for Anopheles mosquito collection, with the aim to study the spatiotemporal distribution of An. stephensi in Ethiopia Random households Purposive households 100 – 200 meter 500 meter 1 kilometer Major aquatic habitat showing the approach used to select habitats and households for Anopheles mosquito collection, with the aim to study al distribution of An. stephensi in Ethiopia Fig. 2 Schematic showing the approach used to select habitats and households for Anopheles mosquito collection, with the spatiotemporal distribution of An. stephensi in Ethiopia Adult mosquito collectionsi p p Four urban centers where An. stephensi was detected were selected for more detailed study: Awash Sebat Kilo (Afar Regional State), Danan (Somali Regional State), Metehara (Oromia Regional State) and Jiga (Amhara Regional State). At each selected site, adult mosquitoes were collected from 50 randomly selected households and their surroundings in December 2022 and Febru- ary 2023. Using a map of urban centers, a household located toward the center of the town was selected first, and then four additional households were selected (at approximately a distance of 100 m, with each of these four dwellings located in a different direction from the first household). This approach was repeated 9 times, to select the remaining 45 households systematically (Fig. 3). Adult Anopheles collections were conducted both indoors and outdoors from the selected house- holds using Centers for Disease Control and Prevention light traps (CDC LTs) and from any structures serving as potential resting places within the household’s prop- erty compound using a Prokopack aspirator (John W. Hock Company, Gainesville, FL, USA). Trained field workers collected adult mosquitoes from 06:00 to 08:00 h and from 17:30 to 19:00 h with Prokopack aspirators. Indoor resting mosquito collections were con- ducted along the walls and ceilings of houses and under or behind household furniture. Outdoor collections were made from vegetation, tree trunks or walls of water con- tainers located outside of the selected structures. The collections were made for 15–30 min in each structure, with the time scaled to the number and size of the struc- tures/areas. The head of the household was requested to avoid activities that could repel mosquitoes on the night before collection, such as smoking inside the house, using repellents or spraying aerosol insecticides [46, 47]. Host-seeking adult mosquitoes were collected both indoors and outdoors using CDC LTs. Indoor traps were set near a sleeping space at the foot edge next to an exist- ing bed-net, and corresponding outdoor traps were set within 5–10 m of each household selected for the indoor collection. Traps were set approximately 1.5 m from the ground, both indoors and outdoors. At approximately 18:00 h, the battery was connected to run the trap, and the following morning, at 06:30 h, the trap was removed. Ashine et al. Parasites & Vectors (2024) 17:166 Page 5 of 18 Fig. 3 Diagrammatic representation of household selection for studying exposure to An. stephensi. Adult mosquito collectionsi Large shaded circles indicate the selected households together with additional structures within a 50-m radius for entomological collection. Shaded circle designated with A indicates the first selected household (in red) located toward the center of the town. Shaded circles designated with B–E indicate 4 additional households that were selected by moving 100 m (blue dotted line) in four directions from the first selected household. The remaining 45 households were selected by repeating this approach 9 times. The numbers in the blue circle indicate different types of structures within a 50-m radius of the selected household for adult or immature-stage mosquito collection Fig. 3 Diagrammatic representation of household selection for studying exposure to An. stephensi. Large shaded circles indicate the selected households together with additional structures within a 50-m radius for entomological collection. Shaded circle designated with A indicates the first selected household (in red) located toward the center of the town. Shaded circles designated with B–E indicate 4 additional households that were selected by moving 100 m (blue dotted line) in four directions from the first selected household. The remaining 45 households were selected by repeating this approach 9 times. The numbers in the blue circle indicate different types of structures within a 50-m radius of the selected household for adult or immature-stage mosquito collection Immature‑stage mosquito collection and rearing The collection cup was tied off securely prior to the trap being switched off and then labeled with the associated household code, whether an indoor/outdoor collection and with the date of collection before being removed from the trap and the mosquitoes retrieved. Aquatic habitats within a 50-m radius of the selected households were surveyed for immature stages, with the collections made using dippers and pipettes [48]. The col- lected immature-stage mosquitoes were subsequently transported to temporary field insectaries in plastic jars where they were transferred into enamel trays contain- ing water from their respective aquatic habitat and larval Ashine et al. Parasites & Vectors (2024) 17:166 Ashine et al. Parasites & Vectors (2024) 17:166 Page 6 of 18 food; the trays were check each day for pupation. Pupae were collected into beakers containing water from the respective aquatic habitat and placed in rearing cages with cotton balls soaked in a 10% sugar solution at the top of the cages. When all the pupae emerged, the beak- ers were removed from the rearing cages. CTATCAAGCAACACGACT3’) universal primers in a total reaction mixture of 25 µl containing 1 µl of DNA template, 0.25 µM each of ST-F and U5.8S-F primers, 0.37 µM of the UD2-R primer, 0.25 µM of dNTPs, 1.5 µM of Mg and 0.5 µM of Taq polymerase). PCR cycling con- ditions were: 95 °C for 30 s; 30 cycles of 95 °C for 30 s, 55 °C for 30 s and 68 °C for 7 min; with a final extension step at 68 °C for 7 min. The identification of specimens as An. stephensi was based on the visualization of a 438-bp band following gel electrophoresis of the PCR products. Morphological identification and preservation of mosquito samples All wild-caught adults and those reared from immature stages were sorted by genus and sex after anesthetiza- tion with 70% alcohol. Only female Anopheles and Aedes mosquitoes were counted and recorded. Female mosqui- toes belonging to the genus Anopheles were identified morphologically to the species level. Anopheles mosqui- toes with speckled legs, with maxillary palpus with two apical pale bands very broad, with speckling on palpus segment three and with second main dark area on wing vein one with two pale interruptions were identified as An. stephensi [49]. Wild-caught female adult Anopheles mosquitoes were sorted based on their abdominal status as freshly fed or unfed, then preserved individually in a 1.5-ml Eppendorf tube with holes at the top and sealed in Ziplock bags containing silica gel. The immature-stage (larvae and pupae) collections were pooled by the house- hold and preserved in absolute ethanol for molecular identification of An. stephensi. Detection of Plasmodium infection Plasmodium parasites in wild-caught Anopheles mosqui- toes were identified either by PCR (AHRI laboratory) or enzyme-linked immunosorbent assay (ELISA) (Jimma University TIDRC laboratory). The PCR used to detect Plasmodium parasites targeted the cytochrome c oxidase subunit 1 (COXI) mitochondrial gene, and the presence of the parasites was based on the visualization of a 540- bp region following gel electrophoresis of the PCR prod- ucts, as described elsewhere [51, 52]. Briefly, the PCR was performed in a total reaction volume of 25 µl containing 3 µl of DNA template from the head and thorax, prim- ers (0.25 µM), dNTPs (0.2 µM), Mg (1.5 µM) and 1 unit of Taq polymerase. For those samples which tested PCR- positive for COXI, a nested PCR was run targeting the small 18S subunit of P. falciparum and P. vivax [53]. The first amplification reaction (nested-1) was performed in a final reaction volume of 25 µl containing 3 µl of DNA template, and the second reaction was performed by using a 5 µl amplicon of nested-1. The presence of P. fal- ciparum and P. vivax was confirmed upon visualization of 205- and 120-bp bands, respectively, following electro- phoresis of the PCR products on 1% agarose gel. Molecular proceduresh Absorb- ance was read at 405–411 nm using an ELISA reader (model ELX800; BioTek, Winooski, VT, USA) after 30 and 60 min of incubation for P. falciparum and P. vivax, respectively. Samples with a value higher than twofold of the mean absorbance value of the negative controls were considered to be positive for Plasmodium parasites. acid]; Kirkegaard and Perry Laboratories [KPL], Gaith- ersburg, MD, USA) was added and the plate incubated for 30 min for P. falciparum and 60 min for P. vivax. Between the addition of each reagent or sample and the incubation, the wells were shaken and washed. Absorb- ance was read at 405–411 nm using an ELISA reader (model ELX800; BioTek, Winooski, VT, USA) after 30 and 60 min of incubation for P. falciparum and P. vivax, respectively. Samples with a value higher than twofold of the mean absorbance value of the negative controls were considered to be positive for Plasmodium parasites. Data management and statistical analysish Data management and statistical analysish Data management and statistical analysis The data were collected using tablets with data forms developed in REDCap [57, 58] and uploaded to the AHRI data server on a daily basis. Data were downloaded and cleaned using Microsoft Excel (Microsoft Corp., Red- mond, WA, USA), and Microsoft Excel and Stata soft- ware release 14 (StataCorp LLC, College Station, TX, USA) were used for analysis. Only Anopheles mosquito catches identified to species level were included in the statistical analyses. Site positivity proportion was deter- mined by dividing the number of sites at which Anoph- eles species were identified in at least one round of entomological surveys by the number of sites surveyed. To estimate catches per method of collection, method- specific catches of species were divided by the total number of that species caught. Relative abundance of Anopheles species was investigated per site as catches of specific species divided by the total adult-stage collec- tions per site. The mean number of catches per trap was determined by considering the number of traps in all col- lection rounds. Household exposure was defined as the presence of either immature or adult stages of An. ste- phensi in a 50-m radius of surveyed households. The level of household-exposure was estimated as the fraction of the surveyed households per number of households detected with An. stephensi. Mixed blood meal sources were included in the nominator to determine the blood meal indices. Detection of blood meal sourcesh The blood meal source of wild-caught, freshly blood- fed female Anopheles mosquitoes was analyzed by PCR (AHRI laboratory) or ELISA (Jimma University TIDRC laboratory). For the PCR, the extracted DNA from the abdominal region of female Anopheles was analyzed by a multiplex PCR assay as previously described [56]. The PCR assay was performed in a reaction volume of 25 µl containing universal vertebrate-specific and species- specific primers for pigs, humans, goats, dogs and cows [0.2 μM of each primer], 1× GoTAQ (Promega, Madison, WI, USA), 1.5 mM MgCl2, 0.2 mM dNTPs and 13.85 µl of molecular-grade water. The PCR amplification condi- tions were: 95 °C for 5 min; 40 cycles of 95 °C for 60 s, 57 °C for 60 s and 72 °C for 60 s; with a final extension of 72 °C for 7 min. The PCR products were confirmed by gel electrophoresis. For the ELISA, the abdomens of freshly fed female Anopheles mosquitoes were homogenized in phosphate- buffered saline (PBS) in a 1.5-ml grinding tube follow- ing a standard protocol [55]. More specifically, 100 μl of homogenized sample or control was loaded added into a well of an ELISA plate and the plate incubated for 2 h at room temperature, following which the wells were washed 3 times with 200 µl of PBS-Tween 20 solution. Then, 50 µl of host-specific peroxidase-labeled mAb of human, bovine and goat (Sigma-Aldrich) was added and incubated for 1 h in the dark, followed by 3 washes with 200 µl of PBS–Tween 20, with the plate shaken 5 times with each wash. Finally, 100 µl of ABTS was added to each well as the substrate for the peroxidase enzyme, and the mixture was incubated for 30 min. The plates were observed visually, and their optical density was read at 405–414 nm using an ELISA reader (model ELX800; BioTek). Samples with a value higher than twofold the mean absorbance value of the negative controls were considered to be positive for Plasmodium parasites. Molecular proceduresh Sporozoite rate was determined as the frac- tion of Anopheles species tested per that species detected with Plasmodium parasite by PCR and ELISA. Molecular proceduresh The head and thorax of mosquitoes were separated from the abdomen for molecular analyses carried out at the Armauer Hansen Research Institute (AHRI) and Jimma University (JU) Tropical Infectious Diseases Research Center (TIDRC) laboratories. The bisected parts or pooled immature-stage materials were homogenized using a BioSpec BeadBeater apparatus (Bead Homoge- nizer 96 Microplate; Biospec Products, Bartlesville, USA) in 150 µl of molecular-grade water with 0.2 mg of beads (diameter: 1.0 mm; composition: zirconia/silica; BioSpec Products) at 3800 rpm for 20 s. DNA was extracted from 50- and 100-µl samples of homogenate according to the manufacturer’s instructions (Qiagen GmbH, Hilden, Germany) and used for the detection of Plasmodium infection (head-thorax), blood meal analysis (abdomen), molecular species identification (immature stages) and confirmation of morphological identification (adult stage). p p g g To detect Plasmodium via ELISA, the head-thorax of each mosquito was separated from the abdomen and ground in blocking buffer containing IGEPAL CA-630 (Sigma-Aldrich, St. Louis, MO, USA) in a 1.5-ml grinding tube. Antibodies against the circumsporozoite protein (CSP) of P. falciparum, P. vivax-210 (Pv-210) and P. vivax- 247 (Pv-247) were detected using a sandwich CSP-ELISA [54, 55]. More specifically, 50 μl of species-specific cap- ture monoclonal antibody (mAb) was added to each well of a micro-ELISA plate. The binding sites were blocked by adding 200 µl of blocking buffer and incubating the plate for 1 h at room temperature. Then, 50 μl of mosquito homogenate or control sample was added to the respec- tive labeled wells. The plates were subsequently covered and incubated for 2 h at room temperature, following which 50 μl of peroxidase-linked mAb were then added to the wells and the plates incubated for 1 h at room tem- perature. Finally, 100 µl of peroxidase substrate solution (ABTS [2,2’-azino-bis 3-ethylbenzothiazoline-6-sulfonic A PCR endpoint assay targeting the internal tran- scribed spacer 2 region (ITS2) was performed for spe- cies identification of An. stephensi as described elsewhere [50]. Briefly, the PCR was performed using the ST-F (5’CGTATCTTTCCTCGCATCCA3’), U5.8S-F (5’ATC ACTCGGCTCATGGATCG3’) and UD2-R (5’GCA Ashine et al. Parasites & Vectors (2024) 17:166 Ashine et al. Parasites & Vectors (2024) 17:166 Page 7 of 18 acid]; Kirkegaard and Perry Laboratories [KPL], Gaith- ersburg, MD, USA) was added and the plate incubated for 30 min for P. falciparum and 60 min for P. vivax. Between the addition of each reagent or sample and the incubation, the wells were shaken and washed. Species identification by molecular methods l f fi ld h d l either not found (Kebri Beyah) or not identified morpho- logically (Yabelo) due to damage during collections. A total of 80 field-caught adult mosquitoes identified as An. stephensi based on morphological characteristics and 28 pooled immature-stage mosquito samples were screened using molecular methods to confirm species identity. Of the 80 adult samples identified morphologi- cally as An. stephensi, the molecular assays confirmed the identity as An. stephensi in 78 samples; in the remaining two samples, the products were either not amplified or were found to be non-An. stephensi. Among the pooled immature-stage mosquito samples, 82.1% (23/28 pools) were confirmed to contain An. stephensi. Detection of blood meal sources Blood meals were analyzed in 784 Anopheles mosqui- toes, of which 386 samples were assayed by multiplex PCR (Additional file 1: Fig. S1) and 398 specimens were analyzed using an ELISA. Among Anopheles mosquitoes identified with a blood meal source, 76.9% (50/65) of An. stephensi and 56.2% (181/322) of An. arabiensis were found with single host blood. The blood meal indices for An. stephensi were 69.2% for bovines (BBI), 32.3% for ovines (OBI) and 24.6% for humans (HBI), including the mixed blood meal sources (Table 4); for An. arabiensis, the blood meal indices were 65.4% for bovines, 46.7% for ovines and 35.8% for humans. Spatiotemporal distribution of An. stephensi In four rounds of entomological surveys, 30.8% (8/26) of the sites were positive for An. stephensi, immature and/or adult stages. Both adult and immature stages of An. ste- phensi were detected at six of these positive sites (Babile, Danan, Dubti, Jiga, Kebri Dehar, and Modjo), while at the remaining two sites either adults (Assosa) or immature stages (Ataye) were detected (Table 2). In four of the An. stephensi-positive sites, adult or immature stages were recorded every round. The mean number of wild-caught adult An. stephensi per trap was 0.15 (CDC LT: 0.04 per trap night; Prokopack aspirator: 0.38 per collection) (Additional file 1: Table S2). Anopheles stephensi lar- vae were collected from a range of artificial and natural aquatic habitats. Results Anopheles mosquito fauna and abundance Among the study sites, 96.2% (25/26) were positive for Anopheles mosquitoes, including An. stephensi, and 99.2% (5353/5398) of the total catches were identified to species level. Of the total adult Anopheles catches, 86.6% (1612/1862) were collected by CDC LTs, and 13.4% (250/1862) were collected by Prokopack aspira- tors (Fig. 4), with 79.5% of adult catches identified as An. arabeinsis, 7.7% as An. stephensi and 7.4% as An. phar- oensis. Anopheles coustani, An. tenebrosus, An. fuenstus and An. rufipes accounted for only 2.5%, 1.8%, 1.1% and 0.1% of adult catches, respectively. Anopheles stephensi was predominant in four sites of the 26 surveyed (Babile, Kebri Dehar, Danan and Modjo), with 77.3–100% rela- tive abundance. Among the immature-stage collections, An. stephensi was predominant in five sites of the 26 sur- veyed (Babile, Kebri Dehar, Danan, Dubti, and Modjo), with 75–100% relative abundance. Anopheles arabiensis was the predominant species across most sites (20/26) (Table 1). In two study sites, Anopheles mosquitoes were Ashine et al. Parasites & Vectors (2024) 17:166 Page 8 of 18 Fig. 4 Anopheles mosquito catches. A total number of Anopheles mosquitoes collected and morphologically identified to species level. B proportion of adult Anopheles catches according to method of collection. CDC LT, U.S. Centers for Disease Control and Prevention light traps i ll d d h l i ll id ifi d i l l B Fig. 4 Anopheles mosquito catches. A total number of Anopheles mosquitoes collected and morphologically identified to species level. B proportion of adult Anopheles catches according to method of collection. CDC LT, U.S. Centers for Disease Control and Prevention light traps Fig. 4 Anopheles mosquito catches. A total number of Anopheles mosquitoes collected and morphologically identified to species level. B proportion of adult Anopheles catches according to method of collection. CDC LT, U.S. Centers for Disease Control and Prevention light traps Species identification by molecular methodsi Household exposure to An. stephensi During additional extensive entomological sampling in and around households, An. stephensi was detected in three of the four urban centers, namely Danan, Awash Sebat Kilo and Metehara. Among the 50 surveyed house- holds, 30% (15/50) were positive for An. stephensi in Danan. The household positivity rates were 26% (13/50) and 18% (9/50) in Awash Sebat Kilo and Metehara, respectively. Anopheles stephensi was not detected in any of the sampled households in Jiga during the sampling period (Table 3). Detection of Plasmodium infection Among the 1847 Anopheles mosquitoes examined for Plasmodium infection, 69.4% (1282/1847) and 30.6% (565/1847) were assessed using PCR (Additional file 1: Fig. S2) and ELISA, respectively (Table 5). None of the 197 samples identified as An. stephensi were found to Ashine et al. Parasites & Vectors (2024) 17:166 Page 9 of 18 Table 1 Distribution and abundance of Anopheles mosquitoes across study urban centers, Ethiopia, from surveys performed in 2021–2023 Site Anopheles species N Relative abundance of species (%) Mean number of Anopheles mosquitoes per trap Method of collection CDC LT PRO n % Mean number of Anopheles mosquitoes per trap n % Mean number of Anopheles mosquitoes per trap Afambo An. arabiensis 634 81.9 7.93 598 94.3 7.48 36 5.7 0.9 An. pharoensis 106 13.7 1.33 105 99.1 1.31 1 0.9 0.03 An. tenebrosus 34 4.4 0.43 34 100 0.43 Akaki An. arabiensis 22 100 0.28 21 95.5 0.26 1 4.5 0.03 Arba Minch An. arabiensis 62 96.9 0.78 62 100 0.78 An. pharoensis 2 3.1 0.03 2 100 0.03 Asendabo An. arabiensis 22 78.6 0.28 10 45.5 0.13 12 54.5 0.3 An. fuenstus 1 3.6 0.01 1 100 0.01 An. coustani 5 17.9 0.06 3 60 0.04 2 40 0.05 Assosa An. arabiensis 8 50 0.1 8 100 0.1 An. coustani 6 37.5 0.08 6 100 0.08 An. pharoensis 1 6.3 0.01 1 100 0.01 An. stephensi 1 6.3 0.01 1 100 0.01 Ataye An. arabiensis 28 80 0.35 24 85.7 0.3 4 14.3 0.1 An. fuenstus 3 8.6 0.04 3 100 0.04 An. coustani 4 11.4 0.05 4 100 0.05 Babile An. stephensi 3 75 0.04 3 100 0.08 An. rufipes 1 25 0.01 1 100 0.01 Bambasi An. arabiensis 3 50 0.04 3 100 0.04 An. coustani 2 33.3 0.03 2 100 0.03 An. pharoensis 1 16.7 0.01 1 100 0.01 Bonga An. Household exposure to An. stephensi arabiensis 8 100 0.1 7 87.5 0.09 1 12.5 0.03 Danan An. stephensi 15 100 0.19 4 26.7 0.05 11 73.3 0.28 Dembiya An. arabiensis 29 100 0.36 18 62.1 0.23 11 37.9 0.28 Dilla An. arabiensis 16 94.1 0.2 16 100 0.2 An. pharoensis 1 5.9 0.01 1 100 0.01 Dubti An. arabiensis 47 70.1 0.59 38 80.9 0.48 9 19.1 0.23 An. coustani 2 3 0.03 2 100 0.03 An. pharoensis 11 16.4 0.14 11 100 0.14 An. stephensi 7 10.4 0.09 7 100 0.09 Ashine et al. Parasites & Vectors (2024) 17:166 Page 10 of 18 Table 1 (continued) Site Anopheles species N Relative abundance of species (%) Mean number of Anopheles mosquitoes per trap Method of collection CDC LT PRO n % Mean number of Anopheles mosquitoes per trap n % Mean number of Anopheles mosquitoes per trap Gambella An. arabiensis 9 42.9 0.11 9 100 0.11 An. fuenstus 4 19 0.05 4 100 0.05 An. pharoensis 8 38.1 0.1 8 100 0.1 Harbu An. arabiensis 132 97.8 1.65 120 90.9 1.5 12 9.1 0.3 An. fuenstus 1 0.7 0.01 1 100 0.01 An. coustani 1 0.7 0.01 1 100 0.03 An. pharoensis 1 0.7 0.01 1 100 0.01 Jiga An. arabiensis 87 76.3 1.09 84 96.6 1.05 3 3.4 0.08 An. coustani 17 14.9 0.21 17 100 0.21 An. pharoensis 3 2.6 0.04 1 33.3 0.01 2 66.7 0.05 An. stephensi 7 6.1 0.09 7 100 0.09 Kebri Dehar An. stephensi 20 95.2 0.25 5 25 0.06 15 75 0.38 An. pharoensis 1 4.8 0.01 1 100 0.01 Kombolcha An. arabiensis 30 78.9 0.38 26 86.7 0.33 4 13.3 0.1 An. fuenstus 7 18.4 0.09 6 85.7 0.08 1 14.3 0.03 An. coustani 1 2.6 0.01 1 100 0.01 Metamma An. arabiensis 17 100 0.21 12 70.6 0.15 5 29.4 0.13 Modjo An. stephensi 91 97.8 1.14 91 100 2.28 An. arabiensis 2 2.2 0.03 2 100 0.03 Omorate An. arabiensis 38 97.4 0.48 34 89.5 0.43 4 10.5 0.1 An. pharoensis 1 2.6 0.01 1 100 0.01 Salamago An. arabiensis 230 100 2.88 226 98.3 2.83 4 1.7 0.1 Shewa Robit An. arabiensis 22 81.5 0.28 16 72.7 0.2 6 27.3 0.15 An. fuenstus 4 14.8 0.05 4 100 0.05 An. coustani 1 3.7 0.01 1 100 0.01 Woreta An. arabiensis 34 81 0.43 29 85.3 0.36 5 14.7 0.13 An. Household exposure to An. stephensi 1: January-May; 2: June-November; 3: October-November; 4: December-January Anopheles stephensi- positive sites Collection roundsa and stages caught Round 1 Round 2 Round 3 Round 4 Adult Immature Adult Immature Adult Immature Adult Immature Assosa − − − − + − − − Ataye − + − − − − − − Babile − +A + − − − − − Danan + +A + +A + +A + + Dubti − +A + +AN + +A + +A Jiga + − + +N + − + +N Kebri Dehar + +A + +A + +A + +A Modjo − +A + +A − +A − − Table 2 Spatiotemporal distribution of Anopheles stephensi across study urban centers, Ethiopia, 2021–2023 Key: −, Negative for both stages; +, positive for wild-caught adults; +N, positive for immature stages in natural habitat; +A, positive for immature stages in artificial habitat; +AN: positive for immature stages in artificial and natural habitat a Round number and associated months of mosquito collections. 1: January-May; 2: June-November; 3: October-November; 4: December-January Key: −, Negative for both stages; +, positive for wild-caught adults; +N, positive for immature stages in natural habitat; +A, positive for immature stages in artificial habitat; +AN: positive for immature stages in artificial and natural habitat a Round number and associated months of mosquito collections. 1: January-May; 2: June-November; 3: October-November; 4: December-January the site positivity of An. stephensi was relatively low (8/26 sampled sites) compared to that reported in pre- vious studies. A study in 2020 that covered 10 sites in eastern Ethiopia reported the presence of An. stephensi at all sites [36]. Similarly, sampling at 21 sites between 2018 and 2020 revealed 61.9% positivity for An. stephensi [37]. Another study conducted by the PMI Vector Link Ethiopia project showed the presence of An. stephensi in 16 urban settings, of which nine (56.3%) sites were newly positive for An. stephensi [60]. One explanation for the differences in results might be the selection of study sites; most of the collection points in these previous studies were purposefully chosen to detect An. stephensi, while the current study followed substantial random steps in the selection of study sites and collection points within sites. Our approach has the advantage of providing unbi- ased distribution estimates, but it does reduce the prob- ability of detection. Household exposure to An. stephensi In addition, some of our study sites were located far from major transportation corridors [61], which are considered as the main invasion routes.i be infected with Plasmodium parasites. Overall, the Plasmodium sporozoite rate of An. arabiensis was 4.3% (62/1434), of which 16.1% (10/62) were P. falciparum and 83.9% (52/62) were P. vivax. In An. pharoensis, the Plas- modium sporozoite rate was 6.7% (8/119), of which 12.5% (1/8) were P. falciparum and 87.5% (7/8) were P. vivax. Two other Anopheles mosquito species were detected with sporozoites of P. vivax: An. coustani (12.8%, 5/39) and An. funestus (11.5%, 3/26). be infected with Plasmodium parasites. Overall, the Plasmodium sporozoite rate of An. arabiensis was 4.3% (62/1434), of which 16.1% (10/62) were P. falciparum and 83.9% (52/62) were P. vivax. In An. pharoensis, the Plas- modium sporozoite rate was 6.7% (8/119), of which 12.5% (1/8) were P. falciparum and 87.5% (7/8) were P. vivax. Two other Anopheles mosquito species were detected with sporozoites of P. vivax: An. coustani (12.8%, 5/39) and An. funestus (11.5%, 3/26). Household exposure to An. stephensi coustani 7 16.7 0.09 7 100 0.09 An. pharoensis 1 2.4 0.01 1 100 0.01 9 4 8 120 1 1 84 17 1 7 5 1 26 6 1 12 2 34 1 226 16 4 1 29 7 1 Page 11 of 18 Ashine et al. Parasites & Vectors (2024) 17:166 Table 1 (continued) Site Anopheles species N Relative abundance of species (%) Mean number of Anopheles mosquitoes per trap Method of collection CDC LT PRO n % Mean number of Anopheles mosquitoes per trap n % Mean number of Anopheles mosquitoes per trap Total An. arabiensis 1480 79.5 0.47 1363 92.1 0.66 117 7.9 0.11 An. coustani 46 2.5 0.01 43 93.5 0.02 3 6.5 An. fuenstus 20 1.1 0.01 19 95 0.01 1 5 An. pharoensis 137 7.4 0.04 134 97.8 0.06 3 2.2 An. rufipes 1 0.1 1 100 An. stephensi 144 7.7 0.05 24 16.7 0.01 120 83.3 0.12 An. tenebrosus 34 1.8 0.01 34 100 0.02 CDC LT U.S. Centers for disease control and prevention light traps, N, total number of catches, n number of catches per method, % proportion of catches per method, PRO Prokopack aspirator Table 1 (continued) Ashine et al. Parasites & Vectors (2024) 17:166 Page 12 of 18 Table 2 Spatiotemporal distribution of Anopheles stephensi across study urban centers, Ethiopia, 2021–2023 Key: −, Negative for both stages; +, positive for wild-caught adults; +N, positive for immature stages in natural habitat; +A, positive for immature stages in artificial habitat; +AN: positive for immature stages in artificial and natural habitat a Round number and associated months of mosquito collections. Discussionh The results of the present study add to the body of avail- able evidence on the distribution and abundance of inva- sive An. stephensi in Ethiopia. Entomological surveillance in 26 urban centers between 2021 and 2023 revealed that An. arabiensis was the predominant Anopheles species in the catches, accounting for 79.5% of all collections, followed by An. stephensi, accounting for 7.7% of the total Anopheles catches. The relative abundance of adult An. stephensi was greater than that of An. arabiensis in Babile, Kebri Dehar, Danan and Modjo. Modjo is located along the main ground transportation route or corridor that connects Ethiopia to Djibouti [36]. Generally, adult An. stephensi collections were low (mean: 0.15 catches/ trap), and most of the immature-stage collections were from artificial aquatic habitats.i In line with the findings of other studies [16, 36], we also noted that An. stephensi was more readily detectable as immature stages than as adults in most of the positive sites. The highest proportion of An. stephensi collections (85.7%) was obtained as immature stages (larvae and pupae) in aquatic habitats. A range of aquatic habitats were positive; for example, in Dubti, immature stages of An. stephensi were detected in both artificial habitats (water tanks, barrels, buckets, tires) and natural habi- tats (ponds, streams, swamps and marshes). It has been reported that An. stephensi can breed in various aquatic habitats with differing physicochemical characteristics, such as salinity and turbidity [62]. In Modjo, Danan, Kebri Dehar and Babile, immature stages of An. stephensi i Since the first detection of An. stephensi in eastern Ethiopia in 2016, new positive sites have been identi- fied in subsequent surveys [36, 37, 59]. In line with these findings, we detected An. stephensi in western Ethiopia (Assosa), in an area bordering Sudan, which might indi- cate the continued spread of this species. We also found An. stephensi at all previously reported sites, as well as at new sites along its purported invasion route. However, Ashine et al. Parasites & Vectors (2024) 17:166 Page 13 of 18 Table 3 Household exposure rates for immature, adult and both stages of Anopheles stephensi across four urban centers, Ethiopia, 2023 AHP Aquatic habitat positivity rate for An. stephensi, CDC LT U.S. Centers for disease control and prevention light traps, HHP household positivity rate for An. stephensi, N, total number of Anopheles caught, n number of habitats or houses positive for An. Discussionh stephensi, PRO Prokopack aspirator Study sites Houses surveyed (n) Wild-caught adult-stage mosquitoes Immature-stage collection Both mosquito stages Total HHP, n (%) N CDC LT PRO HHP, n (%) Aquatic habitats AHP, n (%) N HHP, n (%) HHP, n (%) Awash S.K. 50 9 9 3 (6) Birka 2 (67) 94 10 (20) 13(26) Plastic drum 8 (80) 158 Danan 50 21 3 18 9 (18) Birka 5 (83) 378 5 (10) 1 (2) 15(30) Plastic tank 1 (100) 20 Jiga 50 29 29 Ground water 3 Metehara 50 26 10 16 2 (4) Birka 5 (83) 564 6 (12) 1 (2) 9(18) Plastic drum 2 (100) 46 Table 3 Household exposure rates for immature, adult and both stages of Anopheles stephensi across four urban centers, Ethiopia, 2023 AHP Aquatic habitat positivity rate for An. stephensi, CDC LT U.S. Centers for disease control and prevention light traps, HHP household positivity rate for An. stephensi, N, total number of Anopheles caught, n number of habitats or houses positive for An. stephensi, PRO Prokopack aspirator be due to well-established populations of An. stephensi in areas where it was first reported since its invasion.i were detected only in artificial habitats. This variation highlights how larval source management of An. ste- phensi, which has been recommended by the WHO [63] and is being implemented by PMI VectorLink and oth- ers, will be more complex than simply targeting container habitats. i p Our findings reveal that An. stephensi prefers non- human vertebrate hosts for their blood meal. The most prevalent blood meal among An. stephensi detected with sources of blood was cattle (69.2%), followed by goats (32.3%). These findings are consistent with results previ- ously reported in Ethiopia [16, 37] and India [67], which showed that most An. stephensi fed on livestock. In the present study, one-third of An. stephensi fed on unidenti- fied blood meal sources, which might be due to a lack of host antibodies or primers for blood meal analysis. It is noteworthy that at some of the study sites, especially in eastern Ethiopia, the most readily available animals were camels and that these sites were where most of the tested An. stephensi were collected. Despite the relatively high non-human vertebrate host blood meal indices, 24.6% of An. stephensi were found with human blood, including mixed blood meal sources. Discussionh The blood meal source of vec- tors might be affected by multiple factors, including host availability and proximity, possibly explaining why 76.9% of the An. stephensi and 56.2% of An. arabiensis fed on a single blood meal source of either an animal or a human host. In the current study, An. arabiensis was the most abun- dant species at 20/26 sites, which is in line with the find- ings of other studies showing that this species is still the predominant malaria vector in different eco-epidemio- logical settings of Ethiopia [20, 25, 64]. Even though An. arabiensis is considered less adapted to urban ecology [65], our findings suggest that it is likely to be the pri- mary malaria vector in urban centers in Ethiopia. The other Anopheles species collected in this study were An. pharoensis, An. coustani, An. funestus, An. tenebrosus and An. rufipes, which together accounted for 12.8% of the total adult Anopheles catches. Of these five species, An. pharoensis and An. funestus have been reported to be secondary or suspected malaria vectors in Ethiopia [66]. We detected An. pharoensis infected with P. falciparum or P. vivax at one and five of our study sites, respectively, while An. coustani and An. funestus were detected with P. vivax sporozoites across four and three of the study sites, respectively. Of the 197 screened An. stephensi, none were detected with Plasmodium parasites. These findings are similar to those of another study in which none of the tested An. stephensi was positive for Plasmodium [36]. However, a study conducted in 2019 in Awash Sebat Kilo reported an infection rate of 2.8% for P. vivax and 1.4% for P. falci- parum, based on analysis of homogenates of whole mos- quitoes [16]. The authors of another study in which the heads and thoraxes was used to detect Plasmodium by ELISA reported that the sporozoite rate was 0.5% in Dire Dawa and 0.3% in Kebri Dehar for P. vivax [37]. The most recent study, from Dire Dawa, implicated An. stephensi The level of household exposure to An. stephensi was heterogeneous across the study sites, with household positivity for both (adults and immature) stages rang- ing from 18% in Metehara to 30% in Danan. The level of household exposure to adult An. Discussionh stephensi was high- est in the region where this species was first reported as an invasive species (Danan) and lower in more central parts of the country (Awash Sebat Kilo and Metehara). A similar trend was observed for household exposure to the immature stages of An. stephensi. These results could Ashine et al. Parasites & Vectors (2024) 17:166 Page 14 of 18 Table 4 Determination of blood meal sources of wild-caught adult Anopheles mosquitoes collected across urban centers in Ethiopia using multiplex PCR and enzyme-linked immunosorbent assay, 2021–2023 BBI Bovine blood meal index, CBI canine blood meal index, ELISA enzyme-linked immunosorbent assay, HBI human blood meal index, OBI ovine blood meal index, N number of Anopheles tested, n number of samples with S, T and UN, respectively, S single blood meal source, T single + mixed blood meal source, UN unidentified blood meal source Site Anopheles species N HBI BBI OBI CBI UN, n (%) S, n (%) T, n (%) S, n (%) T, n (%) S, n (%) T, n (%) S, n (%) T, n (%) Assayed using multiplex PCR Afambo An. arabiensis 211 15 (7.1) 28 (26.2) 56 (26.5) 72 (34.1) 10 (4.7) 20 (9.5) 5 (2.4) 8 (3.8) 104 (49.3) An. pharoensis 22 2 (9.1) 4 (40) 4 (18.2) 6 (27.3) 1 (4.5) 2 (9.1) 1 (4.5) 12 (54.5) An. tenebrosus 6 1 (16.7) 1 (20) 3 (50) 3 (50) 1 (16.7) 1 (16.7) 1 (16.7) Akaki An. arabiensis 13 2 (15.4) 3 (23.1) 1 (7.7) 10 (76.9) Arba Minch An. arabiensis 1 1 (100) 1 (100) Awash S. K An. stephensi 7 1 (14.3) 3 (42.9) 4 (57.1) 3 (42.9) Danan An. stephensi 52 1 (1.9) 9 (23.7) 25 (48.1) 33 (63.5) 4 (7.7) 4 (7.7) 14 (26.9) Kebri Dehar An. stephensi 22 2 (9.1) 2 (14.3) 6 (27.3) 6 (27.3) 6 (27.3) 6 (27.3) 8 (36.4) Metehara An. stephensi 3 1 (33.3) 2 (66.7) 1 (33.3) 2 (66.7) Omorate An. arabiensis 18 3 (16.7) 4 (66.7) 2 (11.1) 2 (11.1) 1 (5.6) 12 (66.7) Salamago An. arabiensis 29 9 (31) 15 (78.9) 1 (3.4) 3 (10.3) 3 (10.3) 7 (24.1) 10 (34.5) Assayed by ELISA Asendabo An. arabiensis 14 7 (50) 2 (14.3) 9 (64.3) 5 (35.7) An. funestus 1 1 (100) 1 (100) An. coustani 2 1 (50) 1 (50) 2 (100) Assosa An. BBI Bovine blood meal index, CBI canine blood meal index, ELISA enzyme-linked immunosorbent assay, HBI human blood meal index, OBI ovine blood meal index, N number of Anopheles tested, n number of samples with S, T and UN, respectively, S single blood meal source, T single + mixed blood meal source, UN unidentified blood meal source BBI Bovine blood meal index, CBI canine blood meal index, ELISA enzyme-linked immunosorbent assay, HBI human blood meal index, OBI ovine blood meal index, N number of Anopheles tested, n number of samples with S, T and UN, respectively, S single blood meal source, T single + mixed blood meal source, UN unidentified blood meal source y y N number of Anopheles tested, n number of samples with S, T and UN, respectively, S single blood meal source, T single + mixed blood m blood meal source Discussionh arabiensis 5 1 (100) 1 (20) 1 (20) 4 (80) An. coustani 6 2 (33.3) 2 (33.3) 4 (66.7) Ataye An. arabiensis 9 2 (22.2) 5 (55.6) 3 (33.3) 6 (66.7) 1 (11.1) An. coustani 2 2 (100) 2 (100) Bambasi An. coustani 1 1 (100) 1 (100) Bonga An. arabiensis 3 1 (33.3) 1 (33.3) 2 (66.7) 1 (33.3) Dembiya An. arabiensis 14 2 (20) 2 (14.3) 7 (50) 3 (21.4) 8 (57.1) 4 (28.6) Dubti An. arabiensis 42 6 (35.3) 2 (4.8) 7 (16.7) 8 (19) 13 (31) 25 (59.5) An. pharoensis 10 1 (10) 1 (10) 2 (20) 8 (80) Gambella An. arabiensis 4 1 (25) 1 (25) 3 (75) An. funestus 4 1 (25) 1 (100) 3 (75) Harbu An. arabiensis 112 10 (8.9) 16 (30.8) 10 (8.9) 34 (30.4) 6 (5.4) 32 (28.6) 60 (53.6) An. pharoensis 1 1 (100) 1 (100) An. coustani 1 1 (100) 1 (100) Jiga An. arabiensis 77 7 (9.1) 29 (50.9) 7 (9.1) 43 (55.8) 5 (6.5) 38 (49.4) 20 (26) An. pharoensis 2 1 (50) 1 (50) 1 (50) 1 (50) An. coustani 13 1 (7.7) 6 (66.7) 7 (53.8) 1 (7.7) 7 (53.8) 4 (30.8) An. stephensi 7 1 (14.3) 3 (50) 5 (71.4) 5 (71.4) 1 (14.3) Kombolcha An. arabiensis 15 1 (6.7) 4 (44.4) 7 (46.7) 1 (6.7) 8 (53.3) 6 (40) An. funestus 5 2 (40) 2 (40) 3 (60) Metemma An. arabiensis 9 1 (11.1) 4 (100) 2 (22.2) 1 (11.1) 5 (55.6) Shewa Robit An. arabiensis 8 1 (12.5) 8 (100) 7 (87.5) An. funestus 4 1 (25) 4 (100) 3 (75) Woreta An. arabiensis 9 1 (11.1) 5 (71.4) 1 (11.1) 6 (66.7) 3 (33.3) 2 (22.2) An. coustani 6 2 (33.3) 2 (100) 4 (66.7) Total An. arabiensis 595 47(7.9) 115 (19.3) 87 (14.6) 209 (35.1) 39 (6.6) 150 (25.2) 8 (1.3) 16 (2.7) 273 (45.9) An. coustani 31 4 (12.9) 9 (29) 13 (41.9) 2 (6.5) 14 (45.2) 12 (38.7) An. funestus 14 1 (7.1) 1 (7.1) 1 (7.1) 7 (50) 6 (42.9) 6 (42.9) An. pharoensis 39 3 (7.7) 5 (12.8) 5 (12.8) 9 (23.1) 2 (5.1) 5 (12.8) 1 (2.6) 23 (59) An. stephensi 99 5 (5.1) 16 (16.2) 31 (31.3) 45 (45.5) 14 (14.1) 21 (21.2) 34 (34.3) An. BBI Bovine blood meal index, CBI canine blood meal index, ELISA enzyme-linked immunosorbent assay, HBI human blood m b f h l d b f l h d l l bl d l l Discussionh tenebrosus 6 1 (16.7) 1 (16.7) 3 (50) 3 (50) 1 (16.7) 1 (16.7) 1 (16.7) Table 4 Determination of blood meal sources of wild-caught adult Anopheles mosquitoes collected across urban centers in Ethiopia using multiplex PCR and enzyme-linked immunosorbent assay, 2021–2023 Ashine et al. Parasites & Vectors (2024) 17:166 Page 15 of 18 Table 5 Sporozoite rate in wild-caught adult Anopheles mosquitoes collected across study urban centers, Ethiopia, 2021–2023 CSP-ELISA Circumsporozoite protein-enzyme-linked immunosorbent assay a Plasmodium species: PF P. falciparum, PV P. vivax. n, Number of infected mosquitoes; T, number tested b Afa, Afambo; Aka, Akaki; Arb, Arba Minch, Awa, Awash Sebat Kilo; Bab, Babile; Dan, Danan; Dil, Dilla; Dah, Kebri Dehar; Met, Metehara; Mod, Modjo; Omo, Omorate; Sal, Salamago; Ase, Asendabo; Ass, Assosa; Ata, Ataye; Bam, Bambasi; Bon, Bonga; Dem, Dembiya; Dub, Dubti; Gam, Gambella; Har, Harbu; Jig, Jiga; Kom, Kombolcha; Sho, Shewa Robit Anopheles species Plasmodium speciesa Mosquito collection sitesb Afa Aka Arb Awa Bab Dan Dil Dah Met Mod Omo Sal Total Assayed using multiplex PCR An. stephensi T 9 3 56 20 3 91 179 PF, n (%) PV, n (%) An. arabiensis T 614 21 61 16 2 34 219 967 PF, n (%) 3 (0.5) 1 (2.9) 2 (0.9) 6 (0.6) PV, n (%) 2 (0.3) 1 (1.6) 2 (0.9) 5 (0.5) An. pharoensis T 98 2 1 101 PF, n (%) 1 (1) 1 (1) PV, n (%) 3 (3.1) 3 (3) An. tenebrosus T 34 34 PF, n (%) PV, n (%) Assayed by CSP-ELISA Ase Ass Ata Bam Bon Dem Dub Gam Har Jig Kom Sho Total An. stephensi T 1 7 7 15 PF, n (%) PV, n (%) An. arabiensis T 24 7 31 4 9 30 55 9 183 89 18 8 467 PF, n (%) 2 (8.3) 2(6.7) 4 (0.9) PV, n (%) 3 (12.5) 1 (14.3) 6 (19.4) 1 (25) 2 (22.2) 4 (13.3) 1 (1.8) 1 (11.1) 18 (9.8) 6 (6.7) 3(16.7) 1 (12.5) 47 (10.1) An. pharoensis T 1 1 11 1 1 3 18 PF, n (%) PV, n (%) 1 (100) 1 (9.1) 1 (100) 1 (33.3) 4 (22.2) An. funestus T 1 3 11 7 4 26 PF, n (%) PV, n (%) 1 (100) 1 (33.3) 1 (14.3) 3 (11.5) An. Discussionh coustani T 5 6 4 3 1 2 1 16 1 39 PF, n (%) PV, n (%) 1 (25) 1 (100) 1 (50) 2 (12.5) 5 (12.8) Ashine et al. Parasites & Vectors (2024) 17:166 Page 16 of 18 Page 16 of 18 collection and transportation. We are also grateful to the federal, regional and district health officers for their collaboration during the study period. collection and transportation. We are also grateful to the federal, regional and district health officers for their collaboration during the study period. in an outbreak and detected a P. falciparum sporozo- ite rate of 1.2% [38]. It should be noted that our study might be limited in its ability to elucidate An. stephensi sporozoite infection, as a large proportion of the adult catches (91/146) were from a single aquatic site using the Prokopack aspirator. Funding This work was supported by the National Institute for Health Research (NIHR) (using the UK’s Official Development Assistance [ODA] Funding) and Wellcome (220870_Z_20_Z) under the National Institutes of Health-Wellcome Partnership for Global Health Research. The views expressed are those of the authors and not necessarily those of Wellcome, the NIHR or the Department of Health and Social Care. Consent for publication Not applicable. Consent for publication Not applicable. Abbreviations AHRI Armauer Hansen Research Institute BBI Bovine blood index CDC LTs U.S. Centers for Disease Control and Prevention light traps COXI Cytochrome c oxidase subunit 1 CSP Circumsporozoite protein ELISA Enzyme-linked immunosorbent assay HBI Human blood index ITS2 Internal transcribed spacer 2 region mAb Monoclonal antibody OBI Ovine blood index PBS Phosphate-buffered saline REDCap Research Electronic Data Capture Consent for publication Not applicable. Abbreviations AHRI Armauer Hansen Research Institute BBI Bovine blood index CDC LTs U.S. Centers for Disease Control and Prevention light traps COXI Cytochrome c oxidase subunit 1 CSP Circumsporozoite protein ELISA Enzyme-linked immunosorbent assay HBI Human blood index ITS2 Internal transcribed spacer 2 region mAb Monoclonal antibody OBI Ovine blood index PBS Phosphate-buffered saline REDCap Research Electronic Data Capture Conclusionsi The datasets reported herein were shared with stakeholders, including the Ministry of Health and the WHO. All the data from the CEASE project will be made publicly available upon completion of the study. Our findings reveal an expansion of An. stephensi into a new western geographical range and its transition to predominant species status in some areas where it was first detected. These results highlight the need for enhanced entomological surveillance with efficient traps to determine the bionomics and relative contribution of An. stephensi for malaria transmission in the region. In the meantime, the plan set forth to limit the spread and contain An. stephensi establishment should be put into action. Author details 1 1 Department of Biology, College of Natural and Computational Sciences, Arba Minch University, Arba Minch, Ethiopia. 2 Malaria and NTD Research Division, Armauer Hansen Research Institute, Addis Ababa, Ethiopia. 3 Tropical and Infectious Diseases Research Center, Jimma University, Jimma, Ethio- pia. 4 Department of Biology, College of Natural Sciences, Jimma University, Jimma, Ethiopia. 5 School of Medical Laboratory Sciences, Institute of Health, Jimma University, Jimma, Ethiopia. 6 Department of Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. 7 Disease Prevention and Control Directorate, Ethiopian Federal Ministry of Health, Addis Ababa, Ethiopia. 8 Lancaster Ecology and Epidemiology Group, Lancaster Medi- cal School, Lancaster University, Lancaster, UK. Supplementary Information Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13071-024-06243-3. Additional file 1: Table S1. Study sites in Ethiopia, 2021–2023. Table S2: Occurrence and abundance of An. stephensi across eight positive urban centers by round and stage of collection, Ethiopia, 2021–2023. Figure S1. Multiplex PCR for blood meal source detection in freshly fed wild-caught female Anopheles mosquitoes. Figure S2. Gel images of COXI and nested PCR for detecting Plasmodium infection. The online version contains supplementary material available at https://doi. org/10.1186/s13071-024-06243-3. The online version contains supplementary material available at https://doi. org/10.1186/s13071-024-06243-3. Additional file 1: Table S1. Study sites in Ethiopia, 2021–2023. Table S2: Occurrence and abundance of An. stephensi across eight positive urban centers by round and stage of collection, Ethiopia, 2021–2023. Figure S1. Multiplex PCR for blood meal source detection in freshly fed wild-caught female Anopheles mosquitoes. Figure S2. Gel images of COXI and nested PCR for detecting Plasmodium infection. Received: 22 December 2023 Accepted: 11 March 2024 Received: 22 December 2023 Accepted: 11 March 2024 Received: 22 December 2023 Accepted: 11 March 2024 Competing interests The authors declare that they have no competing interests. References 1. WHO. World malaria report 2023. 2023. https://www.who.int/teams/ global-malaria-programme/reports/world-malaria-report-2023. Accessed 10 Dec 2023. Author contributions MJD, DW, EG, DY, ALW, TA and AE conceived the study. MJD, DW, EG, DY, ALW, AR, YA, TA, AE, ES, EZ, FM, LS, AEp and AZ designed the study. TA and AE drafted the manuscript. MJD, DW, EG, DY, ALW, AR, AEp, YA, ES, FM, EE, NN, AZ, GA, DD and HS contributed to the finalization of the manuscript. TA, AE, YA, AK, NN, EE, AD, EA, KW, EZ and MA conducted the field data collection and morphological identification of the mosquitoes. TA, AE, YA, NN, TT, SWB, AD and JDD conducted the molecular laboratory work. TA, AE, MGB, BL and FAK performed the data management and analysis. AR, YA, KW and ES managed the project. All authors read and approved the final version of the manuscript. There are a number of limitations to our study. The lim- ited number of An. stephensi adults caught indicates the need for further studies to investigate more efficient trap- ping methods. We employed both PCR and ELISA for detecting blood meal sources and Plasmodium infection in Anopheles mosquitoes, which might limit the direct comparability of our findings across study sites. The col- lection rounds did not directly coincide with the malaria transmission seasons in Ethiopia, and some of the sites were excluded due to civil unrest. Ethics approval and consent to participate Ethical approval was received from the Institutional Review Board (IRB) of the Institute of Health of Jimma University (JUIH/IRB/575/23), AHRI/ALERT ethics committee (PO/16/21), the National Research Ethics Review Committee (NRERC) of the Federal Ministry of Education of Ethiopia (Reference: 7/1-229/ m259/35) and the Liverpool School of Tropical Medicine (Reference: 21-013). Written informed consent to participate in the study was obtained from the heads of household (or their designates) for all participating households. 2. WHO. World malaria report 2020: 20 years of global progress and chal- lenges. 2020. https://www.who.int/publications/i/item/9789240015791. Accessed 22 Dec 2021. Acknowledgements The authors would like to thank the study households and the field research team, including the entomology technicians, the community facilitators and the drivers at AHRI and Jimma University, for their support during sample Ashine et al. 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https://openalex.org/W2601424403	https://europepmc.org/articles/pmc5393286?pdf=render	English	null	Environmental exposure to arsenic and chromium in an industrial area	Environmental science and pollution research international	2,017	cc-by	7,157	Environmental exposure to arsenic and chromium in an industrial area Luigi Vimercati1 & Maria F Gatti1 & Tommaso Gagliardi1 & Francesco Cuccaro2 & Luigi De Maria1 & Antonio Caputi1 & Marco Quarato1 & Antonio Baldassarre1 Luigi Vimercati1 & Maria F Gatti1 & Tommaso Gagliardi1 & Francesco Cuccaro2 & Luigi De Maria1 & Antonio Caputi1 & Marco Quarato1 & Antonio Baldassarre1 Received: 8 November 2016 /Accepted: 13 March 2017 /Published online: 20 March 2017 # The Author(s) 2017. This article is published with open access at Springerlink.com Abstract Arsenic and chromium are widespread environ- mental contaminants that affect global health due to their tox- icity and carcinogenicity. To date, few studies have investigat- ed exposure to arsenic and chromium in a population residing in a high-risk environmental area. The aim of this study is to evaluate the exposure to arsenic and chromium in the general population with no occupational exposure to these metals, resident in the industrial area of Taranto, Southern Italy, through biological monitoring techniques. We measured the levels of chromium, inorganic arsenic, and methylated metab- olites, in the urine samples of 279 subjects residing in Taranto and neighboring areas. Qualified health staff administered a standardized structured questionnaire investigating lifestyle habits and controlling for confounding factors. The biological monitoring data showed high urinary concentrations of both the heavy metals investigated, particularly Cr. On this basis, it will be necessary to carry out an organized environmental monitoring program, taking into consideration all exposure routes so as to correlate the environmental concentrations of these metals with the biomonitoring results. Keywords Arsenic . Chromium . Carcinogens . Environmental exposure . Biological monitoring . Public health Keywords Arsenic . Chromium . Carcinogens . Environmental exposure . Biological monitoring . Public health Environ Sci Pollut Res (2017) 24:11528–11535 DOI 10.1007/s11356-017-8827-6 Environ Sci Pollut Res (2017) 24:11528–11535 DOI 10.1007/s11356-017-8827-6 RESEARCH ARTICLE 2 Health Local Unit of Barletta-Andria-Trani, 76121 Barletta, Italy 1 Interdisciplinary Department of Medicine, Occupational Medicine BB. Ramazzini^, University of Bari Medical School, Giulio Cesare Square 11, 70124 Bari, Italy Introduction Arsenic and chromium (Cr) are widespread environmental contaminants that affect global health due to their toxicity and carcinogenicity (Centeno et al. 2006; IARC 2012a, 2012b; Tsai et al. 2003; Lai et al. 1994; Lee et al. 2002; Chakraborti et al. 2003). Both exist ubiquitously in the envi- ronment and they are commonly present in air, water, soil, and sediments and could find their routes into the human body through inhalation, ingestion, and skin absorption (ATSDR, Agency for Toxic Substances and Disease Registry 2007). Arsenic exists in organic and inorganic forms and in differ- ent oxidation or valence states. The valence states of arsenic compounds relevant to human health are the trivalent (AsIII) and pentavalent (AsV) states. These arsenic species include * Luigi Vimercati luigi.vimercati@uniba.it Antonio Caputi antonio.caputi@uniba.it Marco Quarato marco.quarato91@gmail.com Antonio Baldassarre antonio.baldassarre@uniba.it Maria F Gatti maria.gatti@uniba.it Tommaso Gagliardi tommaso.gagliardi@uniba.it Francesco Cuccaro francesco_cuccaro@hotmail.com Luigi De Maria luigi.demaria@uniba.it 11529 Environ Sci Pollut Res (2017) 24:11528–11535 (Bhattacharyya and Shekdar 2003). Finally, cement factories produce dusts during raw material processing, grinding of clinker, and packaging of finished product: this matter is com- posed of several elements, among which different species of chromium and arsenic and it is deposited to many hundreds of meters from the site of release (Gbadebo and Bankole 2007). arsenates (compounds containing AsO4 3−), arsenites (com- pounds containing AsO3 3−), and the mono-methyl (MMA) and di-methyl (DMA) metabolites. Arsenic species in the tri- valent (III) state including arsenous acid (commonly arsenite), dimethylarsinous acid (DMAIII), and monomethylarsonous acid (MMAIII) are estimated more toxic at lower doses than those of other arsenic species (ATSDR 2007; Drobna et al. 2009). Results from animal and epidemiological studies have shown that inorganic arsenic (iAs) compounds can be catego- rized as carcinogens (group 1) or potential carcinogens (group 2B) such as DMA and MMA, while arsenobetaine and other organoarsenicals have not been categorized as carcinogens (group 3) (IARC 2012a, 2012b). In food and dietary supplements, chromium is mainly Cr(III) and is considered to play a key role in human metab- olism, otherwise in drinking, water is primarily Cr(VI). In contrast with arsenic, is not so clear if chromium can cause cancer following ingestion via drinking water. According to this hypothesis, two ecological studies conduct- ed in China (IARC 1990; Beaumont et al. 2008) and in Greece (Linos et al. Introduction Cr(0) is mainly in its metallic form as a component of iron-based alloys such as stainless steel. Trivalent chromium (Cr(III)) is primarily of geological or- igin and hexavalent chromium (Cr(VI)) is derived mainly from industrial processes (Zhitkovich 2011), but can also orig- inate from the oxidation of naturally occurring Cr(III) by min- erals containing Mn-oxides (Oze et al. 2007). As for chromium, the precise mechanisms that acute or chronic exposure to arsenic performs to induce cancer are not yet understood. Recent studies have shown that the toxic- ity of arsenic depends on several factors: exposure amount, length, and frequency, biological species, age, sex, individual susceptibility, genetics, and nutrition (Abernathy et al. 1999). Different hypotheses regarding the toxic mechanism be- hind arsenic have been suggested, including induced chromo- some abnormalities, promotion/progression, oxidative stress, suppression of p53, altered DNA repair, enhanced cell prolif- eration, altered DNA methylation patterns, altered growth fac- tors, and gene amplification (Hong et al. 2014). As for chromium, the precise mechanisms that acute or chronic exposure to arsenic performs to induce cancer are not yet understood. Recent studies have shown that the toxic- ity of arsenic depends on several factors: exposure amount, length, and frequency, biological species, age, sex, individual susceptibility, genetics, and nutrition (Abernathy et al. 1999). Inter alia, since 1960s, Cr(0) is mainly used in its metallic form by steel factories as a component of iron-based alloys such as stainless steel and tin-free steel, less expensive than tin steel. During these processes, metallic powder containing chromium ions and chromium oxides is produced and, despite the presence of particular filters, it is poured into the air. Moreover, factories’ blast furnaces melt metal scrap contain- ing chromium and other ions to obtain steel. Due to high temperature, Cr(III) and Cr(VI) evaporate and are output via the furnaces’ chimneys (Nicodemi 1994). Chromium also is a waste product in oil refining processes (on average, 27–80 mg per kg of oily sludge): a wrong waste management could lead to a substantial soil pollution around the refinery plant Different hypotheses regarding the toxic mechanism be- hind arsenic have been suggested, including induced chromo- some abnormalities, promotion/progression, oxidative stress, suppression of p53, altered DNA repair, enhanced cell prolif- eration, altered DNA methylation patterns, altered growth fac- tors, and gene amplification (Hong et al. 2014). Introduction 2011) estimated lung and stomach cancer mortal- ity associated with prolonged oral consumption of water con- taminated with Cr(VI). The primary source of arsenic exposure in human is drink- ing water (NRC 1999; NRC 2001; Smith et al. 2002; Watanabe et al. 2003). Organic arsenic compounds (e.g., arsenobetaine) are mainly found in fish, which thus may give rise to human exposure, whereas inorganic arsenic is found in groundwater used for drinking in different areas of the world (such as Asia and South America), although it has been long recognized that arsenic exposure from drinking water is caus- ally related to cancer in the lungs, kidney, bladder, and skin (WHO 2001). Water reservoirs can be contaminated by oil refining wastes: indeed, conventional waste-treatment tech- niques are not effective in the removal of the same arsenical species contained in crude oil (e.g., As(III) and As(V)) (Tonietto et al. 2010). In addition, contaminated soils are a source of arsenic exposure (WHO 2001). Soil can be contam- inated by arsenic-rich steel plants’ emissions, due to the melt- ing of metal scrap containing this metalloid in blast furnaces to obtain recycled iron. Neighboring land may be polluted for decades after plant closure (Lambert et al. 2011). Like for arsenic, it has long been established that inhalation of chromium, in particular hexavalent chromium (CrVI), can cause human lung cancer (IARC 1990). The specific mechanism of chromium carcinogenicity re- mains unclear; however, there is an abundance of data supporting the genotoxicity and mutagenicity of Cr(VI) in vivo and in vitro. Particularly, chromium in its hexavalent form is considered to be a pro-carcinogen. Cr(VI) manages to enter the cell through molecular mimicry mechanism as an oxyanion followed by its metabolic reduction to Cr(V), Cr(IV), and to the final reduced trivalent form. These reduced forms have been shown to induce a wide range of genomic DNA damage, which may lead to DNA replication inhibition (Salnikow and Zhitkovich 2008; Alexander and Aaseth 1995; O’Brien et al. 2001). Moreover, Cr(VI) is thought to be able to induce DNA double-stranded breaks selectively on euchroma- tin and to accumulate ubiquitinated forms of histone H2AX: these two kinds of damage lead to suppressed upregulation of inducible genes and help to explain the high genotoxic poten- tial of this metal (DeLoughery et al. 2015). Cr(0), Cr(III), and Cr(VI) are used commercially and are present in the environment. Materials and methods Between January 2010 and April 2012, a cross-sectional study was conducted to measure the urinary excretion of inorganic arsenic and its methylated metabolites monomethyl arsenic acid (MMA) and dimethylarsinic acid (DMA), chromium as well as urinary creatinine, which was used both to confirm the acceptability of urine samples and to adjust the metal concentrations. First-void urine (FVU) was sampled from each participant into clean conical 50-mL polypropylene tubes, which were then immediately sealed with O-ring screw caps and packed into coolers with frozen ice packs. Samples were sent to the laboratory and then stored at −20 °C and analyzed within 1 month. After analysis, we excluded 47 (13.4%) subjects from the study because of creatinine excretion values <0.3 g/L or >3.0 g/L. The analysis of the urine samples was performed by atomic absorption spectrophotometry (PerkinElmer Corp.—Model 5100 PC—PerkinElmer Inc.—Wellesley, MA, USA), employing the hydrides (arsine) generation technique for de- termining As and the graphite furnace method for Cr accord- ing to the NIOSH analytical methods (NIOSH 2003); an au- tomated kinetic Jaffe technique using alkaline picric acid was used to measure creatinine. Urine samples were not processed for metal concentrations if the creatinine excretion was not within the range of 0.3–3.0 g/L (ACGIH 2010). After these exclusions, the final sample consisted of 279 subjects, including 135 males and 144 females aged between 18 and 77 years (mean age 46.0 ± 13.12 SD). Of the 279 study subjects, 179 resided in the city of Taranto; they were subdivided into three district areas: BPaolo VI^ (N 39), BTamburi—Old Town^ (N 50), or BNew Town^ (N 90). A total of 55 subjects were residents of the nearby Statte munic- ipality and 45 resided in the Laterza municipality (Table 1). An internal quality control (IQC), according to manufac- turer instructions, was used systematically to verify the repro- ducibility and the repeatability of the data that were obtained using the standard curve prepared by the operator. Comparisons among groups were made employing non- parametric techniques (rank sum Wilcoxon-Mann-Whitney test and Kruskal-Wallis test). We also performed a multivari- ate analysis through a linear regression model, investigating the association of the urinary concentrations of As and Cr with the explanatory variables obtained by questionnaire. Introduction The aim of this study was to assess arsenic and chromium exposure in the general population with no occupational ex- posure to these metals, resident in the municipalities of Taranto and Statte, site of a large integrated cycle steel found- ry, a refinery, and a cement factory, and in the municipality of Environ Sci Pollut Res (2017) 24:11528–11535 11530 Laterza, 54-km driving distance from Taranto, considered as a non-polluted area because no significant industrial plants are present. would be treated in an anonymous and collective way, with scientific methods and for scientific purposes in accordance with the principles of the Helsinki Declaration. In fact, the land close to Taranto industrial plant is polluted by many heavy metals, as well as PAHs (Campo et al. 2012): the water samples also taken from aquifers contain high arse- nic and chromium concentrations (ARPA 2009). The steel plant in Taranto, for 2005, has emitted into the air 3800.8 kg of chromium and compounds, into the water 20,407.3 kg of chromium and compounds, and 1172.1 kg of arsenic and com- pounds (APAT - INES 2005). Moreover, in Taranto Gulf, soil contamination is partially linked to air pollution and both could be not homogeneously distributed in closer sites (e.g., Paolo VI, Statte) due to meteorological conditions (Mangia et al. 2013). After obtaining informed consent from each participant qualified health staff it was administered a standardized struc- tured questionnaire. Each questionnaire included personal da- ta of the study participants and information of lifestyle habits. In particular, the questionnaire has investigated the residential history, housing exposure (intensity of car traffic, the presence of fireplace inside their home, proximity to industrial areas), environmental exposure (use of pesticides, paints, wood pre- servatives), and occupational exposure (company, type of job, use of PPE). In addition, they were investigated regarding eating habits especially if they had consumed seafood in the last 48–72 h before urine collection and were evaluated with other confounding factors including quantity and type of wa- ter consumed (tap or bottled mineral water) and smoking habits (type, cigarettes/day, years of smoking, use of cigars, pipe). The questionnaire also investigates the presence of dis- eases and use of drugs. Table 1 Flying distance from the industrial site Results The median urinary concentration in the entire study popula- tion was within the SIVR reference limit whereas the 95th percentile was higher than that of the upper limit (Table 2). Considering the municipalities, the 95th percentile was higher than the reference value only in Statte (Table 2). Moreover, the median urinary concentration of iAs + MMA + DMA was significantly higher in Statte than that in Taranto or Laterza, although it was still within the range limits (Table 2). In the different districts in the city of Taranto, the 95th percentile and median urinary values remained within range limits (Table 3). The median urinary concentration in the entire study popula- tion was within the SIVR reference limit whereas the 95th percentile was higher than that of the upper limit (Table 2). Considering the municipalities, the 95th percentile was higher than the reference value only in Statte (Table 2). Moreover, the median urinary concentration of iAs + MMA + DMA was significantly higher in Statte than that in Taranto or Laterza, although it was still within the range limits (Table 2). In the different districts in the city of Taranto, the 95th percentile and median urinary values remained within range limits (Table 3). When analyzing the urinary excretion of iAs + MMA + DMA in relation to the variables considered in the study popu- lation, similar median values were obtained in both sexes. In subjects who drank tap water, urinary iAs + MMA + DMA values (3.6 μg/L) were higher than in those who drank bottled mineral water (2.5 μg/L). Slightly higher values were found in smokers (4.1 μg/L) than those in non-smokers (3.8 μg/L). Statistically significant differences were found when comparing the urinary concentrations in those who had eaten shellfish and/ or seafood in the 48–72 h before sampling (9.8 vs 3.8 μg/L) (Table 4). Table 2 shows the urinary levels of iAs + MMA + DMA, and Cr measured in the overall population of 279 subjects residing in Taranto and neighboring areas. It was carried out a comparison of the urinary Cr and As in study groups with the range proposed by the Italian Reference Values Society (SIVR). Table 3 shows the urinary levels of iAs + MMA + DMA, and Cr measured in the different districts of Taranto (Paolo VI, Tamburi-Old Town, New Town). Results When analyzing the urinary excretion of iAs + MMA + DMA in relation to the variables considered in the study popu- lation, similar median values were obtained in both sexes. In subjects who drank tap water, urinary iAs + MMA + DMA values (3.6 μg/L) were higher than in those who drank bottled mineral water (2.5 μg/L). Slightly higher values were found in smokers (4.1 μg/L) than those in non-smokers (3.8 μg/L). Statistically significant differences were found when comparing the urinary concentrations in those who had eaten shellfish and/ or seafood in the 48–72 h before sampling (9.8 vs 3.8 μg/L) (Table 4). Materials and methods The main variables included in the model were age, sex, body mass index, drinking water, smoking habits, city of residence, The research was conducted with 350 subjects residing in Taranto and the surrounding area for at least 10 years; they were randomly selected from the Regional Assisted Care Registry to reduce the possibility of bias in self-selection. The response rate was high (93.1%). All of the subjects were contacted in accordance with pro- cedures agreed upon by local general practitioners, who had previously been invited to a dedicated meeting at which they were fully informed about the aims of the study and asked whether they would be willing to collaborate. All subjects agreed to the processing of their personal data and understood that this information was categorized as Bsensitive data^. All subjects were informed that data from the research protocol Table 1 Flying distance from the industrial site Laterza 34 km Statte 2.3 km Paolo VI 3 km Tamburi—Old Town 200 m New Town 2.7 km Laterza 34 km Statte 2.3 km Paolo VI 3 km Tamburi—Old Town 200 m New Town 2.7 km 11531 Environ Sci Pollut Res (2017) 24:11528–11535 The multivariate analysis showed the following results. For Cr, we found an association with the city of residence; specif- ically, we found a higher concentration in the people living in Statte vs Taranto (p = 0.001), and this analysis is not influ- enced by principal investigated confounding factors (Table 5). dwelling site, consumption of fish, crustaceans, and shellfish in the 48–72 h before collection, presence of dental fillings, and use of fireplaces in homes. dwelling site, consumption of fish, crustaceans, and shellfish in the 48–72 h before collection, presence of dental fillings, and use of fireplaces in homes. A p value ≤0.05 was considered significant. Statistical analysis was conducted using packages SAS (v. 9.0) and STATA (v. 11). Statistically significant Urinary chromium The median value was 0.3 μg/L, which was comparable to the upper limit of the range proposed by the SIVR. The 95th percentile was significantly above the reference value limits (Table 2). The differences in the measured median values among the municipalities were significant, with the highest values found in Statte (Table 2). In the different districts of Taranto, the highest median values of urinary concentrations were found in the Paolo VI district (Table 3). In our study, no association between the use of pesticides and urinary concentrations of As was found. Moreover, we investi- gated the association between having a fireplace in home and the urinary excretion of As, without finding any association. The median values of urinary chromium were comparable in both sexes, in all age classes, among smokers and no smokers, regardless of whether they drank tap or bottled min- eral water, and seafood consumption (Table 4). The multivariate analysis showed the following results. For As, we found an association with the city of residence; spe- cifically, we found a higher concentration in the people living in Statte vs Taranto (p = 0.001) and this analysis is not influ- enced by principal investigated confounding factors (Table 5). In our study, we investigated the association between hav- ing a fireplace in home and the urinary excretion of Cr, with- out finding any association. Discussion Tobacco smoke is known to contain chromium (VI), and indoor air polluted by cigarette smoke can contain hundreds of times the amount of chromium (VI) found in outdoor air (IARC 2012a, 2012b). However, other different exposure routes, such as transdermal way (as well as inhalation and in- gestion), are possible but their contribution to overall chromi- um intake is not clear and cannot be investigated in this study. To date, few studies have investigated exposure to arsenic and chromium in a population residing in a high-risk environmen- tal area such as Taranto, Apulia Region (Southern Italy) (Iavarone et al. 2012). Many years have passed since the WHO first included the Taranto area among those at high environmental risk and underlined the increased mortality rates, as compared to Italy as a whole, for bladder, liver, and lung cancer, as well as cancer of the pleura and non-Hodgkin lymphoma (WHO 1997). The subjects who had eaten seafood and/or shellfish 48– 72 h before urine collection had higher levels of urinary ex- cretion of arsenic (9.8 vs 3.8 μg/L). In fact, diet is the main source of non-occupational exposure to arsenic (Vimercati et al. 2009). Foods with the highest content of arsenic include some marine organisms, such as shellfish and crustaceans (Argese et al. 2005; Fattorini et al. 2004; Lopez et al. 1994; WHO 2001). However, some authors did not find a positive association between the consumption of seafood and/or shellfish and the urinary concentrations of As. In particular, Hsueh et al. (2002) found no differences in the urinary con- centrations of the various As species before and after refraining from eating seafood for 3 days, respectively. The median value for chromium (0.3 μg/L) was the upper limit value of the relative SIVR range, while the 95th percentile was actually higher than the proposed SIVR upper limit. There were no significant differences in urinary excretion by sex, age, type of water drunk, and number of smoked cigarettes, unlike in other reports in literature (EPA 1984; SIVR 2011; Zhitkovich 2002). Moreover, our study found higher urinary levels of chro- mium and arsenic in people living close to industrial plants. It has been generally accepted that low or moderate doses of orally ingested Cr(VI) are non-carcinogenic, but, more re- cently, the potential risks of Cr(VI) exposure by ingestion in drinking water have come under increased scrutiny (Nickens et al. 2010). Urinary chromium Table 2 Mean, median, and 95th percentile of the urinary levels of As and Cr (μg/L) by municipality Municipality Metal mean SD p5 p50 (median) p95 n SIVR reference values Whole study population iAs + MMA + DMA 6.1 8.6 1.4 3.8 16.8 279 2.0–15 Cr 0.5 0.5 0.1 0.3 1.3* 279 0.05–0.35 Taranto iAs + MMA + DMA 5.2 8 1.5 3.8 11.1 179 2.0–15 Cr 0.4 0.3 0.1 0.3 1* 179 0.05–0.35 Laterza iAs + MMA + DMA 3.2 2.3 0.9 2.7 8.5 45 2.0–15 Cr 0.4 0.4 0.1 0.3 1.2* 45 0.05–0.35 Statte iAs + MMA + DMA 11.5 11 2.5 8.8 27.1* 55 2.0–15 Cr 0.9 1 0.2 0.5 2.5* 55 0.05–0.35 Statistically significant Table 2 Mean, median, and 95th percentile of the urinary levels of As and Cr (μg/L) by municipality Environ Sci Pollut Res (2017) 24:11528–11535 11532 Table 3 Mean, median, and 95th percentile of the urinary levels of As and Cr (μg/L) in the different districts in the city of Taranto District Metal mean SD p5 p50 (median) p95 n SIVR reference values New Town iAs + MMA + DMA 4.3 2.4 1.7 3.8 9.7 90 2.0–15 Cr 0.3 0.3 0.1 0.3 0.9 90 0.05–0.35 Paolo IV iAs + MMA + DMA 4 3.7 1.4 2.7 9.1 39 2.0–15 Cr 0.4 0.2 0.1 0.4 0.8* 39 0.05–0.35 Tamburi—Old Town iAs + MMA + DMA 7.8 14.3 1.4 4.6 14.3 50 2.0–15 Cr 0.4 0.4 0.1 0.3 1.2* 50 0.05–0.35 Statistically significant , and 95th percentile of the urinary levels of As and Cr (μg/L) in the different districts in the city of Taranto We also found a lower concentration in the people living in Laterza vs Taranto (p = 0.037) and a statistically significant association with the consumption of crustaceans in the 48– 72 h before collection (p = 0.019). The chromium concentration limit in drinking water ap- plied both in Italy (Legislative Decree no. 31/2001) and in the USA. (US Environmental Protection Agency—EPA) is 50 μg/L (ATSDR 2007). However, in a study conducted in California (USA), 38% of municipal sources of drinking water reportedly showed higher levels of chromium (VI) than the detection limit of 1 μg/L (Sedman et al. 2006). Environ Sci Pollut Res (2017) 24:11528–11535 Table 5 Urinary excretion of As and Cr metals (μg/L) in relation to the variables listed in Statte municipality Sex Smoking habit Water Seafood consumption Male Female Yes No Tap Bottled Yes No IAs + MMA + DMA 10.4 (27) 7.0 (28) 8.2 (16) 9.9 (39) 8.4 (28) 9.4 (27) 11.8 (4) 8.5 (51) Cr 0.6 (27) 0.5 (28) 0.5 (16) 0.5 (39) 0.5 (28) 0.7 (27) 1 (4) 0.5 (51) () = n value Table 5 Urinary excretion of As and Cr metals (μg/L) in relation to the variables listed in Statte municipality () = n value in Europe, and the emissions of Hg in the same year was 510 kg. There are no available data concerning the emissions of the other metals. In the future, therefore, we believe it will be necessary to carry out an organized environmental monitoring program, taking into consideration all exposure routes to corre- late the environmental concentrations of these metals with the biomonitoring results. However, our study suffers from some limitations due to the small population sample and data analy- sis. In fact, the questionnaire results could be influenced by subjects’ personal replies. Moreover, chromium analysis could be affected by redox reactions interference, during the ions determination (Jiang et al. 2013). Furthermore, for arsenic spe- ciation, best sensitivity was shown for As(III) with respect to MMA, DMA, and As(V) (Moreno et al. 2000). amount of inorganic arsenic-based pesticides has led to serious environmental arsenic contamination (Datta and Sarkar 2005). Given the notorious adverse effects of arsenic exposure in humans, the US Environmental Protection Agency (EPA) banned the use of many inorganic arsenic-based pesticides during the late 1980s and early 1990s (Quazi et al. 2013). Although some countries have issued documents to phase out organo-arsenical pesticides from the market, large agricultural sites contaminated by years of organo-arsenical pesticide appli- cation still exist. These agricultural lands might pose significant health risks in the present and in the future (Li et al. 2016). About that, contribution of transdermal route in overall As intake is hard to estimate and thus cannot be investigated in this study. In our study, no association was found between the use of pesticides and urinary concentrations of As. There were sig- nificant differences between those who drank tap water and those who habitually drank bottled mineral water. Environ Sci Pollut Res (2017) 24:11528–11535 The con- tamination of the main water supply remains a major source of exposure to inorganic As in many parts of the world (IARC 2004), despite the fact that in 1993, the WHO recommended that levels of As in drinking water should not exceed 10 μg/L (WHO 1993). On the other hand, a recent analysis of 40 dif- ferent labels of bottled mineral water on sale in Italy demon- strated higher levels of total As than the legal limit in five of them (Signorile et al. 2007). In contrast, in the surveys of water in the Apulian aqueduct over the period 2004–2006, total As values were consistently below 1 μg/L. In any case, the data we obtained, which may be further confirmed by larger population studies, are sufficient to war- rant the expectation that local and national institutions should be required to adopt preventive measures to reduce the envi- ronmental exposure of the general population to heavy metals, especially lead and chromium. Such actions could help to reduce the health risks, including those of a carcinogenic na- ture, posed to populations residing in areas with a known high environmental impact. Discussion Moreover, despite the notorious toxic effects of arsenic in humans, it is still used in both agriculture and industry (Park et al. 2010; Kumaresan and Riyazuddin 2001). This large Table 4 Urinary excretion of As and Cr metals (μg/L) in relation to the variables listed Sex Smoking habit Water Seafood consumption Male Female Yes No Tap Bottled Yes No IAs + MMA + DMA 4.1 (135) 3.8 (144) 4.1 (73) 3.8 (206) 3.6 (93) 2.5 (186) 9.8* (35) 3.8 (244) Cr 0.4 (135) 0.3 (144) 0.5 (73) 0.3 (206) 0.5 (93) 0.4 (186) 0.4 (35) 0.3 (244) *Statistically significant () = n value Table 4 Urinary excretion of As and Cr metals (μg/L) in relation to the variables listed 11533 Conclusions Argese E, Bettiol C, Rigo C, Bertini S, Colomban S, Ghetti PF (2005) Distribution of arsenic compounds in Mytilus galloprovincialis of the Venice lagoon (Italy). Sci Total Environ 348:267–277 Acknowledgments The authors thank the general practitioners who participated to the study (Dr. Basile, Dr. Carone, Dr. Catucci, Dr. Colucci, Dr. De Sabato, Dr. Dell’Aquila, Dr. Guarino, Dr. Mancino, Dr. Ostillio, Dr. Perrone, Dr. Poretti, Dr. Zizza) and Dr. Michele Conversano and Dr. Giovanni Caputi of the Department of Prevention—Taranto Health Local Organization. Acknowledgments The authors thank the general practitioners who participated to the study (Dr. Basile, Dr. Carone, Dr. Catucci, Dr. Colucci, Dr. De Sabato, Dr. Dell’Aquila, Dr. Guarino, Dr. Mancino, Dr. Ostillio, Dr. Perrone, Dr. Poretti, Dr. Zizza) and Dr. Michele Conversano and Dr. Giovanni Caputi of the Department of Prevention—Taranto Health Local Organization. ARPA (2009) (Agenzia Regionale per la Prevenzione e la Protezione dell’Ambiente) Relazione sui dati ambientali dell’area di Taranto. Available: http://www.arpa.puglia.it/c/document_library/get_file? uuid=96dc386e-2a6d-4758-8c47-e4d15d367c70&groupId=10125 (last access 13/01/2017) ATSDR (Agency for Toxic Substances and Disease Registry) (2007) Toxicological profile for arsenic. U.S. Department of Health and Human Services, Public Health Service, Atlanta Authors’ contributions LV is the principal investigator, planned and designed the study, and drafted the manuscript; MFG is the principal investigator, administered questionnaires, and helped to draft the manu- script; TG carried out analysis of urine samples and measured metals’ concentrations; FC performed statistical and epidemiological analysis; LD performed statistical and epidemiological analysis; AC carried out the data entry; MQ carried out the data entry; AB is the principal inves- tigator, performed statistical and epidemiological analysis, and helped to draft the manuscript. All authors read and approved the final manuscript. Human Services, Public Health Service, Atlanta Beaumont JJ, Sedman RM, Reynolds SD, Sherman CD, Li LH, Howd RA, Sandy MS, Zeise L, Alexeeff GV (2008) Cancer mortality in five villages in China with hexavalent chromium-contaminated drinking water. Epidemiology 19:12–23 Bhattacharyya JK, Shekdar AV (2003) Treatment and disposal of refinery sludges: Indian scenario. Waste Manag Res 21(3):249–261 Campo L, Vimercati L, Carrus A, Bisceglia L, Pesatori AC, Bertazzi PA, Assennato G, Fustinoni S (2012) Environmental and biological monitoring of PAHs exposure in coke-oven workers at the Taranto plant compared to two groups from the general population of Apulia, Italy. Conclusions The importance of investigating the exposure of the general population to As and Cr lies in their ubiquitous nature, since they are also widely distributed in nature, as well as in their harmful effects on human health. We conducted a study to evaluate the exposure to heavy metals in the industrial city of Taranto and the surrounding area in Southern Italy through biological monitoring techniques. Moriske et al. found higher concentrations of heavy metals in indoor air pollution in houses with coal burning and open fireplaces than those in homes with central heating (Moriske et al. 1996). In our study, we investigated the association be- tween having a fireplace in the home and the urinary excretion of heavy metals, but did not find any association. Overall, the biological monitoring data reveal high urinary concentrations of both heavy metals investigated, above all in Statte municipality. We measured the levels of chromium, inorganic arsenic, and its methylated metabolites in the urine samples of 279 subjects residing in Taranto and neighboring areas. However, in our study, it was not possible to correlate the biological monitoring data with the environmental data because the information collected by the official institutions and/or those in the literature were incomplete and only provided by the European Monitoring and Evaluation Programme (EMEP). Our study results showed high urinary concentrations of both heavy metals investigated. It would be appropriate to search the causes of this finding and deepen the impact of industrial plants present in that area. This is important in developing a comprehensive risk assess- ment and management program in order to adopt preventive For the whole province of Taranto, the value of emissions of Pb into the atmosphere in 2009 was 38 tons, one of the highest 11534 Environ Sci Pollut Res (2017) 24:11528–11535 Alexander J, Aaseth J (1995) Uptake of chromate in human red blood cells and isolated rat liver cells: the role of the anion carrier. 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https://openalex.org/W1574073844	https://www.scielo.br/j/epec/a/M3YCG49X3CWZkqLn9WWx3dJ/?lang=pt&format=pdf	Portuguese	null	Uma revisão sobre as investigações construtivistas nas últimas décadas: concepções espontâneas, mudança conceituai e ensino de ciências	Ensaio	2,004	cc-by	11,326	1 Professor Associado Livre Docente do Departamento de Educação e do Programa de Pós-graduação em Educação para a Ciência. (nardi@fc.unesp.br). Resumo A abordagem construtivista no ensino de Ciências tornou-se influente a partir da década de 70, com a grande ênfase dada as investigações sobre as concepções espontâneas de estudantes sobre diversos conteúdos de ciências. As tentativas de levar os resultados das investigações para a sala de aula originaram as discussões sobre mudança conceitual, seguidas de inúmeros estudos que até hoje têm procurado interferir no ensino de sala de aula. Neste capítulo procuramos mostrar alguns trabalhos sobre concepções espontâneas em física realizados no Brasil naquele período e fazer uma revisão das principais publicações que se seguiram após os trabalhos pioneiros de Posner e colaboradores. Palavras-chave: Construtivismo, Concepções espontâneas, Mudança conceitual, Ensino de Ciências 2 Doutora em Educação pela Faculdade de Educação da Universidade Estadual de Campinas, São Paulo, Brasil. Grupo de Pesquisa em Ensino de Ciências. Faculdade de Ciências da Universidade Estadual Paulista – UNESP – Campus de Bauru, São Paulo, Brasil. (steodoro@hotmail.com). Uma revisão sobre as investigações construtivistas nas últimas décadas: concepções espontâneas, mudança conceitual e ensino de ciências A review of the constructivist research in the last decades: spontaneous reasoning, conceptual change and science education Roberto Nardi1 Sandra Regina Teodoro Gatti 2 Keywords: Constructivism, Spontaneous reasoning, Conceptual change, Science Teaching 115 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 Abstract The constructivist approach in Science Education became a strong influence in the middle of the 70s, with a great emphasis on investigating students’ conceptions of a variety of science contents. The attempts to take these results into the science classes originated the research on conceptual change followed by many studies that are still today trying to interfere in classroom teaching. In this chapter we look for presenting some research about spontaneous reasoning in physics carried out in Brazil in that period and to review the main research that followed the pioneer paper on conceptual change published by Posner and his collaborators. 115 A partir da década de 70, pôde-se observar entre os investigadores em Ensino de Ciências um grande empenho em estudar mais profundamente as noções que os estudantes trazem para a sala de aula, previamente ao ensino formal. Os trabalhos de Doran (1972), Viennot (1979), Driver (1985) e Watts e Zylbertajn (1981), foram algumas das investigações pioneiras nessa linha e denominaram tais noções de “conceitos espontâneos”, “conceitos intuitivos”, “formas espontâneas de raciocínio”, “estruturas alternativas” e outras denominações semelhantes. Essas investigações, versando sobre as idéias dos estudantes em relação aos diversos conceitos científicos, revelaram que tais noções podem diferir substancialmente da ciência que se pretende ensinar, influenciam a aprendizagem futura e podem ser resistentes a mudanças. (Driver, 1989). As principais características de tais concepções segundo Coll et. al. (1998) são apresentadas na figura abaixo: Possuem coerência do ponto de vista do aluno, não do ponto de vista científico. São bastante estáveis e resistentes à mudança. São construções pessoais. Possuem um caráter implícito. São descobertos nas atividades ou previsões (“teorias em ação”). Procuram a utilidade mais do que a “verdade”. São compartilhadas por outras pessoas, sendo possível reunir em tipologias. Características dos conhecimentos prévios. Possuem coerência do ponto de vista do aluno, não do ponto de vista científico. São construções pessoais. São bastante estáveis e resistentes à Possuem um caráter implícito. São descobertos nas atividades ou previsões (“teorias em ação”). Procuram a utilidade mais do que a “verdade”. São compartilhadas por outras pessoas, sendo possível reunir em tipologias. Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 116 Figura 1: Características dos conhecimentos prévios. (Coll, Pozo, Sarabia e Valls, 1998, p. 41). Abstract Esses trabalhos contribuíram para o fortalecimento de um então chamado “paradigma construtivista” na investigação sobe o ensino e a aprendizagem e propiciaram a contestação dos chamados modelos de aprendizagem por aquisição conceitual, centrados na transmissão de conhecimentos por parte do professor e não no respeito aos conhecimentos prévios dos estudantes. Destacamos aqui algumas investigações nesta linha realizadas nesse período no Brasil, particularmente na área de ensino de Física, e ainda algumas das idéias que surgiram como conseqüência das investigações iniciais de Posner e colaboradores que, pela primeira vez, trazem à comunidade de investigadores em Educação em Ciências um modelo de ensino por “mudança conceitual”. Concluímos observando que a complexidade das discussões que se seguiram aparecem no esforço de levar em consideração outros fatores presentes nas relações de ensino e aprendizagem não considerados no modelo inicial de Posner. Entendemos, por fim, a necessidade de encorajar a continuidade da investigação nessa área, procurando envolver nessa tarefa futuros docentes, ainda em seu processo de formação profissional, e os docentes em exercício com a finalidade de trazer para a academia as reflexões ocorridas em sala de aula para a verificação dos modelos que vêm sendo propostos. Alguns estudos sobre concepções espontâneas e psicogênese de conceitos realizados no Brasil Muitos dos estudos sobre concepções espontâneas e também sobre psicogênese de conceitos neste chamado “paradigma construtivista” foram realizados nas décadas anteriores no Brasil. A título de exemplificação, destacamos algumas dessas investigações, envolvendo conceitos de Ciências, particularmente da Física. Laburú (1987) estudou como os alunos descrevem e compreendem o conceito de aceleração, a fim de re-elaborar suas concepções prévias. A coleta de dados foi baseada em 117 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 trabalhos de Piaget e Thowbridge-McDermott onde foram entrevistados 34 alunos numa faixa etária compreendida entre 11 e 16 anos de idade, distribuídos entre 6ª e a 8ª séries do Ensino Fundamental e a 2ª série do Ensino Médio3. As entrevistas foram desencadeadas a partir de três tarefas-experimento onde foram mostrados movimentos acelerados. Na primeira tarefa o entrevistador utilizou “... uma pequena madeira sobre a qual se prendiam dois grampos, através dos quais se passava uma fita branca a ser puxada pelo entrevistador. Esta fita partia do repouso e daí por diante sua velocidade era aumentada continuamente. Entre os grampos, e mantendo sempre o mesmo lugar, o entrevistado batia de forma cadenciada uma caneta de tipo pincel atômico, de maneira a imprimir pontos sobre a fita, tentando manter o ritmo constante”.(Laburú,1993, p. 62). trabalhos de Piaget e Thowbridge-McDermott onde foram entrevistados 34 alunos numa faixa etária compreendida entre 11 e 16 anos de idade, distribuídos entre 6ª e a 8ª séries do Ensino Fundamental e a 2ª série do Ensino Médio3. As entrevistas foram desencadeadas a partir de três tarefas-experimento onde foram mostrados movimentos acelerados. Na primeira tarefa o entrevistador utilizou “... uma pequena madeira sobre a qual se prendiam dois grampos, através dos quais se passava uma fita branca a ser puxada pelo entrevistador. Esta fita partia do repouso e daí por diante sua velocidade era aumentada continuamente. Entre os grampos, e mantendo sempre o mesmo lugar, o entrevistado batia de forma cadenciada uma caneta de tipo pincel atômico, de maneira a imprimir pontos sobre a fita, tentando manter o ritmo constante”.(Laburú,1993, p. 62). No final do experimento a fita assemelhava-se à figura 2. No final do experimento a fita assemelhava-se à figura 2. No final do experimento a fita assemelhava-se à figura 2. 3 Ensino Fundamental, no Brasil, corresponde aos primeiros anos de escolarização formal, posteriores à Educação Infantil.O Ensino Médio, corresponde a três anos de escolarização, posterior ao Ensino Fundamental, equivalente à “High School” nos Estados Unidos. Segundo a legislação brasileira, toda a escolarização (Educação Infantil, Ensino Fundamental e Ensino Médio corresponde à Educação Básica). 118 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 Alguns estudos sobre concepções espontâneas e psicogênese de conceitos realizados no Brasil Figura 2 - Tarefa-experimento utilizada na primeira situação desencadeadora das entrevistas (Fonte : Laburú, 1993, p.62) Figura 2 - Tarefa-experimento utilizada na primeira situação desencadeadora das entrevistas (Fonte : Laburú, 1993, p.62) A segunda tarefa consistia de um experimento “... no qual se fazia uso de um caderno especial, em cujas espirais o entrevistador passava uma régua fazendo estalos. A régua era passada pelo entrevistador de forma a produzir ora movimentos uniformes, ora acelerados. Esta tarefa procurava explorar o aumento ou a diminuição do intervalo de tempo em iguais intervalos de espaço”. Na terceira tarefa foram apresentados (vide Figura 3) “... dois carrinhos em duas pistas distintas e paralelas. (...) Esta tarefa objetivava a confrontação das acelerações dos dois carrinhos. Ela poderia ser resolvida comparando-se as velocidades finais nulas e dos 118 seus respectivos tempos, ou as velocidades no instante de ultrapassagem, considerando-se os us respectivos tempos, ou as velocidades no instante de ultrapassagem, considerando-se os pectivos tempos iguais”.(Laburú,1993, p. 63). Figura 3 - Tarefa-experimento utilizada na terceira situação desencadeadora das entrevistas (Fonte: Laburú, 1993, p.64). respectivos tempos iguais”.(Laburú,1993, p. 63). Figura 3 - Tarefa-experimento utilizada na terceira situação desencadeadora das entrevistas (Fonte: Laburú, 1993, p.64). Os resultados obtidos neste estudo identificaram três padrões diferentes de concepções: nas diferentes faixas etárias, estavam presentes: a aceleração como aumento de velocidade; no Ensino Médio, principalmente, a aceleração é entendida como variação de velocidade; apenas no Ensino Médio a aceleração foi entendida como razão. O estudo mostra que predominou a identificação da aceleração com a própria velocidade na 6ª série, mas esta também foi encontrada nos três níveis de escolaridade investigados. O autor percebe também que a passagem dos alunos pelo ensino formal do conceito de aceleração não influenciou na diminuição das idéias intuitivas de aceleração como critério de velocidade. Recomenda que o emprego de um vocabulário do senso comum deve ser discutido, pois este contribui para uma interpretação incorreta do conceito de aceleração. Observa também que as concepções de alguns alunos se aproximam do que pensava Galileu, ou seja, “uma concepção de aceleração como incremento de velocidade por unidade de espaço”.(Laburú, 1993, p.71). Outro estudo na área da Física foi realizado por Goulart e colaboradores (1989), sobre os conceitos espontâneos de fenômenos relativos à luz numa amostra de estudantes de Ensino Fundamental e Médio em escolas públicas, na sua maioria entre seis e dez anos. Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 Alguns estudos sobre concepções espontâneas e psicogênese de conceitos realizados no Brasil As entrevistas foram realizadas individualmente e os protocolos foram baseados em questões- chave e atividades elaboradas depois de um contato prévio, onde se levantou a linguagem utilizada pelas crianças para explicar os fenômenos físicos do cotidiano. Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 119 Os aspectos investigados referiam-se à natureza da luz; suas propriedades; a relação dos três fatores intervenientes no processo de visão: fonte, receptor, objeto-reflexão difusa por objetos opacos; reflexão; transmissão e absorção da luz branca com o uso de filtros e as cores. Dentre as atividades elaboradas foi utilizada uma situação exemplo a fim de obter dados de “como a criança explica o processo de visão” (Figura 4). Figura 4: Seqüência de situações utilizadas para investigar os modelos de visão da criança. Figura 4: Seqüência de situações utilizadas para investigar os modelos de visão da criança. Das conclusões constam recomendações dos autores sobre algumas implicações para o desenvolvimento curricular e para a capacitação de professores de Ciências: “deve-se oferecer mais tempo e esforço aos estudantes para ‘investigações próprias’; reconhecer o nível de abstração que é necessário para um dado entendimento e que a criança pode atingir um dado ponto; organizar o currículo de tal maneira que as situações e conceitos mais simples sejam apresentados primeiro”. (Goulart et al., 1989, p. 17). Os autores citam ainda a importância de se levar estes resultados obtidos aos professores em exercício nas escolas de Ensino Fundamental e Médio, objetivando levá-los a investigar em sua prática de sala de aula, detectando estes conceitos entre seus estudantes a fim de possivelmente substituí-los por conceitos próximos aos considerados científicos. A psicogênese de conceitos, também foi objeto de estudo, nesse período, no Brasil. Esses estudos procuraram mostrar como as idéias evoluem no decorrer do tempo na mente do aluno. Para tanto, essas investigações utilizaram amostras de sujeitos em diversas faixas etárias, visando verificar como ocorre a gênese e/ou a evolução do referido conceito. A Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 120 investigação sobre o conceito de velocidade realizada por Silva (1988) e sobre o conceito de campo de força (Nardi, 1990) são exemplos desses estudos psicogenéticos 4. 4 Esses estudos foram realizados na década de 80 por componentes do Grupo de Estudos Psicogenéticos de Conceitos Científicos da Faculdade de Educação da Universidade de São Paulo, sob coordenação da Profa. Dra. Anna Maria Pessoa de Carvalho. Alguns estudos sobre concepções espontâneas e psicogênese de conceitos realizados no Brasil investigação sobre o conceito de velocidade realizada por Silva (1988) e sobre o conceito de campo de força (Nardi, 1990) são exemplos desses estudos psicogenéticos 4. Silva utilizou-se do método de entrevistas clínicas para identificar como crianças e adolescentes (53 sujeitos) de diversas faixas etárias de uma escola estadual, constroem as noções de velocidade linear e angular. As entrevistas foram desencadeadas a partir de três questões básicas: na primeira delas, os alunos são questionados sobre dois carros que partem no mesmo instante do ponto O (Figura 5), realizam uma volta no mesmo tempo e chegam juntos novamente em O. A questão é a seguinte: Qual dos dois carros, A ou B, têm maior velocidade? Justifique sua resposta. o A B o Figura 5 - Situação desencadeadora da primeira questão básica da entrevista (Fonte: Silva, 1990). Figura 5 - Situação desencadeadora da primeira questão básica da entrevista (Fonte: Silva, 1990). Na segunda questão é apresentado um cone (figura 6), que está girando. Dois pontos são marcados sobre sua superfície. A questão desencadeadora é a seguinte: Qual desses pontos, A ou B, têm maior velocidade? Justifique. A B Figura 6 - Situação desencadeadora da segunda questão básica da entrevista (Fonte: Silva, 1990). Figura 6 - Situação desencadeadora da segunda questão básica da entrevista (Fonte: Silva, 1990). 4 Esses estudos foram realizados na década de 80 por componentes do Grupo de Estudos Psicogenéticos de Conceitos Científicos da Faculdade de Educação da Universidade de São Paulo, sob coordenação da Profa. Dra. Anna Maria Pessoa de Carvalho. 121 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 121 Na última das três questões, é mostrado ao aluno um dispositivo onde aparecem duas polias interligadas (Figura 7) e a questão é: Se o sistema de polias (P1 e P2) estiver girando, qual das duas polias têm maior velocidade? Justifique. Sobre o mesmo sistema é perguntado também: Se marcarmos dois pontos quaisquer sobre a correia que liga as duas polias, qual desses dois pontos tem maior velocidade? A f1 B f2 Figura 7 - Situação desencadeadora da terceira questão básica da entrevista. (Fonte: Silva, 1990.) A investigação apontou que os estudantes passam por três estágios diferentes com f2 Figura 7 - Situação desencadeadora da terceira questão básica da entrevista. (Fonte: Silva, 1990.) Figura 7 - Situação desencadeadora da terceira questão básica da entrevista. 122 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 Alguns estudos sobre concepções espontâneas e psicogênese de conceitos realizados no Brasil (Fonte: Silva, 1990.) A investigação apontou que os estudantes passam por três estágios diferentes com Figura 7 Situação desencadeadora da terceira questão básica da entrevista. (Fonte: Silva, 1990.) A investigação apontou que os estudantes passam por três estágios diferentes com A investigação apontou que os estudantes passam por três estágios diferentes com A investigação apontou que os estudantes passam por três estágios diferentes com relação à noção de velocidade linear e angular. O autor mostra assim suas conclusões: “Os sujeitos de um primeiro estágio não possuem as noções relativas aos conceitos de velocidade. No seu processo de construção do conhecimento físico, passam a se apoderar da razão espaço-tempo na descrição da natureza e na medida em que evoluem, através de abstrações reflexivas, vão generalizando essa idéia para as situações mais e mais complexas. A partir daí, passam a construir uma contradição entre velocidade angular e a idéia de velocidade linear, que se centra na observância dos pontos, e começam, também, a observar o conjunto todo em movimento, ao redor de um eixo de rotação. Essa contradição faz-se equilibrada no momento em que a idéia de velocidade deixa de ser única, aparecendo, assim, a velocidade angular”.(Silva, 1990, p. 12). relação à noção de velocidade linear e angular. O autor mostra assim suas conclusões: “Os sujeitos de um primeiro estágio não possuem as noções relativas aos conceitos de velocidade. No seu processo de construção do conhecimento físico, passam a se apoderar da razão espaço-tempo na descrição da natureza e na medida em que evoluem, através de abstrações reflexivas, vão generalizando essa idéia para as situações mais e mais complexas. A partir daí, passam a construir uma contradição entre velocidade angular e a idéia de velocidade linear, que se centra na observância dos pontos, e começam, também, a observar o conjunto todo em movimento, ao redor de um eixo de rotação. Essa contradição faz-se equilibrada no momento em que a idéia de velocidade deixa de ser única, aparecendo, assim, a velocidade angular”.(Silva, 1990, p. 12). No outro estudo psicogenético, Nardi (1990) procura investigar as noções que os alunos apresentam sobre o conceito de campo de força e sua relação com o desenvolvimento cognitivo. Para tanto, são entrevistados 45 sujeitos escolhidos aleatoriamente entre alunos de Ensino Fundamental e Médio, a partir de algumas situações desencadeadoras, utilizando ímãs, 122 Rev. Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 Alguns estudos sobre concepções espontâneas e psicogênese de conceitos realizados no Brasil Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 122 objetos metálicos, limalha de ferro e uma foto onde aparecia um astronauta no espaço, fora de uma nave, conforme figura 8, abaixo. FIGURA 8 - Reprodução do diapositivo utilizado na situação desencadeadora sobre o espaço, forma e campo gravitacional da Terra. (Fonte: Nardi, 1991, p. 72). A partir da análise das entrevistas clínicas efetuadas, o autor classifica os sujeitos FIGURA 8 - Reprodução do diapositivo utilizado na situação desencadeadora sobre o espaço, forma e campo gravitacional da Terra. (Fonte: Nardi, 1991, p. 72). g ( , , p ) A partir da análise das entrevistas clínicas efetuadas, o autor classifica os sujeit A partir da análise das entrevistas clínicas efetuadas, o autor classifica os sujeitos em três níveis, desde as explicações menos elaboradas (nível 1) até as explicações consideradas mais elaboradas (nível 3). Observa que indivíduos com 13 anos de idade, já começam a ter noções relativamente elaboradas sobre o conceito de campo de força e, em alguns casos específicos, as idades não correspondem ao nível que a maioria pertence. O autor conclui ainda que o conceito de campo de força “pode ser introduzido na 8a série do Ensino Fundamental, pois, nesta faixa etária, os alunos já começam a perceber propriedades de um campo físico e generalizar as diversas situações diferentes através de uma mesma explicação causal: o modelo de campo”.(Nardi, 1991, p.129). A investigação mostra ainda uma analogia entre os modelos apresentados pelos estudantes e a evolução histórica das idéias que culminaram com o conceito de campo de força, as concepções dos estudantes sobre a forma, espaço e campo gravitacional do planeta Terra e ainda a evolução do que chamou de "léxico científico" dos sujeitos da amostra, ou seja, o vocabulário utilizado pelos estudantes de acordo com a faixa etária (Nardi, 1994). 123 Especificamente com relação às concepções sobre o campo gravitacional terrestre, foi possível identificar as idéias dos sujeitos sobre a forma do planeta Terra, e classificá-las em noções que variam de uma visão mais egocêntrica, para uma visão mais conceitual, segundo terminologia empregada por Nussbaum (1979). A seqüência de desenhos da Figura 8, abaixo, resume essa “evolução”, revelando um comparativo com resultados de outros estudos. Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 Alguns estudos sobre concepções espontâneas e psicogênese de conceitos realizados no Brasil Figura 9: a) Seqüência que mostra os desenhos (cópias reduzidas) dos quatro modelos principais representativos das noções apresentadas pelos sujeitos da amostra, classificando-as da menos (noção 1) para a mais elaborada conceitualmente (noção 4). (Fonte: Nardi, 1991, p.122). b) Várias noções de Terra apresentadas por crianças israelenses (Nussbaum 1979 p 83) a) b) c) a) b) b) b) c) Figura 9: a) Seqüência que mostra os desenhos (cópias reduzidas) dos quatro modelos principais representativos das noções apresentadas pelos sujeitos da amostra, classificando-as da menos (noção 1) para a mais elaborada conceitualmente (noção 4). (Fonte: Nardi, 1991, p.122). b) Várias noções de Terra apresentadas por crianças israelenses. (Nussbaum, 1979, p. 83). c) A evolução das concepções de crianças sobre o tópico “A Terra no espaço e campo gravitacional” (adaptado de Baxter,1989, p. 505). Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 124 De maneira similar aos estudos acima, foram realizados inúmeros outros em todo o mundo. Um dos levantamentos mais completos sobre as investigações realizadas nesta linha foi elaborado por Pfundt e Duit (1994), que conseguiu abranger a maioria dos trabalhos de investigação realizado nas últimas décadas em diversas áreas do conhecimento, conforme mostra o Quadro 1 abaixo (Pfundt e Duit, 1994, p. XXVI). ÁREA GERAL Nº DE ESTUDOS TÓPICOS ESPECÍFICOS MECÂNICA 421 Força e movimento; trabalho, força, energia, velocidade, aceleração, gravidade, pressão, densidade, flutuação, afundamento. ELETRICIDADE 218 Circuitos simples e ramificados; estruturas topológicas e geométricas; modelos de fluxo de corrente; corrente, voltagem e resistência, eletromagnetismo; perigo da eletricidade. PARTÍCULAS 119 Estruturas da matéria; explicações de fenômenos (por ex. calor, estados da matéria); concepções de átomo; radioatividade. ÓTICA 111 Luz; propagação da luz; visão; cor. ENERGIA 103 Transformação de energia; conservação; degradação. CALOR 100 Calor e temperatura; transferência de calor; dilatação; aquecimento; mudança de estado; ebulição; solidificação; explicação dos fenômenos do calor no modelo de partículas. ASTRONOMIA 59 Forma da Terra; concepções do universo; características da atração gravitacional; satélites. FÍSICA “MODERNA” 35 Física quântica; relatividade especial. BIOLOGIA 336 Nutrição vegetal; fotossíntese; osmose; vida, origem da vida; evolução; sistema circulatório humano; genética; saúde; crescimento. QUÍMICA 225 Combustão, oxidação, reações químicas, transformações de substâncias, equilíbrio químico; símbolos, fórmulas, conceito de mol, eletroquímica. Quadro 1: Número de estudos sobre concepções de estudantes em diferentes áreas da Ciência (Pfundt & Duit, 1994, p. Alguns estudos sobre concepções espontâneas e psicogênese de conceitos realizados no Brasil xxvi) Quadro 1: Número de estudos sobre concepções de estudantes em diferentes áreas da Ciência (Pfundt & Duit, 1994, p. xxvi) Quadro 1: Número de estudos sobre concepções de estudantes em diferentes áreas da Ciência (Pfundt & Duit, 1994, p. xxvi) Esses estudos, levantados até 1994, eram em maior número na área de Física, conforme as porcentagens mostradas no gráfico da figura 9. 20% 14% 66% QUÍMICA BIOLOGIA FÍSICA Figura 9: Porcentagens de estudos sobre concepções dos estudantes em Biologia, Química e Física (Pfundt & Duit, 1994, p. xxvii). 125 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 Figura 9: Porcentagens de estudos sobre concepções dos estudantes em Biologia, Química e Física (Pfundt & Duit, 1994, p. xxvii). Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 125 O modelo de “mudança conceitual” e os estudos decorrentes dessa proposta O modelo de “mudança conceitual” e os estudos decorrentes dessa proposta O modelo de “mudança conceitual” e os estudos decorrentes dessa proposta Os dados obtidos em estudos como os acima descritos levaram os investigadores a estudá-los, não só no intuito de aplicá-los a situações de ensino, mas também à procura de referenciais teóricos em que se respaldar. As investigações conduzidas por Nussbaum e Sharoni-Dagan (1983), Driver e Erickson (1983), Hewson e Hewson (1984) foram alguns dos trabalhos que objetivaram, assim, encontrar bases empíricas para as constatações decorrentes das estruturas conceituais dos alunos, detectadas a partir do grande volume de investigações produzidas, conforme mostra Pfundt e Duit no levantamento acima citado. Assim, na espera de encontrar modelos que propiciassem condições necessárias para que o aluno, a partir de suas “concepções espontâneas”, pudesse rejeitá-las em favor das “concepções cientificamente aceitas” pelo ensino formal, os investigadores passam a desenvolver propostas dos chamados “modelos de mudança conceitual”. O primeiro modelo nesse sentido, e que se constituiu em marco importante para a investigação em Educação em Ciências foi o proposto Posner e colaboradores (1982) - chamado de modelo PSHG em função dos nomes dos autores: Posner, Strike, Hewson e Gertzog. Estas investigações, embasadas em estudos de filósofos da ciência como Kuhn (1970), Lakatos (1970) e Toulmin (1972), assumiam a aprendizagem como uma atividade racional, fundamentalmente voltada para a compreensão e aceitação de idéias que pareçam ser inteligíveis e racionais (Teodoro, 2000). Procuravam responder como as concepções espontâneas iniciais dos estudantes seriam transformadas em concepções científicas, a partir da apresentação de novas ideais e evidências. Esses autores partiram da idéia que existem exemplos análogos de mudança conceitual na Ciência e no ensino de Ciências. Ao utilizar conceitos já conhecidos para analisar um novo fenômeno, os alunos estariam usando o que autores chamaram de assimilação. Na maioria das vezes, entretanto, as concepções dos estudantes mostraram-se 126 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 126 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 126 inadequadas para permitir a compreensão de um novo fenômeno de maneira satisfatória; neste caso, o sujeito necessita de substituir ou reorganizar seus conceitos centrais. Neste caso, os autores consideram a ocorrência de uma forma mais radical de mudança, chamada de acomodação, importante no trabalho dos autores. Segundo Teodoro (2000), os trabalhos de Posner e colaboradores expressam a teoria de acomodação em resposta à duas questões: Sob quais condições um conceito central acaba sendo substituído por outro? 5 Segundo Teodoro (2000), os autores utilizam a metáfora da ecologia conceitual de Toulmim (1972) para explicar as concepções, crenças e valores dos estudantes. Nesta perspectiva, os conceitos estão estruturados em uma rede inter-relacionada de tal forma que a mudança em um conceito afeta os demais. Outro aspecto importante é que como os conceitos ocupam “nichos” conceituais distintos, existe a possibilidade de uma competição entre eles. 127 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 O modelo de “mudança conceitual” e os estudos decorrentes dessa proposta E ainda: Quais aspectos da ecologia conceitual5 (ou seja, das concepções que os indivíduos possuem) governam a seleção de novos conceitos? Nesse modelo proposto, para que ocorra a acomodação, quatro condições são necessárias: Em primeiro lugar, deve haver insatisfação com as concepções existentes, já que é improvável que cientistas e estudantes realizem uma mudança profunda em suas concepções sem que tenham perdido a fé em sua capacidade de resolução de problemas. Depois, essa nova concepção deve ser inteligível, ou seja, deve ser compreensível em sua estrutura cognitiva. Num próximo passo, a nova concepção deve parecer inicialmente plausível. Qualquer nova concepção adotada deve ao menos parecer ter capacidade para resolver os problemas gerados por suas predecessoras, caso contrário não parecerá uma escolha plausível. E, finalmente, a nova concepção deve sugerir a possibilidade de um frutífero programa de investigação, ou seja, deve ser útil, resolver problemas e abrir novas perspectivas. Mellado e Carracedo (1993; apud. Cunha, 1999) mostra, conforme o Quadro 2 , a influência de filósofos como Kuhn, Toulmin e Bachelard nas propostas construtivistas surgidas no período. 127 REVOLUCIONISMO (Kuhn) EVOLUCIONISMO (Toulmin) TRADIÇÕES DE INVESTIGAÇÃO (Laudan) PROGRAMAS DE PESQUISA (Lakatos) FALSEACIONISMO (Popper) O conhecimento se constrói. É importante o conhecimento anterior. Construtivismo: O estudante constrói de forma ativa seu próprio conhecimento partindo do conhecimento anterior que tem para ele coerência e utilidade. As teorias científicas são entes complexos que não podem ser rejeitados por falsificação. Não existem experimentos cruciais. Uma teoria científica se rejeita por um experimento crucial que a contradiz. A mudança de paradigma científico se produz em momentos de crise de forma total e revolucionária As teorias científicas evoluem gradualmente por pressão seletiva. Coexistem conceitos das velhas teorias e das novas A mudança de tradição se produz quando existe mudança ontológica, metodológica e de teorias. O progresso se produz por competência entre programas. Os programas de investigação têm núcleos centrais resistentes à mudanças. Os estudantes formulam hipóteses que os levam a conclusões provisórias A mudança conceitual dos alunos se produz provocando conflitos. As teorias dos estudantes são resistentes à mudanças. A mudança conceitual se produz nos alunos quando tem insatisfações com as teorias prévias e as novas lhes resultam inteligíveis, plausíveis e úteis. Aprendizagem de ciências como mudança conceitual, atitudinal e metodológica. A mudança conceitual dos estudantes é gradual. Incorporam idéias novas e mantém algumas das anteriores. O modelo de “mudança conceitual” e os estudos decorrentes dessa proposta A mudança conceitual dos alunos se produz como substituição em certos momentos de crise. REVOLUCIONISMO (Kuhn) EVOLUCIONISMO (Toulmin) TRADIÇÕES DE INVESTIGAÇÃO (Laudan) PROGRAMAS DE PESQUISA (Lakatos) FALSEACIONISMO (Popper) O h i t t ói É i t t h i t t i REVOLUCIONISMO (Kuhn) EVOLUCIONISMO (Toulmin) TRADIÇÕES DE INVESTIGAÇÃO (Laudan) PROGRAMAS DE PESQUISA (Lakatos) FALSEACIONISMO (Popper) O conhecimento se constrói. É importante o conhecimento anterior. Construtivismo: O estudante constrói de forma ativa seu próprio conhecimento partindo do conhecimento anterior que tem para ele coerência e utilidade. As teorias científicas são entes complexos que não podem ser rejeitados por falsificação. Não existem experimentos cruciais. Uma teoria científica se rejeita por um experimento crucial que a contradiz. A mudança de paradigma científico se produz em momentos de crise de forma total e revolucionária As teorias científicas evoluem gradualmente por pressão seletiva. Coexistem conceitos das velhas teorias e das novas A mudança de tradição se produz quando existe mudança ontológica, metodológica e de teorias. O progresso se produz por competência entre programas. Os programas de investigação têm núcleos centrais resistentes à mudanças. Os estudantes formulam hipóteses que os levam a conclusões provisórias A mudança conceitual dos alunos se produz provocando conflitos. As teorias dos estudantes são resistentes à mudanças. A mudança conceitual se produz nos alunos quando tem insatisfações com as teorias prévias e as novas lhes resultam inteligíveis, plausíveis e úteis. Aprendizagem de ciências como mudança conceitual, atitudinal e metodológica. A mudança conceitual dos estudantes é gradual. Incorporam idéias novas e mantém algumas das anteriores. A mudança conceitual dos alunos se produz como substituição em certos momentos de crise. adro 2: Analogias entre as Escolas Construtivistas e a Aprendizagem de Ciências. (Mellado & Carracedo, 1993; apud. Cunha, 1999). 128 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 O conhecimento se constrói. É importante o conhecimento anterior. Quadro 2: Analogias entre as Escolas Construtivistas e a Aprendizagem de Ciências. (Mellado & Carracedo, 1993; apud. Cunha, 1999). 128 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 As críticas ao modelo de Posner e colaboradores foram importantes para a evolução das discussões que se seguiram. A crítica de Hashweh (1986), por exemplo, considerou o modelo proposto por Posner et. al. O modelo de “mudança conceitual” e os estudos decorrentes dessa proposta coerente, mas assinalou que ele falhava em não enfatizar adequadamente as distinções entre os processos psicológicos que conduziam à estabilidade, aqueles que conduziam à mudança e as distinções entre estes processos psicológicos e as condições externas relacionadas a eles. Para este autor, o problema da explicação da mudança conceitual resumia-se na identificação dos fatores que: afetam a persistência das pré-concepções; afetam a aquisição de novas concepções e, que afetam a reestruturação cognitiva. Outros autores buscaram definir e explicar a mudança. O quadro abaixo ilustra alguns modelos de aprendizagem conceitual, procurando relacionar os termos utilizados em cada um dos estudos. MODELOS DE APRENDIZAGEM CONCEITUAL AQUISIÇÃO CONCEITUAL MUDANÇA CONCEITUAL ESTUDOS Memorização por rotina Captura conceitual Troca conceitual Hewson (1991) Crescimento Afinação Resolução conceitual Rumelhard & Norman (1981) ____ Mudança evolucionária Mudança revolucionária West (1982) ____ Assimilação Acomodação PSHG (1982) Strike & Posner (1982;1986) Desenvolvimento conceitual Resolução conceitual Troca conceitual Pines & West (1986) Acumulação Adaptação Reestruturação Tiberghien (1988) Quadro 3: Designações atribuídas aos modelos de aprendizagem conceitual: sua correspondência. (Santos, 1991, p.181). Quadro 3: Designações atribuídas aos modelos de aprendizagem conceitual: sua correspondência. (Santos, 1991, p.181). Quadro 3: Designações atribuídas aos modelos de aprendizagem conceitual: sua correspondência. (Santos, 1991, p.181). Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 129 Várias críticas foram feitas a esse modelo. Uma das perguntas mais freqüentes procurou esclarecer se o abandono das noções alternativas era realmente possível. As investigações posteriores passaram a apontar que a aquisição de um conceito científico não é necessariamente acompanhada da eliminação de antigas concepções. Solomon (1983), por sua vez, argumentou que as noções cotidianas que os estudantes sustentam tem origem no convívio social. No discurso diário e através dos meios de comunicação de massa, nossas crianças são confrontadas com suposições implícitas sobre como os objetos se movem, sua energia e suas propriedades, que podem estar em conflito direto com a explicação científica que aprendem na escola. Fora do laboratório escolar, esses adolescentes estão sendo continuamente socializados em um repertório completo de explicações não científicas. Um exame de reportagens de jornal e da linguagem cotidiana torna clara a disseminação deste processo subversivo. (Solomon, 1983: 49). Segundo a autora, não haveria meios para extinguir as noções cotidianas, assinalando que os estudantes deveriam ser capazes de pensar e operar em dois diferentes domínios de conhecimento6 e distinguir entre eles. 6 Nesta perspectiva, as noções alternativas e modelos científicos seriam coexistentes. Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 O modelo de “mudança conceitual” e os estudos decorrentes dessa proposta Os trabalhos posteriores, de Hewson e Hewson (1992) levaram em consideração os graus de mudança conceitual conforme três variedades: no primeiro tipo, o entendimento prévio do sujeito é extinto e substituído por uma nova concepção; no segundo, a mudança envolve a aprendizagem de conceitos novos que deverão estabelecer conexões com o que o aluno já sabe. É a “captura conceitual”; na terceira variedade, uma concepção é escolhida. Os autores utilizaram para ilustrar essa situação, a analogia da eleição para um cargo político na qual, entre dois candidatos, um torna-se prefeito, mas ambos continuam a viver na cidade. Segundos esses autores, muitas mudanças na visão dos estudantes assemelham-se ao último tipo, em que os indivíduos lembram continuamente da concepção mais fácil. 130 Por sua vez, Chi (1991) passa a discutir a possibilidade da coexistência de dois sentidos para o mesmo conceito, os quais devem ser utilizados em contextos apropriados. Mortimer (1994) destaca que alguns autores têm tentado demonstrar a dificuldade por parte dos estudantes em abandonarem suas noções cotidianas. O trabalho de Galili & Bar (1992), por exemplo, mostra que os mesmos estudantes que tiveram um bom desempenho em problemas familiares sobre força e movimento revertem a um raciocínio pré-newtoniano de “movimento requer força” em questões não familiares. Os autores concluem que “essa ‘regressão’ a visões ingênuas pelos mesmos sujeitos é uma evidência a mais de que o processo de substituição de crenças ingênuas por novos conhecimentos adquiridos nas aulas de Física é complicado e muitas vezes inconsistente .” (Galili & Bar, 1992, p. 78, apud. Mortimer, 1994, p.64). De maneira semelhante, Scott (1987), ao estudar o desenvolvimento de idéias sobre matéria entre alunos da escola secundária, conclui que ‘mudança conceitual’ não parece um título muito apropriado para o que se observa no processo. “No lugar de mudança conceitual parece haver um desenvolvimento paralelo de idéias sobre partículas e das idéias já existentes (...) O desenvolvimento paralelo de idéias resulta em explicações alternativas que podem ser empregadas no momento e situação apropriados. Não há mudança conceitual do tipo referido por Posner et. al. (1992) como uma acomodação” (Scott, 1987, p. 417; apud Mortimer, 1994, p.64). O modelo de “mudança conceitual” e os estudos decorrentes dessa proposta A partir da noção de perfil epistemológico de Bachelard, Mortimer sugeriu que a noção de “perfil conceitual” fornecia elementos para o entendimento da permanência das idéias prévias entre indivíduos que passaram pelo ensino formal, deslocando a expectativa em relação ao destino de tais noções, já que se admite que não serão abandonadas. Esta noção procurou descrever a diversidade de um conceito quando utilizado em circunstâncias particulares. O autor considerou que a noção de perfil conceitual poderia ser definida como [...] um sistema supra-individual de formas de pensamento que pode ser atribuído a qualquer indivíduo dentro de uma mesma cultura. Apesar de cada indivíduo possuir um perfil diferente, as categorias pelas quais ele é traçado são as mesmas para cada conceito. A noção de perfil conceitual é, portanto, dependente do contexto, uma vez que é fortemente influenciado pelas 131 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 131 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 131 experiências distintas de cada indivíduo; e dependente do conteúdo, já que para cada conceito em particular tem-se um perfil diferente. (Mortimer, 1994, p. 70). experiências distintas de cada indivíduo; e dependente do conteúdo, já que para cada conceito em particular tem-se um perfil diferente. (Mortimer, 1994, p. 70). Salientava ainda que não se deveria interpretar a ausência de mudanças radicais como um fracasso, pois as noções cotidianas sempre integrarão o perfil conceitual do indivíduo. Cunha (1999), reforça esta idéia ao assinalar que a Cunha (1999), reforça esta idéia ao assinalar que a Mudança conceitual raramente envolve um abandono completo de uma noção a favor de uma outra. Do contrário, com freqüência envolve adição de novas noções, retenção de noções existentes e aquisição de um sentido do contexto no qual a nova noção é mais apropriada. (Cunha, 1999: p.87). Mas as críticas ao modelo de mudança conceitual proposto por Posner e colaboradores, não atingiram apenas a possibilidade de total abandono das noções alternativas. Outros trabalhos discutiram o excesso de racionalidade que permeava o processo. Por conta dessas críticas, Posner e Strike (1992) reavaliaram a proposta inicial, buscando articular de forma mais coerente o modelo. 132 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 O modelo de “mudança conceitual” e os estudos decorrentes dessa proposta Neste sentido, algumas modificações foram introduzidas, que foram assim justificadas: a) uma enorme gama de fatores devem ser considerados na tentativa de descrever a ecologia conceitual dos estudantes; motivos e objetivos e suas fontes institucionais e sociais devem ser consideradas (concepções científicas e alternativas são partes da ecologia conceitual dos estudantes; assim, elas devem interagir com outros componentes); b) concepções e concepções errôneas podem existir em diferentes modelos de representação e diferentes níveis de articulação; c) uma visão progressiva e interacionista da ecologia conceitual é necessária. Os autores destacaram aí que a tentativa de descrição da mudança conceitual não possuía relações lineares e implicações diretas para o processo de ensino e este não era o objetivo do trabalho desenvolvido em 1982. Além disso, assinalaram que em nenhum 132 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 132 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 132 momento afirmaram que todos os tipos de mudança conceitual ocorrem como uma acomodação. As críticas ao modelo de mudança conceitual proposto por Posner e colaboradores (1982), segundo Pintrich, Marx e Boyle (1993), basearam-se nos seguintes aspectos: 1) as concepções espontâneas dos estudantes desempenhavam um papel paradoxal no processo de mudança; de um lado, podiam impedir que ela ocorresse e, ao mesmo tempo, formariam uma base a partir da qual o indivíduo julgaria a validade da nova informação; 2) a metáfora da ecologia conceitual apresentava várias limitações; 3) as quatro condições para que a mudança ocorresse não levavam em consideração os aspectos motivacionais; 4) questionavam a validade de se admitir o aluno como um cientista, já que este último está inserido em uma comunidade que o impele a produzir algo a partir de certos parâmetros, como problemas, métodos, normas e valores que não são compartilhados por um grupo de estudantes. A proposta de Pintrich et. al. (1993) visava ampliar o campo da mudança conceitual, inserindo aspectos do domínio sobre fatores motivacionais. Os autores destacaram que os [...] modelos cognitivos são relevantes e úteis para conceitualizar a aprendizagem dos estudantes, mas sua crença em um modelo acadêmico de ensino frio e puramente cognitivo [...] pode não ser adequado para descrever o ensino no contexto de sala de aula. (Pintrich et. al., 1993. O modelo de “mudança conceitual” e os estudos decorrentes dessa proposta p.167) [...] modelos cognitivos são relevantes e úteis para conceitualizar a aprendizagem dos estudantes, mas sua crença em um modelo acadêmico de ensino frio e puramente cognitivo [...] pode não ser adequado para descrever o ensino no contexto de sala de aula. (Pintrich et. al., 1993. p.167) As investigações passam a sugerir que o processo de mudança conceitual não poderia ser descrito como puramente vinculado aos aspectos lógicos e cognitivos. Se de um lado, nas comunidades de ciências naturais o conteúdo das teorias aceitas é freqüentemente determinado por fatores lógicos e empíricos, a mudança conceitual individual em um contexto de sala de aula não operaria da mesma forma. (Pintrich. et. al., 1993, p. 170). Os autores sugerem, assim, um modelo mais subjetivo (hot) de mudança conceitual individual, a partir de uma posição construtivista de que tal ação seria influenciada por processos pessoais, motivacionais, sociais e históricos. Os fatores subjetivos e a 133 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 133 motivação são aí encarados como forma de criar um clima de comprometimento do aluno com o trabalho escolar e o conhecimento. Fatores contextuais de sala de aula Fatores motivacionais Fatores cognitivos Condições para a mudança conceitual Estruturas da tarefa Autêntica Desafiadora Estruturas de autoridade Escolha mais favorável Desafio mais favorável Estruturas de avaliação Baseada no crescimento Erro encarado de maneira positiva Gerenciamento da sala de aula Uso do tempo Normas de engajamento Modelo do professor Pensamento científico Disposições científicas Suporte do professor Cognição Motivação Objetivos principais Crenças epistêmicas Interesse pessoal Valor de utilidade Importância Auto-eficácia Crenças de controle Atenção seletiva Ativação do conhecimento prévio Processamento profundo Elaboração Organização Resultado e solução do problema Avaliação metacognitiva e controle Controle eletivo e regulação Insatisfação Inteligibilidade Plausibilidade Caráter frutífero Quadro 4: Fatores contextuais de sala de aula, motivacionais e cognitivos relacionados ao processo de mudança conceitual. (Pintrich et. al., 1993, p.175). Quadro 4: Fatores contextuais de sala de aula, motivacionais e cognitivos relacionados ao processo de mudança conceitual. (Pintrich et. al., 1993, p.175). Quadro 4: Fatores contextuais de sala de aula, motivacionais e cognitivos relacionados ao processo de mudança conceitual. (Pintrich et. al., 1993, p.175). O Quadro 4 apresentado acima, fornece uma visão geral da análise e revela a proposta do modelo sugerido pelos autores. O modelo de “mudança conceitual” e os estudos decorrentes dessa proposta Os seguintes pontos foram evidenciados: a) as relações entre os fatores cognitivos, motivacionais e de sala de aula e as quatro condições para a mudança conceitual seriam interativas e dinâmicas; b) os processos cognitivos podem ser 134 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 134 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 134 influenciados por crenças motivacionais; c) As crenças motivacionais poderiam ser melhor definidas como situações ou contextos específicos, contrastando com as orientações anteriores em que a motivação era considerada um traço estável da personalidade. Pintrich e colaboradores sugeriram ainda algumas condições que deveriam ser respeitadas no trabalho em sala de aula: 1) as tarefas deveriam ser desafiadoras, significativas e autênticas no sentido de possuir alguma importância para a vida do indivíduo fora da escola; 2) o trabalho deveria ser estruturado de forma que o aluno pudesse fazer escolhas e controlar suas próprias atividades; 3) os procedimentos de avaliação deveriam valorizar outros aspectos que não fossem remetidos à competição, à comparação social ou à recompensas externas. Novas perspectivas passaram também a considerar a metacognição na discussão da mudança conceitual. Gunstone (1991), Gunstone e Northfield (1992) passam a defender que a mudança conceitual pudesse ser encarada em termos de reconhecimento, avaliação e reconstrução, ou seja, o indivíduo precisaria reconhecer a existência e a natureza de suas noções e deveria então decidir se tais concepções deveriam ser mantidas ou reconstruídas. Toda a argumentação dos autores baseou-se em três afirmações: cada um de nós, individualmente, constrói seu próprio significado a partir da experiência; isto implica que a compreensão é individual, e enquanto construções individuais, são diferentes (apesar de freqüentemente apresentarem pontos em comum); grande parte das construções que fazemos enquanto geramos nosso entendimento envolve relacionar novas idéias e experiências com aquilo que nós já sabemos e acreditamos. Gunstone, por sua vez, assinalou a importante complementaridade entre metagognição e construtivismo: Por metacognição eu quero dizer amalgama do conhecimento do estudante, consciência e controle, relevantes para sua aprendizagem. [...] uma aprendizagem metacognitiva apropriada é a que pode efetivamente assegurar um processo construtivista de reconhecimento, avaliação e, quando necessário, reconstrução das idéias existentes. (Gunstone, 1991, pp. 135 –136). 135 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 135 Rev. O modelo de “mudança conceitual” e os estudos decorrentes dessa proposta Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 135 A discussão de Dushl (1995) acerca da mudança conceitual, ao reconhecer no docente o papel de facilitador da aprendizagem, propôs três classes de informação acerca de como os indivíduos aprendem, constroem e desenvolvem o conhecimento científico e habilidades que poderiam servir como subsídios para a ação docente. Tais aspectos são mostrados no Quadro 5 abaixo. CAMPO PERGUNTA BÁSICA Conhecimento epistemológico/ científico Que conhecimentos, provas ou dados decidimos utilizar e com que objetivo o fazemos? Habilidades cognitivas/ pensamento Quais estratégias de raciocínio e de construção de significado analisamos e empregamos? Habilidades sociais/ de comunicação Quais são as ações que favorecem a obtenção de informação sobre o conhecimento científico, habilidades de pensamento e habilidades de comunicação? Quadro 5: Campos de informação (Duschl, 1995, p. 4). CAMPO Conhecimento epistemológico/ científico Habilidades cognitivas/ pensamento Habilidades sociais/ de comunicação Quadro 5: Campos de informação (Duschl, 1995, p. 4). Dushl e Gitomer (1991) e Dushl (1995) sugeriram que as essas informações poderiam ser utilizadas para o planejamento, a realização e avaliação de tarefas e atividades de ensino bem como para acompanhamento e avaliação da capacidade dos alunos para construir significados, adquirir e comunicar o conhecimento científico e para compreender a natureza da ciência. Dessa forma, as atividades permitiriam o aparecimento de idéias e explicações por parte dos alunos, a discussão e compreensão de tais idéias, bem como a avaliação e uma retroalimentação do processo. Os argumentos dos autores eram no sentido de que a chave para uma aprendizagem significativa estaria alicerçada na noção de informação útil. A informação útil não se obtém com tarefas de ensino que tenham todos os estudantes trabalhando para obter exatamente os mesmos resultados. A informação útil não se obtém a partir de atividades (tarefas escritas ou perguntas formuladas pelo professor) nas quais se pede aos estudantes que trabalhem com escolhas limitadas [...]. E o que é mais importante, a informação útil não é obtida a partir de atividades nas quais os alunos não compreendem os objetivos [...] 1 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 136 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 136 E ainda: [...] as tarefas de ensino se baseiam em soluções a problemas autênticos e com sentido. [...] quando se consegue que os estudantes participem na estruturação do problema e na produção de afirmações que descrevam sua percepção e seu significado. [...] quando se dá aos alunos a possibilidade de analisar e discutir as produções, afirmações e idéias de cada um deles sobre os processos de investigação.[...] quando a atividade na aula se estrutura a partir de práticas científicas empregadas na comunicação, argumentação e explicação de afirmações sobre conhecimento científico, de procedimentos metodológicos e objetivos de investigação. (Duschl, 1995, p.10). Para que essa mudança de postura ocorresse em sala de aula, Duschl (1995) propôs uma seqüência instrucional com cinco passos, denominada conversação avaliativa. O objetivo seria envolver estudantes e professores em um diálogo, numa seqüência de conflitos com o propósito de gerar acordos e desacordos. Um elemento chave dessa proposta estava nas chamadas conversações avaliativas que surgiriam a partir da análise e da discussão da informação útil produzida pelos estudantes (portifólio cultural). 1. Fazer com que os sujeitos ou grupos participem das tarefas que produzam uma diversidade ou gama de resultados. 2. Conduzir apresentações em público para dar uma idéia precisa da diversidade de esforços e significados. 3. Analisar e discutir as características de tal diversidade concentrando- se no objetivo. 4. Síntese em grupo: Empregar discussões para, na medida do possível, obter uma opinião consensual ou ao menos uma diminuição da diversidade original, fazendo uso dos critérios do objetivo da tarefa. 5. Aplicar o que foi aprendido em uma situação diferente. Analisar novamente uma tarefa já realizada ou empregá-la a outras novas. Quadro 6: Cinco passos em uma conversação avaliativa. (Duschl, 1995, p. 10). 1. Fazer com que os sujeitos ou grupos participem das tarefas que produzam uma diversidade ou gama de resultados. 2. Conduzir apresentações em público para dar uma idéia precisa da diversidade de esforços e significados. 3. Analisar e discutir as características de tal diversidade concentrando- se no objetivo. 4. Síntese em grupo: Empregar discussões para, na medida do possível, obter uma opinião consensual ou ao menos uma diminuição da diversidade original, fazendo uso dos critérios do objetivo da tarefa. 5. Aplicar o que foi aprendido em uma situação diferente. Analisar novamente uma tarefa já realizada ou empregá-la a outras novas. Quadro 6: Cinco passos em uma conversação avaliativa. (Duschl, 1995, p. 10). Rev. Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 E ainda: Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 137 portifólio cultural através de uma comparação com a visão tradicional de ensino. Cultura Tradicional de Ciência Cultura de Portifólio de Ciência Visão de Ciência Método científico estritamente hipotético- dedutivo. Epistemologia lógico-positivista Distinção sustentável (válida) entre observação e teoria. Método científico parcial Concepção epistemológica Distinção observação/teoria não persistente (sustentável). Papel do estudante Baixo “input” do estudante/imagem não ativa Significados científicos recebidos Baixo nível de reflexão Uso de estratégias desenvolvidas pelo estudante Alto “input” do estudante /imagem ativa Significados científicos negociados Alto grau de reflexão Uso de conhecimento estratégico /fundamentado Papel do Professor Disseminador de Conhecimento científico Não participante na construção do conhecimento científico Rigorosa aderência ao currículo prescrito Artífice do conhecimento científico participante na construção do conhecimento sobre ciência Modifica e adapta o currículo prescrito Objetivos do Currículo Conhecimento científico: o que sabemos Conhecimento sobre ciência: como e porque nós sabemos Enfatiza explicações inteiramente prontas Enfatiza o crescimento do conhecimento e o desenvolvimento da explicação Amplitude do conhecimento Profundidade do conhecimento Conhecimento científico básico Conhecimento científico contextualizado Unidades curriculares discretas (distintas) Unidades curriculares relacionadas Quadro 7: Contrastando entre a visão tradicional de ensino e a proposta de portifólio cultural. (Duschl Gitomer, 1991: 849). ortifólio cultural através de uma comparação com a visão tradicional de ensino. Cultura Tradicional de Ciência Cultura de Portifólio de Ciência Visão de Ciência Método científico estritamente hipotético- dedutivo. Epistemologia lógico-positivista Distinção sustentável (válida) entre observação e teoria. Método científico parcial Concepção epistemológica Distinção observação/teoria não persistente (sustentável). Papel do estudante Baixo “input” do estudante/imagem não ativa Significados científicos recebidos Baixo nível de reflexão Uso de estratégias desenvolvidas pelo estudante Alto “input” do estudante /imagem ativa Significados científicos negociados Alto grau de reflexão Uso de conhecimento estratégico /fundamentado Papel do Professor Disseminador de Conhecimento científico Não participante na construção do conhecimento científico Rigorosa aderência ao currículo prescrito Artífice do conhecimento científico participante na construção do conhecimento sobre ciência Modifica e adapta o currículo prescrito Objetivos do Currículo Conhecimento científico: o que sabemos Conhecimento sobre ciência: como e porque nós sabemos Enfatiza explicações inteiramente prontas Enfatiza o crescimento do conhecimento e o desenvolvimento da explicação Amplitude do conhecimento Profundidade do conhecimento Conhecimento científico básico Conhecimento científico contextualizado Unidades curriculares discretas (distintas) Unidades curriculares relacionadas Quadro 7: Contrastando entre a visão tradicional de ensino e a proposta de portifólio cultural. (Dusch Gitomer, 1991: 849). E ainda: Quadro 7: Contrastando entre a visão tradicional de ensino e a proposta de portifólio cultural. (Duschl e Gitomer, 1991: 849). Frente a tantos modelos e discussões, o desafio do professor no ensino de Ciências parece ser o de confrontar o indivíduo com suas concepções e conceitos que a produção científica oferece, propiciando momentos de reflexão e escolha. Silveira (1992 apud Peduzzi, 1998) salienta que um “(...) indicador de que a consolidação de uma nova teoria se deu no aluno é a sua capacidade de responder a situações problemáticas de ambas as formas, de acordo “(...) indicador de que a consolidação de uma nova teoria se deu no aluno é a sua capacidade de responder a situações problemáticas de ambas as formas, de acordo 138 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 138 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 138 com as concepções alternativas e de acordo com a nova teoria, verbalizando a consciência de que essas respostas estão assentadas sobre teorias diversas”. (Silveira, 1992. apud Peduzzi, 1998, p. 73). Ainda que o papel do ensino formal não seja o de alterar simplesmente as “teorias” dos sujeitos, traduzidas em suas concepções, pela teoria científica através de uma mudança radical, este deveria ser capaz de proporcionar aos indivíduos a compreensão consciente de ambas, permitindo a aprendizagem do conceito científico através de sua diferenciação das noções cotidianas. Isto revela a necessidade de que o docente seja preparado para atuar em uma nova perspectiva e adquira conhecimentos teóricos sobre a aprendizagem de Ciências. Assim, a promoção de discussões sistemáticas sobre os resultados de pesquisas referentes aos modelos de mudança conceitual e às concepções alternativas parece ser uma das atividades que facilitam a construção de uma concepção de ensino como mudança conceitual (Marion et. al. ,1999). Neste sentido, Hewson e t. al. (1999) argumentam que aceitar que os estudantes possuem concepções que necessitam ser mudadas nos processos de ensino e aprendizagem é uma coisa, porém totalmente diversa de se atribuir ao docente a responsabilidade de se engajar no ensino construtivista. Tal postura desconsidera o fato de que os docentes também possuem concepções prévias sobre os processos de ensino e aprendizagem, baseadas em suas experiências enquanto alunos e em outras experiências de ensino formal, incluindo os cursos de formação. E ainda: Dessa forma, “(...) os futuros docentes necessitarão passar por uma mudança conceitual com respeito às suas concepções sobre ensino, aprendizagem, ciência e/ou natureza do conhecimento”. (Hewson et. al., 1999, p. 254). “(...) os futuros docentes necessitarão passar por uma mudança conceitual com respeito às suas concepções sobre ensino, aprendizagem, ciência e/ou natureza do conhecimento”. (Hewson et. al., 1999, p. 254). A perspectiva construtivista exige competências que a formação inicial do docente não têm conseguido desenvolver, a começar pela aceitação de novas metodologias de ensino. A revisão acima mostra o surgimento do “modelo de mudança conceitual” proposto por Posner e colaboradores a partir da intensificação das investigações sobre concepções espontâneas surgidas a partir da década de 70, como algumas exemplificadas no caso brasileiro, bem como as conseqüentes discussões que se sucederam. Embora essa revisão tenha destacado importantes contribuições dos autores sobre a temática, é apenas um recorte dos autores, estando longe, portanto, de esgotar ou representar a imensa literatura, as diversas 139 Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 139 tendências que se constituíram a partir desses estudos e os resultados produzidos por estudos realizados com a finalidade de aplicar esses modelos e tendências em situações de ensino de sala de aula. Rev. Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 Considerações Finais A revisão acima pode mostrar, feitas as ressalvas anteriores, a importante contribuição que as investigações dentro desse “paradigma construtivista” trouxeram para a Educação em Ciências. Observa-se a complexidade do caminho que as discussões sobre “mudança conceitual” experimentaram ao procurar atender as novas demandas que se fizeram presentes quando se passou considerar e a contemplar vários outros fatores que intervém nos processos de ensino e aprendizagem. Essa contribuição também é mostrada em outras reflexões sobre o construtivismo como as de Matthews (2000, p. 501), que mostra que os estudos nesta linha têm auxiliado a educação em ciências ao “re-enfatizar a importância do conhecimento prévio para o processo de aprendizagem de novos conteúdos, ao realçar a importância da compreensão como objeto do ensino de Ciências, ao promover o comprometimento do aluno na aula e outros avanços.” Esses estudos parecem também ter levado os docentes à consciência para a “dimensão Essa contribuição também é mostrada em outras reflexões sobre o construtivismo como as de Matthews (2000, p. 501), que mostra que os estudos nesta linha têm auxiliado a educação em ciências ao “re-enfatizar a importância do conhecimento prévio para o processo de aprendizagem de novos conteúdos, ao realçar a importância da compreensão como objeto do ensino de Ciências, ao promover o comprometimento do aluno na aula e outros avanços.” Esses estudos parecem também ter levado os docentes à consciência para a “dimensão humana presente na Ciência, sua possibilidade de falha, sua conexão com a cultura e interesses, o lugar da convenção na teoria científica , a historicidade dos conceitos os procedimentos complexos da avaliação da teoria e muito mais”. Esses estudos parecem também ter levado os docentes à consciência para a “dimensão humana presente na Ciência, sua possibilidade de falha, sua conexão com a cultura e interesses, o lugar da convenção na teoria científica , a historicidade dos conceitos os procedimentos complexos da avaliação da teoria e muito mais”. Da mesma forma, Duit e Treagust (2003, p. 684), entendem “a necessidade de acabar com a distância entre a teoria e prática pelo menos até certo ponto” e o que a investigação em mudança conceitual não pode ser transposta diretamente para a sala de aula, mas, os programas de capacitação docente podem auxiliar na mudança as concepções sobre o ensino e a aprendizagem de professores e conseqüentemente suas práticas. Para tanto, é Rev. Considerações Finais Ensaio \| Belo Horizonte \| v.06 \| n.02 \| p.115-144 \| jul-dez \| 2004 14 Rev. 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https://openalex.org/W4312679019	https://ph.pollub.pl/index.php/acs/article/download/3355/2954	English	null	ANALYSIS OF THE USABILITY AND ACCESSIBILITY OF WEBSITES IN VIEW OF THEIR UNIVERSAL DESIGN PRINCIPLES	Applied Computer Science	2,022	cc-by	7,726	* Department of Computer Science, Lublin University of Technology, Lublin, Poland, blazej.badzio@pollub.edu.pl, agnieszka.bodziak@pollub.edu.pl, bartek.brodawka@gmail.com karol.buchajczuk@pollub.edu.pl, maria.paszkowska@pollub.pl, m.dzienkowski@pollub.pl, p.powroznik@pollub.pl Applied Computer Science, vol. 18, no. 3, pp. 63–85 doi: 10.35784/acs-2022-22 Submitted: 2022-06-14 \| Revised: 2022-09-05 \| Accepted: 2022-09-14 Applied Computer Science, vol. 18, no. 3, pp. 63–85 doi: 10.35784/acs-2022-22 Submitted: 2022-06-14 \| Revised: 2022-09-05 \| Accepted: 2022-09-14 Applied Computer Science, vol. 18, no. 3, pp. 63–85 doi: 10.35784/acs-2022-22 Keywords: accessibility, usability, universal design, eye tracking, WCAG Błażej BADZIO, Agnieszka BODZIAK, Bartłomiej BRODAWKA, Karol BUCHAJCZUK, Maria SKUBLEWSKA-PASZKOWSKA [0000-0002-0760-7 Mariusz DZIEŃKOWSKI [0000-0002-1932-297X], Paweł POWROŹNIK [0000-0002-5705-4785] * Department of Computer Science, Lublin University of Technology, Lublin, Poland, 1. INTRODUCTION Access to the Internet has become much more common nowadays – about 60% of the Earth's population has it (Digital Around the World, n.d.). News services are the main, and often the only source of news from the world, used by millions of people. Among the users, a significant percentage of them are people with disabilities, often with defects that prevent proper vision or hearing. This problem is marginalized and, as research shows, many websites do not take into account users with special needs, which excludes them from the social space (Leszczyńska, 2019). Therefore, in order to enable all users to have equal access to all available information, special solutions are implemented to make it easier for people to receive content published in the Internet. The main institution dedicated to spread awareness about web accessibility is the World Wide Web Consortium – W3C (W3C, 2022). This year it has come up with the Web Accessibility Initiative, or WAI (Initiative (WAI), W3C Web Accessibility, n.d.). In addition to educational activities, it is also involved in the creation of new guidelines, the development of evaluation tools and conducting research, the effect of which is to improve the accessibility of graphical user interfaces (GUIs). The field that deals with equality in access to products and the environment is universal design (UD). It assumes access for as many users as possible, who can use them independently (Centre for Excellence in Universal Design, n.d.). It should be noted, however, that facilitating access to content via the Internet for people with disabilities is only one of many aspects of universal design. Another, equally important, is to facilitate access to information for technically excluded people. The needs of these people require taking into consideration specific functionalities at the interface design level in order to comfortably use the Internet resources. One of the above-mentioned groups are people with visual impairment. Depending on the type of visual impairment, they require additional GUI functionalities, such as: the ability to magnify the text, listening to content, or entering it by voice. One of the key issues for the interface to be clear and easy to use is the way elements are arranged on the page. For their correct implementation, the WCAG guidelines (Web Content Accessibility Guidelines 2.1., n.d.) are used. It is a set of rules and recommendations aimed at guaranteeing solutions to the largest group of users. Abstract act Universal design is a strategic approach for planning and designing both the products and their environment, aimed at making a given product available to the widest number of possible users. It ensures equality for all of them and the opportunity to participate in the society. This concept is also crucial in the process of designing and developing software. The research was conducted with the use of four services, three of them were implemented for the purpose of this study. Two of them took into consideration the principles of universal design, while the others did not. The aim of the study was verification of the level of usability and accessibility of services by means of three independent methods: the LUT (Lublin University of Technology) checklist, an assessment taking into account WCAG 2.0 (Web Content Accessibility Guidelines) standards using the automatic WAVE evaluation tool (Web Accessibility Evaluation Tool) and a device allowing to track the movement of the eye while performing various tasks on websites. The websites were assessed by twenty experts in the field of creating web application interfaces, using the LUT checklist. The time to the first fixation (TTFF) that it took respondents to look at specific website elements was measured using the eye tracker device and iMotions software. All websites were checked by means of the WAVE tool to detect irregularities and non-compliance with universal design standards. The analysis performed clearly indicated that websites that follow the universal design guidelines were more useful, intuitive and accessible for users. It might be concluded that interfaces allow to find necessary information and perform desired actions in a shorter time when prepared in accordance with the principles of universal design. 63 2. LITERATURE REVIEW Accessibility for people with various disabilities, broadly understood, has been a frequently analyzed scientific issue recently. In the article (Stasiak & Dzieńkowski, 2021) university websites were examined in terms of the level of their accessibility for people with disabilities. The study consisted of two parts. The first one uses a 13-question checklist, while the second one uses five automatic tools (Lighthouse, ACE, MAUVE ++, FAE, Utilitia) to evaluate websites in terms of their compliance with the WCAG standard. On the basis of the obtained results, the authors concluded that the analyzed websites required additional functionalities, such as the possibility of changing the interface colours and implementing the mobile version of the website. The availability of library pages for people using screen readers was analyzed in (Yoon et al., 2016). Research was carried out, consisting of a survey part, in which participants reported accessibility problems, and a part in which automated testing tools were used. The results showed that the library sites are not accessible to users with visual impairments who use screen readers. The most frequently encountered accessibility barriers were due to problems with navigation and semantics, not to coding errors. The article (Fogli, Provenza & Bernareggi, 2014) presents the developed language of design patterns ensuring accessibility also for blind designers, the creation of which was preceded by a three-stage analysis: heuristic evaluation, a survey and an analysis of created websites. The paper (Pascual et al., 2014) analyzed the impact of barriers on the mood of website users depending on the accessibility of information contained on websites. The study took into account time of performed tasks, efficiency as a way to understand the usefulness of the website through the percentage of positively completed tasks and the final satisfaction of each task. The results showed that the WCAG 2.0 compliant site had better performance, effectiveness and user satisfaction scores. The availability of the Norwegian Broadcasting Corporation's news services to ensure equal access to information for different social groups was analyzed in (Sanderson, Chen & Kessel, 2015). The study consisted of interviews with participants, analysis of functionality, structure and navigation of websites. Based on the answers provided and the heuristic assessment, it was concluded that the websites that participated in the study did not meet the standards compliant with WCAG 2.0. 1. INTRODUCTION The aim of this paper was to build two websites, taking into consideration the WCAG guidelines, and then compare them with real equivalents existing in the Internet that do not meet the accessibility requirements. The principles of universal design were implemented during the development of both websites. In the case of the implementation of the former, the emphasis was placed on the placement of GUI elements, while in the case of the latter – on the contrast settings. An experiment was prepared for the analysis, in which three different research methods were used. The eye tracking technique was used in the first analysis, the WAVE validator in the second, and the LUT (Lublin University of Technology) checklist in the last one. The impact of the placement of elements on the website on the speed of their location by the user, the number of errors according to the WCAG 2.0 standard and the ergonomics of the developed user interfaces were examined. As part of the work, two research hypotheses were formulated: "an information service limited by access barriers for people with visual impairment is less perceived and more difficult to use than an accessible service" and "a service made in accordance with the principles and guidelines of universal design is more intuitive to use". 64 2. LITERATURE REVIEW The availability of websites of universities, corporations and federal institutions in the USA, with particular emphasis on people with visual impairments, has been investigated in (Michalska et al., 2014). The results of more than ten years of research presented there proved that the analyzed websites were unavailable in terms of consistency and transparency according to the WCAG guidelines. The main cause of these problems was the constant change of information on pages, the rotation of people responsible for the appearance of the sites and too fast maintenance of the system, not taking into consideration the issues of compliance with WCAG rules. A similar study was presented in (Harper & Chen, 2012), where over 6,000 websites over 10 years were analyzed in terms of the accessibility standards used. The results showed that only 10% of the websites surveyed follow the WCAG guidelines. The article (Pivetta et al., 2013) analyses a number of validation tools to verify the Moodle learning platform for compliance with WCAG 2.0 standards. The WAVE tool (WAVE Web Accessibility Evaluation Tool, n.d.) proved to be the most effective automatic validator. In some situations, an audit using one evaluation tool may turn out to be insufficient, as evidenced by the results of the study presented in (Kumar, Shree DV 65 & Biswa, 2021). The authors analyzed the availability of two news services using 10 of the most popular evaluation tools. This article (Acosta-Vargas, Acosta & Luján-Mora, 2018) analyzed 348 major Latin American university sites for WCAG 2.0 compliance errors using seven evaluation tools. The results showed that almost all of the analyzed websites were characterized by a significant number of errors, mainly concerning the contrast of elements on the page. The study (Ismail, Kuppusamy & Paiva, 2020) analyzed 59 websites of univer- sities in Portugal in terms of the accessibility of websites for people with disabilities. The analysis was performed with the use of three tools for website validation according to the W3C standard: WAVE, AChecker and aXe. Based on the collected statistical data, it was found that the main problems of the pages concerned the contrasts of elements on the pages, links without visible text, alternative texts for graphics and buttons. It should be noted that the analysis with automatic evaluation tools may often turn out to be insufficient. The book (Abascal, Arrue & Valencia, 2019) compared solutions based on automated scripts and manual crowdsourcing solutions. 2. LITERATURE REVIEW The results showed that manual analysis was necessary to adapt the website for people with disabilities. The graphical user interface design is one of the most important aspects when creating and implementing a website. The GUI must not only be readable, but also follow established conventions, such as the arrangement of individual elements on the page. Otherwise, it may be difficult to find the information one is interested in. A study (Alonso-Virgós et al., 2020) proved that there were "learned" behaviors, both for users and web developers, regarding page layout. A similar issue was addressed in (Isa et al., 2016), which analyzed the accessibility of the Malaysian tourism site using the Achecker evaluation tool, and developed a series of guidelines that made it possible to adapt the website and make it more accessible to people with disabilities. The research carried out in this paper is unique due to the lack of research on websites using eye tracker focusing on the elements responsible for the availability of websites for people with visual impairment, such as farsightedness, myopia or colour blindness. 3. PROTOTYPE TEST APPLICATIONS The WordPress tool (Narzędzie do blogowania, platforma wydawnicza i system zarządzania treścią witryny, n.d.) was used to create the first application, which allowed the maximum fulfilment of WCAG guidelines and thus managed to minimize the number of errors during testing with the WAVE tool. Wordpress made it easy to manage the content and modify elements directly in the code or with the use of appropriate plugins. The second application was created using three web technologies: HTML hypertext markup language, CSS stylesheet and PHP language. Such a choice was dictated by the desire to gain more control over each fragment of the website, which was important when examining the arrangement of elements. 3.1. Service for testing the contrast and size of elements In this case, the layout of the graphical user interface has the characteristic features of an Internet information service which contains the most current and most important data on the home page. It is displayed as the largest tile on the page (Fig. 1). Conversely, less important or older messages are displayed below as small tiles. The website also has archival entries, 66 a search by category and a tool for changing the size and type of font, as well as for changing the contrast of colours displayed on the page. It also allows the user to log in/create an account and add comments under articles, in the appropriate section. The user panel also offers the option of editing personal data, changing the password, profile photo and deleting the account on the website. Fig. 1. The main page of the service Fig. 1. The main page of the service Fig. 1. The main page of the service 3.2. Service for examining the arrangement of elements 3.2. Service for examining the arrangement of elements The second website has been designed in such a way that its elements responsible for the implementation of the accessibility aspects are arranged in a way resembling other, similar, most popular websites. For this version of the website, prepared in accordance with the principles of universal design (Fig. 2), a CSS file was produced, which ensured the correct arrangement of appropriate elements. Fig. 2. Main page of UD compatible service Fig. 2. Main page of UD compatible service 67 The version of the website inconsistent with the universal design guidelines for the arrangement of elements, created for the purpose of this study, is presented in Fig. 3. Fig. 3. Main page of UD incompatible service Fig. 3. Main page of UD incompatible service 4. RESEARCH METHODOLOGY Four experiments were conducted to investigate the quality and accessibility of the web application interface. Two of them involved the use of the eye tracker tool to examine the contrast and the placement of elements on the page. The next one was to use the WAVE validator to verify errors and irregularities according to the WCAG 2.0 standard. For the last study, the LUT (Lublin University of Technology) checklist (Miłosz, 2014) was used, by means of which a subjective assessment of the quality of the analyzed websites was made. Four websites were used for the study. Three developed for the purposes of the research. The first two (Fig. 1 and 2) were compatible with the UD guidelines, while the third one (Fig. 3) was not. The fourth website, available in the Internet (TVP Info, n.d.), also was not compatible with the UD guidelines. Twenty students of Computer Science with extensive experience in designing and implementing applications participated in the study. All of them took part in the eye tracking study. The participants were divided into two separate groups for assessment of the interfaces using the LUT checklist. They assessed the websites that were and were not compliant with the Universal Design guidelines. Ten of the participants had visual impairments: farsightedness, myopia and colour blindness. They all signed the consent for the study. 68 4.1. Assessment methods The first part of the experiment was performed using the Gazepoint GP3 HD eye tracker (GP3 HD Eye-Tracking Device – Take Advantage Of Research-Grade Equipment, n.d.), which uses a camera to monitor the activity of the eyeball (blinks, stops or fast movements) at a frequency of 150 Hz. The iMotions 9.0 (iMotions: Unpack Human Behavior, n.d.) software was used to do the research using the eye tracker. Each participant was shown charts with instructions and individual views of the tested visualizations. The task of the participants was to locate specific elements. The data obtained from the experiment were exported and a statistical analysis was conducted. The measures used in the study were: the number of fixations, fixation duration and the time to the first fixation. Each participant completed 10 tasks for each website. In the second part of the study, each participant, in order to get acquainted with the websites, received a list of eleven commands to be executed, the content of which was related to the interfaces of two sites with UD and without UD principles. The commands concerned the layout of the interface components and the GUI transparency. Based on their experience, after interacting with websites, they filled in the LUT checklist, using a rating scale from 1 to 5. This measure is based on the Nielsen heuristics. The WUP measure was calculated from the obtained results (Miłosz, 2014). To determine the availability of websites, the Internet tool "wave.webaim.org" was used to analyze the compliance of websites with the WCAG guidelines. Additionally, this tool allowed to verify the level of the website and set the direction of actions to improve the website / web application. The validator indicated errors that were often unnoticeable and could seriously affect the functioning of the website. The choice of the WAVE tool was preceded by thorough analyses of similar solutions, and was determined by such issues a high assessment of specialists, a wide spectrum of analysis (not only HTML and CSS validation), free license, an attractive way of presenting results, the verification of the correctness of the source code and contrast testing. The research was carried out by two independent experts in the field of universal design and computer science. 5. RESULTS The first part of the experiment involved conducting an eye tracking study. Each participant was displayed alternately a panel with the content of the task to be performed (Tab. 1) and a view of the website. The static analysis was performed with the use of libraries and a tool created in the R language. In order to verify whether the distribution of the samples is similar to the normal distribution, the Shapiro-Wilk test was performed. Additionally, the Levene's test and the Student's t-distribution were performed to see if the data were significantly different. 69 Tab. 1. Set of tasks to study contrast, size and placement of elements using an eye tracker Task no. Contrast Size of elements Element placement 1 Locate the item to search for. Locate the "Contact" tab. Locate the item that allows you to print the article. 2 Locate the comments section. Locate the article tags. Locate the item with user comments. 3 Locate the article tags. Locate the article category. Locate the login button item. 4 Locate the news service department/editorial section in the contact tab. Locate the source of the article. Locate the item with the currency rate widget. 5 Locate the article publication date field. Locate the "Culture" categories in the Category Selection section. Locate the item that allows to turn on the night mode. 6 Locate the source of the article. Locate the author of the article. Locate the item with a bar with scrolling article titles. 7 Locate the “Culture” categories in the Category Selection section. Locate the item to search for. Locate the item with the contact number. 8 Locate the “Poland” categories in the Category Selection section. Locate the "Poland" categories in the Category Selection section. Locate the marked location on the map. 9 Locate the author of the article. Locate the comments section. Locate the location of the registration button. 10 Locate the "World" categories in the Category Selection section. Locate the article published date field. Locate the location of the password field. 5.1. Eye tracking study – the Time to the First Fixation 5.1. Eye tracking study – the Time to the First Fixation The Time to the First Fixation (TTFF) determines the average time to localize a specific area of interest (AOI). This indicator provides information on how specific aspects of the visual scene are prioritized. This metric is useful for evaluating the performance of two or more areas on the same website, application interface, or for comparing similar GUI elements (iMotions: Unpack Human Behavior, n.d.). A short TTFF means that the searched element was found quickly and indicates its strong visibility through, for example, flashy colour, location in the center of the screen, a large size or visual differentiation from other objects. A longer TTFF may be due to the fact that a given element is not located in the typical place or it is only slightly visible. In Tab. 2. the TTFF was presented for contrast study, on pages with and without UD guidelines. 70 Tab. 2. TTFF for page contrast study UD website Time (ms) No-UD website Time (ms) Screen 1 556.8 3546.8 Screen 2 120.3 3657.0 Screen 3 591.3 5043.0 Screen 4 897.0 5692.0 Screen 5 627.5 3436.8 Screen 6 524.5 2828.1 Screen 7 919.2 3191.5 Screen 8 1150.3 4940.5 Screen 9 732.4 3259.4 Screen 10 1298.6 3690.1 Mean 741.79 3928.52 Variance 115107.325 899549.006 Standard deviation 339.274 948.445 Confidence intervals 210.281 587.841 Shapiro-Wilka test 0.869 0.090 Levene’s test 0.09296116 T-test 8.872 * 10-9 The next step was to examine the influence of the element size on two separate websites on the searching time for an element in the GUI. The results are presented in Tab. 3. The next step was to examine the influence of the element size on two separate websites on the searching time for an element in the GUI. The results are presented in Tab. 3. Tab. 3. TTFF for size of elements on page study UD website Time (ms) No-UD website Time (ms) Screen 1 780.6 4549.3 Screen 2 438.9 2357.1 Screen 3 877.1 5187.6 Screen 4 528.4 3948.8 Screen 5 838.6 3312.5 Screen 6 673.5 1601.1 Screen 7 651.4 3762.3 Screen 8 810.5 2693.2 Screen 9 549.9 3597.7 Screen 10 579.1 2631 Mean 672.8 3364.06 Variance 22190.375 1155253.394 Standard deviation 148.964 1074.827 Confidence intervals 92.327 666.172 Shapiro-Wilka test 0.635 0.992 Levene’s test 0.001565083 T-test 2.084 * 10-5 Tab. 3. TTFF for size of elements on page study 71 To analyze the influence of the arrangement of elements, tests were performed on two created services. 5.1. Eye tracking study – the Time to the First Fixation The results of these tests are presented in Tab. 4. To analyze the influence of the arrangement of elements, tests were performed on two created services. The results of these tests are presented in Tab. 4. Tab. 4. TTFF for placement of elements on page study UD website Time (ms) No-UD website Time (ms) Screen 1 2171.9 5021.3 Screen 2 655.1 1483.4 Screen 3 1541.1 6872.1 Screen 4 1196.8 2531.8 Screen 5 741.3 4488.1 Screen 6 789.7 4937.1 Screen 7 2236.4 5417.2 Screen 8 638.0 2693.3 Screen 9 1509.3 4819.7 Screen 10 512.6 4593.5 Mean 1199.22 4285.75 Standard deviation 640.7257 1592.372 Variance 410529.441 2535648.596 Confidence intervals 397.1186 986.9442 Shapiro-Wilk test 0.4365 0.1032 Levene’s test 0.1366 T-test 2.16·10-5 Tab. 4. TTFF for placement of elements on page study A significant time difference can be noticed in locating all test items in all views used in the experiment. The results of the statistical analysis show that the samples have a normal distribution, however, the samples in the size tests have a non-uniform variance, and the samples in the contrast tests have a homogeneous variance. This means that the analysed samples differ significantly between the websites with and without universal design guidelines. 5.2. Eye tracking study – the number of fixations Shapiro- Wilk 1 7.6 2.186 4.778 0.958 0.434 18.65 5.742 32.976 2.516 0.35 0.834 1.00210-9 2 5.2 2.261 5.115 0.991 0.144 17.7 6.821 46.536 2.989 0.216 0.207 2.23310-9 3 11.95 6.27 39.313 2.747 0.227 20.2 8.799 77.431 3.856 0.142 0.673 1.53210-3 4 6.6 1.569 2.463 0.687 0.218 19.2 10.149 103.01 4.448 0.17 0.738 2.87910-6 5 6.95 1.19 1.418 0.521 0.112 17.7 8.202 67.273 3.594 0.422 0.464 1.06810-6 6 6.3 2.273 5.168 0.996 0.115 16.95 4.999 24.997 2.191 0.176 0.575 1.53210-10 7 5 2.555 6.526 1.120 0.402 13.6 6.193 38.357 2.714 0.215 0.742 1.29110-6 8 5.5 1.504 2.263 0.659 0.153 17.25 7.202 51.881 3.156 0.081 0.938 1.58510-8 9 6.7 3.294 10.853 1.444 0.151 18.7 10.250 105.063 4.492 0.746 0.746 1.39310-5 10 5.1 1.889 3.568 0.827 0.368 15.9 8.528 72.726 3.737 0.078 0.202 2.51510-6 Scr. UD website No-UD website Levene’s test T-test Mean Std. dev. Variance Conf. int. Shapiro- Wilk Mean Std. dev. Variance Conf. int. Shapiro- Wilk 1 4.25 2.022 4.092 0.886 0.2932 13.75 6.773 45.881 2.968 0.1821 0.2873 5.51710-7 2 7.9 4.089 16.726 1.792 0.0861 16.4 8.556 73.200 3.750 0.1593 0.6547 2.75210-4 3 5.95 2.928 8.576 1.283 0.08333 13.65 6.149 37.818 2.695 0.1545 1 1.11510-5 4 11.1 8.668 75.147 3.799 0.0609 25.95 10.708 114.681 4.693 0.0650 0.7282 2.32410-5 5 7 4.267 18.210 1.870 0.3782 15.9 6.307 39.778 2.764 0.0991 0.4259 6.52510-6 6 5.85 2.996 8.976 1.313 0.3123 16.15 9.343 87.292 4.094 0.0512 0.5738 3.42810-5 7 6.75 3.275 10.724 1.435 0.5321 19.05 10.555 111.418 4.626 0.0763 0.3990 1.42510-5 8 6.7 3.840 14.747 1.683 0.3627 18.6 8.543 72.989 3.744 0.0512 0.4630 1.55610-6 9 5.95 2.724 7.418 1.194 0.2119 16.5 6.939 48.157 3.041 0.0552 0.7548 2.01610-7 10 7.95 4.236 17.945 1.857 0.2455 15.4 7.576 57.410 3.320 0.0558 0.9282 4.55110-4 Tab. 6. Fixation number for size of elements on the page study Scr. UD website No-UD website Levene’s test T-test Mean Std. dev. Variance Conf. int. Shapiro- Wilk Mean Std. dev. Variance Conf. int. 5.2. Eye tracking study – the number of fixations The number of fixations is correlated to the total dwell time (Holmqvist et al., 2011). The higher the total number of fixations, the lower the participant's search ability or the worse the structure of displayed stimuli. This means that if the informativeness of stimuli is high (in other words, the structure of stimuli supports the information retrieval process), the number of fixations decreases (Grobelny et al., 2006). Then, the analysis of the number of fixations for services in individual tasks was carried out. For the results, the mean, standard deviation, variance and confidence intervals were determined, and the Shapiro-Wilk, the Levene’s and the t-Student tests were performed. The results for the study of the contrast, size and distribution of elements are presented in Tabs. 5, 6 and 7, respectively. 72 Tab. 5. Fixation number for the page contrast study Tab. 5. Fixation number for the page contrast study Tab. 5. Fixation number for the page contrast study Scr. UD website No-UD website Levene’s test T-test Mean Std. dev. Variance Conf. int. Shapiro- Wilk Mean Std. dev. Variance Conf. int. Shapiro- Wilk 1 4.25 2.022 4.092 0.886 0.2932 13.75 6.773 45.881 2.968 0.1821 0.2873 5.51710-7 2 7.9 4.089 16.726 1.792 0.0861 16.4 8.556 73.200 3.750 0.1593 0.6547 2.75210-4 3 5.95 2.928 8.576 1.283 0.08333 13.65 6.149 37.818 2.695 0.1545 1 1.11510-5 4 11.1 8.668 75.147 3.799 0.0609 25.95 10.708 114.681 4.693 0.0650 0.7282 2.32410-5 5 7 4.267 18.210 1.870 0.3782 15.9 6.307 39.778 2.764 0.0991 0.4259 6.52510-6 6 5.85 2.996 8.976 1.313 0.3123 16.15 9.343 87.292 4.094 0.0512 0.5738 3.42810-5 7 6.75 3.275 10.724 1.435 0.5321 19.05 10.555 111.418 4.626 0.0763 0.3990 1.42510-5 8 6.7 3.840 14.747 1.683 0.3627 18.6 8.543 72.989 3.744 0.0512 0.4630 1.55610-6 9 5.95 2.724 7.418 1.194 0.2119 16.5 6.939 48.157 3.041 0.0552 0.7548 2.01610-7 10 7.95 4.236 17.945 1.857 0.2455 15.4 7.576 57.410 3.320 0.0558 0.9282 4.55110-4 Tab. 6. Fixation number for size of elements on the page study Scr. UD website No-UD website Levene’s test T-test Mean Std. dev. Variance Conf. int. Shapiro- Wilk Mean Std. dev. Variance Conf. int. 5.2. Eye tracking study – the number of fixations Shapiro- Wilk 1 7.6 2.186 4.778 0.958 0.434 18.65 5.742 32.976 2.516 0.35 0.834 1.00210-9 2 5.2 2.261 5.115 0.991 0.144 17.7 6.821 46.536 2.989 0.216 0.207 2.23310-9 3 11.95 6.27 39.313 2.747 0.227 20.2 8.799 77.431 3.856 0.142 0.673 1.53210-3 4 6.6 1.569 2.463 0.687 0.218 19.2 10.149 103.01 4.448 0.17 0.738 2.87910-6 5 6.95 1.19 1.418 0.521 0.112 17.7 8.202 67.273 3.594 0.422 0.464 1.06810-6 6 6.3 2.273 5.168 0.996 0.115 16.95 4.999 24.997 2.191 0.176 0.575 1.53210-10 7 5 2.555 6.526 1.120 0.402 13.6 6.193 38.357 2.714 0.215 0.742 1.29110-6 8 5.5 1.504 2.263 0.659 0.153 17.25 7.202 51.881 3.156 0.081 0.938 1.58510-8 9 6.7 3.294 10.853 1.444 0.151 18.7 10.250 105.063 4.492 0.746 0.746 1.39310-5 10 5.1 1.889 3.568 0.827 0.368 15.9 8.528 72.726 3.737 0.078 0.202 2.51510-6 Tab. 6. Fixation number for size of elements on the page study 73 73 Tab. 7. Fixation number for placement of elements on the page study Tab. 7. Fixation number for placement of elements on the page study Scr. UD website No-UD website Levene’s test T-test Mean Std. dev. Variance Conf. int. Shapiro- Wilk Mean Std. dev. Variance Conf. int. Shapiro- Wilk 1 8.4 4.453 19.831 1.952 0.1635 11.55 3.394 11.523 1.487 0.0756 0.7573 0.0162 2 7.45 3.605 12.997 1.580 0.5233 10.35 4.568 20.871 2.002 0.0868 0.0314 0.0318 3 6.95 3.845 14.786 1.685 0.4442 9.85 3.232 10.45 1.416 0.4293 0.2085 0.0138 4 7.2 4.456 19.853 1.953 0.1196 10.3 2.993 8.958 1.312 0.1174 0.6147 0.0138 5 7.15 4.171 17.397 1.828 0.0558 10 2.991 8.947 1.310 0.8289 0.4082 0.0175 6 10.5 3.886 15.105 1.703 0.8084 15.3 7.138 50.957 3.128 0.1645 0.4801 0.0119 7 7.05 3.790 14.365 1.661 0.1631 9.7 2.003 4.010 0.877 0.1167 0.1648 0.0088 8 6.6 3.604 12.989 1.580 0.0909 9.4 3.235 10.463 1.418 0.6025 0.7808 0.0137 9 6.05 3.236 10.471 1.418 0.9138 8.75 3.226 10.407 1.413 0.2718 0.5531 0.0119 10 6.9 3.697 13.673 1.621 0.1472 9.8 3.412 11.642 1.495 0.6052 0.6286 0.0139 Based on the above results, a statistical analysis was analogously performed using the same tools. The results show that the data are normally distributed and that they differ significantly. In order to better understand the results, the graphs shown below have been created. The graphs presented in Figs. 5.2. Eye tracking study – the number of fixations 4, 5, 6, 7, 8, 9 show the number of fixations with their statistical data (quarter range, median) depending on the view for studies on contrast, size and placement of elements on the website. Fig. 4. Number of fixations – element contrast on the website without UD Fig. 4. Number of fixations – element contrast on the website without UD 74 Fig. 5. Number of fixations – element contrast on the UD-enabled website Fig. 5. Number of fixations – element contrast on the UD-enabled website Fig. 6. Number of fixations – element size on the website without UD Fig. 6. Number of fixations – element size on the website without UD 75 75 Fig. 7. Number of fixations – element size on the UD-enabled website Fig. 7. Number of fixations – element size on the UD-enabled website Fig. 8. Number of fixations – element placement on the website without UD Fig. 8. Number of fixations – element placement on the website without UD 76 Fig. 9. Number of fixations – element placement on the UD-enabled website Fig. 9. Number of fixations – element placement on the UD-enabled website Fig. 9. Number of fixations – element placement on the UD-enabled website 5.3. Eye tracking study – fixation duration The last analysis of the results concerned the duration of the eye fixation during the execution of the command on individual views. The results of the research and analyses are presented in Tabs. 8, 9 and 10. The duration of fixation can be applied to both individuals and groups. This measure is interesting in the analysis of various stimuli, i.e., various websites or web applications. In the case of the conducted research, the entire page area was the area of interest, and the stimulus exposure time was related to the time of finding the searched object. The interpretation of this measure may be such that the longer the fixation time, the more time the participants spent on it, which means that the displayed scene was more complicated for the respondent. Therefore, this parameter indicates the difficulty or ease of extracting information (Just & Carpenter, 1976). Tab. 8. Fixation duration for size of elements on the page study Scr. UD website fixation duration (ms) No-UD website fixation duration (ms) Levene’s test Mean Std. dev. Variance Mean Std. dev. Variance 1 2227.028 1766.995 3122270.715 5449.899 1769.524 3131214.445 0.581 2 1626.917 723.749 523813.327 5127.623 1857.711 3451091.645 0.857 3 3604.962 2010.896 4043702.245 6079.612 2051.412 4208292.572 0.207 4 1797.686 792.573 628172.486 5421.189 2640.315 6971261.691 0.257 5 1973.372 801.254 642007.503 5095.739 2147.701 4612618.320 0.535 6 1660.673 766.029 586799.958 4886.513 2715.365 7373205.503 0.548 7 1506.419 623.700 389001.543 4950.125 1959.981 3841524.830 0.903 8 2073.643 856.457 733518.380 5756.947 3622.310 13121127.490 0.228 9 2099.837 1139.046 1297425.948 5455.836 2535.253 6427506.842 0.782 10 2029.934 1576.429 2485128.774 4597.603 2274.366 5172741.132 0.428 Tab. 8. Fixation duration for size of elements on the page study 77 Tab. 9. Fixation duration for the page contrast study Tab. 9. Fixation duration for the page contrast study Scr. UD website fixation duration (ms) No-UD website fixation duration (ms) Levene’s test Mean Std. dev. Variance Mean Std. dev. 5.3. Eye tracking study – fixation duration Variance 1 1452.918 659.319 434702.145 4512.119 1886.928 3560495.866 0.484 2 2533.579 1814.358 3291894.103 4978.360 2730.994 7458329.946 0.712 3 1855.812 1194.277 1426297.826 4593.170 2625.858 6895127.762 0.954 4 3199.884 2715.384 7373309.636 7532.596 4730.115 22373987.380 0.836 5 2422.547 1342.039 1801069.752 5195.723 3177.848 10098718.730 0.825 6 1653.871 819.697 671903.248 4800.330 2172.514 4719816.377 0.928 7 2225.216 1016.202 1032667.433 5446.231 2518.568 6343187.245 0.986 8 2260.856 1366.621 1867652.940 6212.158 4636.144 21493827.830 0.479 9 1929.815 676.483 457629.680 4162.068 2687.036 7220162.087 0.541 10 2517.727 1068.469 1141626.572 4202.766 1914.743 3666240.791 0.944 7 2225.216 1016.202 1032667.433 5446.231 2518.568 6343187.245 0.986 8 2260.856 1366.621 1867652.940 6212.158 4636.144 21493827.830 0.479 9 1929.815 676.483 457629.680 4162.068 2687.036 7220162.087 0.541 10 2517.727 1068.469 1141626.572 4202.766 1914.743 3666240.791 0.944 Tab. 10. Fixation duration for placement of elements on the page study Scr. UD website fixation duration (ms) No-UD website fixation duration (ms) Levene ‘s test Mean Std. dev. Variance Mean Std. dev. Variance 1 3363.627 1966.889 3868653.098 3777.575 1645.394 2707324.495 0.8842 2 3004.432 1809.038 3272619.664 3178.353 1843.847 3399775.061 0.5235 3 2017.958 901.001 811804.268 2987.958 2094.494 4386908.777 0.3271 4 2690.388 1685.690 2841553.226 3337.922 1853.905 3436965.436 0.0350 5 2629.681 1627.812 2649772.994 2866.858 1452.316 2109224.619 0.9331 6 3860.748 2556.886 6537671.03 4762.058 2570.120 6605519.337 0.8462 7 2958.867 2297.602 5278976.192 2626.115 1170.858 1370909.47 0.9674 8 2111.905 1126.478 1268953.877 2530.234 1295.602 1678585.633 0.3995 9 2208.277 1103.397 1217486.821 2465.513 1424.831 2030144.452 0.7891 10 2543.359 1304.938 1702863.719 2802.539 1502.594 2257791.467 0.6698 Fig. 10. Duration of fixations – element contrast on the website without UD Tab. 10. Fixation duration for placement of elements on the page study p p g y Scr. UD website fixation duration (ms) No-UD website fixation duration (ms) Levene ‘s test Mean Std. dev. Variance Mean Std. dev. Variance 1 3363.627 1966.889 3868653.098 3777.575 1645.394 2707324.495 0.8842 2 3004.432 1809.038 3272619.664 3178.353 1843.847 3399775.061 0.5235 3 2017.958 901.001 811804.268 2987.958 2094.494 4386908.777 0.3271 4 2690.388 1685.690 2841553.226 3337.922 1853.905 3436965.436 0.0350 5 2629.681 1627.812 2649772.994 2866.858 1452.316 2109224.619 0.9331 6 3860.748 2556.886 6537671.03 4762.058 2570.120 6605519.337 0.8462 7 2958.867 2297.602 5278976.192 2626.115 1170.858 1370909.47 0.9674 8 2111.905 1126.478 1268953.877 2530.234 1295.602 1678585.633 0.3995 9 2208.277 1103.397 1217486.821 2465.513 1424.831 2030144.452 0.7891 10 2543.359 1304.938 1702863.719 2802.539 1502.594 2257791.467 0.6698 Fig. 10. Duration of fixations – element contrast on the website without UD Fig. 10. Duration of fixations – element contrast on the website without UD 78 Fig. 11. Duration of fixations – element contrast on the UD-enabled website Fig. 11. 5.3. Eye tracking study – fixation duration Duration of fixations – element contrast on the UD-enabled website In the analysis of the duration, in addition to the standard measures such as mean, standard deviation and variance, the Levene test was also performed, on the basis of which it can be concluded that the samples have a homogeneous variance. Only one sample has a heterogeneous variance. Similarly, as in the case of the number of fixations, due to the large amount of data, the graphs presented in Figures 10, 11, 12, 13, 14, 15 were made. Fig. 12. Duration of fixations – element size on the UD-enabled website Fig. 12. Duration of fixations – element size on the UD-enabled website 79 Fig. 13. Duration of fixations – element size on the website without UD Fig. 14. Duration of fixations – element placement on the UD-enabled website Fig. 13. Duration of fixations – element size on the website without UD Fig. 13. Duration of fixations – element size on the website without UD Fig. 13. Duration of fixations – element size on the website without UD Fig. 14. Duration of fixations – element placement on the UD-enabled website Fig. 14. Duration of fixations – element placement on the UD-enabled website 80 80 Fig. 15. Duration of fixations – element placement on the website without UD Fig. 15. Duration of fixations – element placement on the website without UD In Levene's tests for all studies, the result indicated heterogeneity of variance (p value ≤0.05). 5.4. LUT checklist The results obtained using the LUT questionnaire were subjected to a statistical analysis, i.e. calculation of the mean, standard deviation, variance and the Levene's test. The results of the analysis are presented in Tab. 11. Tab. 11. LUT checklist results – contrast, size and placements of website elements Participant WUP score of the UD-enabled service with contrast and size of elements No-UD service with contrast and size of elements -WUP score UD-enabled service with placement of elements - WUP score No-UD service with placement of elements - WUP score 1 4.382 2.902 4.967 2.532 2 4.5 2.902 4.86 2.67 3 4.42 2.841 4.86 2.35 4 4.348 2.896 4.833 3.897 5 4.31 2.94 4.872 3.107 6 4.35 2.893 4.507 3.052 7 4.368 2.86 4.727 1.476 8 4.265 2.87 4.983 1.316 9 4.397 2.908 4.605 1.557 10 4.543 2.905 4.668 2.472 Mean 4.388 2.892 4.788 2.443 Std. dev. 0.083 0.0028 0.815 0.156 Variance 0.007 0.001 0.665 0.024 Levene’s test 0.045 0.0081 In Levene's tests for all studies the result indicated heterogeneity of variance (p valu Tab. 11. LUT checklist results – contrast, size and placements of website elements 81 81 5.5. WAVE – the validator for automatic evaluation of web interfaces The results of the study with the WAVE evaluation tool are graphically represented by pie charts in Figures 16 and 17. harts in Figures 16 and 17. Fig. 16. WAVE analysis results chart – contrast and size of website elements without UD Fig. 16. WAVE analysis results chart – contrast and size of website elements without UD Fig. 17. WAVE analysis results chart – contrast and size of website elements with UD Fig. 17. WAVE analysis results chart – contrast and size of website elements with UD 82 6. DISCUSSION AND CONCLUSIONS Based on the collected results of the statistical analysis, it was found that the results of the research proved the correctness of the hypotheses: "an information service limited by access barriers for people with visual impairment is worse perceived and more difficult to use than a fully accessible service" and "websites made in accordance with generally accepted principles and the universal design guidelines are more intuitive to use”. The average time to search for an element, the duration of the eye fixation and the number of fixations on the views of websites designed according to the universal design guidelines was significantly lower by 93%, 342% and 114%, respectively (Tabs. 3-10). This indicates that the user was able to locate the selected GUI element much faster, and thus the website was easier to use and more intuitive. The WUP indicator of the LUT checklist confirms that particular areas of websites with universal design are more useful than those that do not support these principles. In the sub- areas concerning layout and colour selection, the mean of the results was respectively higher by 80% and 137% for both websites created in accordance with universal design (Tab. 11). The smallest differences were in the areas related to entering data and forms, at the level of 34% and 72%, respectively. The participants indicate better intuitiveness, readability and accessibility of websites compliant with WCAG 2.0 standards. Websites commonly used show a higher number of structural errors and contrast with the use of the WAVE tool (3766% and 5350%, respectively). Moreover, the ratio of errors to guidelines on the created website supporting the principles of universal design is clearly lower than on the website that does not comply with the above principles (Figs. 16, 17). The conducted 3-element analysis confirmed that taking into consideration the principles of universal design while creating websites improves their intuitiveness, usability and accessibility. The application becomes available to a wide range of users, also to people with disabilities, e.g., with visual impairment. The results of the research are complied with the results obtained in the analyzed literature (Acosta-Vargas et al., 2018; Ismail et al., 2020; Pivetta et al., 2013). They clearly indicate the lack of correct implementation of universal design guidelines on many popular websites. Acknowledgments This study has been conducted as part of the “Lublin University of Technology – Universal Design” project, funded by the European Social Fund under the PO WER program. Grant Agreement No. POWR.03.05.00-00-PU32/19-00. The research programme titled “Usability and ergonomics of interfaces study”, was approved by the Commission for Research Ethics, No. 9/2019 dated. 27.05.2019. 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https://openalex.org/W2409697110	https://www.revistas.ufg.br/philosophos/article/download/37530/20508	Portuguese	null	FAZENDO DE CONTA QUE VULCANO NÃO EXISTE	Filósofos/Revista philósophos	2,016	cc-by	7,523	FAZENDO DE CONTA QUE VULCANO NÃO EXISTE 1 Sagid Salles (PPGLM)2 sagidsalles@gmail.com Resumo: Meu objetivo neste artigo é apresentar e analisar algumas versões de teorias do faz de conta para existenciais negativas. Vou rapidamente apre- sentar a perspectiva de Evans e, em maior detalhe, as perspectivas de Walton e Kroon. Sustentarei que as duas primeiras não fornecem um tratamento adequado do fenômeno das existenciais negativas singulares, e que a perspec- tiva de Kroon é melhor do que ambas. Contudo, argumentarei que todas as três têm um mesmo problema, que chamo de o problema da motivação semân- tica. Palavras-chave: existenciais negativas singulares; teorias do faz de conta; pro- blema da motivação semântica. 1 Recebido: 02-09-2015/Aceito: 25-02-2016/Publicado on-line: 06-03-2016. 2 2 Sagid Salles é Doutorando no Programa de Pós-Graduação Lógica e Metafísica da UFRJ (PPGLM), com sanduíche na Universidade de Miami, Rio de Janeiro, RJ, Brasil. INTRODUÇÃO Uma frase como “João não é calvo” é verdadeira se, e so- mente se, o objeto referido por “João” não é calvo. Frases como: ARTIGO ORIGINAL ARTIGO ORIGINAL ARTIGO ORIGINAL DOI: DOI: 1) Vulcano não existe. parecem ser exatamente do mesmo modo, uma frase da forma sujeito-predicado que é verdadeira quando o objeto referido pelo termo sujeito possui a propriedade expressada pelo predicado, e falsa de outro modo. Agora, suponha que PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 1 171 Sagid Salles 1 é verdadeira. Neste caso, ela descreve corretamente o ob- jeto referido por “Vulcano”. Assim, tem de existir um objeto referido pelo nome em questão, um objeto que pos- sa ser corretamente descrito pela frase. Este objeto, é claro, é Vulcano. Consequentemente, Vulcano existe. Se Vulcano existe, então 1 é falsa. A conclusão surpreendente é que: se 1 é verdadeira, então 1 é falsa. 1 é verdadeira. Neste caso, ela descreve corretamente o ob- jeto referido por “Vulcano”. Assim, tem de existir um objeto referido pelo nome em questão, um objeto que pos- sa ser corretamente descrito pela frase. Este objeto, é claro, é Vulcano. Consequentemente, Vulcano existe. Se Vulcano existe, então 1 é falsa. A conclusão surpreendente é que: se 1 é verdadeira, então 1 é falsa. O problema é que o mesmo tipo de inconsistência sur- girá sempre que tentarmos sustentar a verdade de frases como 1. Tais frases são chamadas de existenciais negativas sin- gulares. São frases existenciais que possuem um termo singular (isto é, nomes, demonstrativos, etc.) na posição de sujeito. O problema de explicar como elas podem ser ver- dadeiras é o problema das existenciais negativas singulares. Atualmente há duas estratégias gerais de solução para este problema, as teorias do objeto e as teorias não comprometi- das com objetos. Grosso modo, no primeiro caso tenta-se solucionar o problema postulando algum tipo novo de ob- jetos. Meinong (1904), por exemplo, aceita que 1 é verdadeira quando descreve o referente de “Vulcano” corre- tamente. Mas rejeita que disto possamos concluir que Vulcano existe. Na verdade, há objetos que possuem a pro- priedade de existir e objetos que não possuem esta propriedade. Vulcano é um objeto tão legítimo quanto qualquer outro, apenas acontece de ele não existir. Aliás, é justamente por isto que 1 é verdadeira. A teoria de Mei- nong é interessante e recebeu muitas sofisticações posteriores (por exemplo, Zalta (2000)), mas não nos ocu- paremos com ela aqui. Meu interesse é no segundo tipo de estratégia. 1) Vulcano não existe. O exemplo clássico de teorias não comprometidas com objetos é a teoria das descrições de Russell (1905) e sua aplica- PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 172 FAZENDO DE CONTA QUE VULCANO NÃO EXISTE ARTIGO ORIGINAL ção para os termos singulares. Neste artigo, contudo, minha preocupação é com outra versão das teorias não comprome- tidas. Nomeadamente, as teorias do faz de conta ou teorias da simulação, que têm em Kendall Walton (1990) a sua exposi- ção clássica, mas também foi defendida por Evans e Fred Kroon. Sustentarei que as versões de Walton e Evans não nos fornecem um tratamento adequado das existenciais ne- gativas singulares. Quanto à versão de Kroon, me parece ser mais promissora, mas ainda enfrenta alguns dos problemas das duas anteriores. O artigo será dividido em cinco partes. Na primeira apresento dois critérios de adequação para qualquer teoria das existenciais negativas singulares. Na segunda, apresento resumidamente a noção de faz de conta, alguns conceitos centrais, e explico rapidamente por que o tratamento de Evans não me parece adequado para frases como 1. Na ter- ceira, apresento o tratamento de Walton e um conjunto de objeções a ele. Na quarta, mostro como Kroon pode resol- ver alguns dos problemas apontados antes. Por fim, concluo que todas possuem um mesmo problema: carecer de moti- vação semântica. 1. DOIS CRITÉRIOS DE ADEQUAÇÃO Intuitivamente, frases como “Vulcano existe” ou “Vulcano não existe” têm a mesma estrutura que: 2) João é legal. e e PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 3) Eliz não é simpática. 3) Eliz não é simpática. 173 PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. Sagid Salles Em outras palavras, são frases da forma sujeito-predicado, contendo um termo singular na posição de sujeito e um predicado que expressa uma propriedade. Alguém que pro- fere 2, por exemplo, está usando o nome para se referir a um objeto e o predicado para atribuir uma propriedade a este objeto. Com isto, quero ressaltar dois aspectos impor- tantes. O primeiro é que o nome está sendo usado, e não ape- nas mencionado. Nomes são artifícios de referência singular, são usados com o intuito de se referir a um objeto ou sele- cionar um objeto determinado. Em 2 o nome “João” é usado para se referir a João, e em 3 o nome “Eliz” é usado para se referir a Eliz. Analogamente, quem profere 1 está usando o nome “Vulcano” para fazer referência a um obje- to determinado, e não apenas mencionando. Eu aceito, seguindo Evans (1982, cap. 10), Kripke (2011:58) e Kroon, que qualquer teoria correta das existenciais negativas deve respeitar esta intuição. Chamaremos isto de o critério do uso (KROON, 2000:98). Antes de entrar no segundo critério, é importante escla- recer uma coisa. Daqui por diante vou pressupor que termos singulares – incluindo aqueles que aparecem em existenciais negativas – são não apenas artifícios de referên- cia singular, mas também que sua única função semântica é introduzir um objeto no discurso. Em outras palavras, vou pressupor que alguma forma de teoria da referência direta está correta. Descritivistas e defensores de teorias da referência indireta discordam disto. Mas eles têm seus próprios meios de solucionar o problema das existenciais negativas singula- res. É para os defensores das teorias da referência direta que o problema surge de modo mais saliente. Além disto, as te- PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 174 FAZENDO DE CONTA QUE VULCANO NÃO EXISTE ARTIGO ORIGINAL orias do faz de conta que considerarei fornecem uma solu- ção ao problema consistente com a tese da referência direta. Isto é, elas nos fornecem um meio de explicar como exis- tenciais negativas singulares são verdadeiras sem, com isto, abandonar a crença de que a função dos nomes é mera- mente introduzir objetos no discurso. O segundo aspecto para o qual quero chamar a atenção diz respeito ao termo “existe”. 3) Eliz não é simpática. Se levarmos a sério a analo- gia intuitiva entre existenciais singulares e frases como 2 ou 3, então teremos de ver “existe” como expressando uma propriedade de primeira ordem. Assim como “simpático” expressa uma propriedade de objetos, “existe” expressa uma propriedade de objetos. A diferença, contudo, é que “exis- te” expressa uma propriedade que é possuída por todas as coisas (como sabemos, nem todo mundo é simpático, mas tudo existe!). Mais especificamente, a ideia seria que “exis- te”, tal como cotidianamente usado, é um predicado de primeira ordem. Isto é muitíssimo controverso, mas será as- sumido aqui, sem maiores razões, que qualquer teoria correta das existenciais negativas tem de satisfazer esta res- trição. Sigo Kroon novamente e chamo a isto de requisito de simplicidade.3 3 Em analogia ao fato de este ser o significado simples e cotidiano de “existe”. 2. TEORIAS DO FAZ DE CONTA: SIMULANDO REFERÊNCIA E PREDICAÇÃO Antes de ver como teorias do faz de conta podem ser usadas para resolver o problema das existenciais negativas singula- res, é necessário dizer algo sobre o que é uma teoria deste tipo. A exposição clássica é o Mimesis as Make-Believe de PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 17 175 Sagid Salles Sagid Salles Kendall Walton. A exposição de Walton é complexa, longa e envolve uma infinidade de conceitos que vão muito além do que é necessário para os propósitos deste artigo. No que segue exponho apenas alguns aspectos que serão importan- tes para o tratamento das existenciais negativas. Nós temos alguma ideia intuitiva do que é um jogo de faz de conta. Jogos de faz de conta são uma espécie de ativi- dade imaginativa na qual simulamos que algumas coisas são o caso e outras não. Um exemplo é o jogo da torta de barro. Neste jogo crianças simulam que certas bolas de barro são tortas. Sabemos o quão longe crianças podem ir nestes jo- gos. Um buraco na terra pode ser o forno, as tortas têm de ser colocadas no forno antes de ingeridas, podem ser ven- didas para o colega em troca de pedrinhas que representam dinheiro, etc. Obviamente, tanto nós quanto as crianças sa- bemos que nem o pedaço de barro é realmente uma torta, nem o forno é realmente um forno, etc. Por outro lado, participar corretamente do jogo envolve compreender o que é o caso naquele jogo de faz de conta. Você sabe que, ao entrar num jogo destes com o seu filho, não deverá chamar as bolas de barro de “bolas de barro”, mas de “tortas”. Isto porque, naquele jogo, as bolas de bar- ro não são meras bolas de barro, mas tortas. Isto ilustra a diferença entre algo ser realmente o caso e algo ser o caso em um jogo de faz de contas. Quando uma criança diz “isto é uma torta”, ela está dizendo algo literalmente falso. Aquilo não é uma torta, é puro barro. Mas está dizendo algo que é verda- deiro no jogo de faz de conta em questão, algo que é ficcionalmente verdadeiro (WALTON, 1990:1.5; 2000:71). Um importante aspecto disto é que não há nada de er- rado em supor que nossas crianças saibam que suas tortas são, na verdade, puro barro. 2. TEORIAS DO FAZ DE CONTA: SIMULANDO REFERÊNCIA E PREDICAÇÃO Quando uma delas diz “esta PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 176 FAZENDO DE CONTA QUE VULCANO NÃO EXISTE ARTIGO ORIGINAL torta é uma delícia”, não precisa estar tentando seriamente se referir a uma torta. Ela apenas simula ou faz de conta que se refere a uma torta. O mesmo ocorre com boa parte de nosso discurso sobre ficção. Quando alguém diz que Sherlock Holmes é um detetive, não está seriamente ten- tando se referir a um indivíduo pelo nome “Sherlock Holmes”. Ao contrário, ele simula usar o nome referenci- almente. Existe uma diferença entre o caso da torta e o de Sher- lock Holmes. Esta diferença é captada pela distinção de Evans (1982:358) entre jogos existencialmente conservadores e jogos existencialmente criativos. Os primeiros são aqueles em que simulamos que algo que realmente existe é outra coisa. No jogo das tortas nós simulamos que as bolas de barro (que realmente existem) eram tortas. Neste contexto, quan- do a criança diz “esta torta é uma delícia” ela simula se referir a uma torta, mas efetivamente se refere a uma bola de barro. Os segundos são aqueles nos quais simulamos que algo existe, sem que de fato exista. Quando dizemos “Sher- lock Holmes é um detetive”, simulamos nos referirmos ao Sherlock Holmes, mas não nos referimos a qualquer coisa de fato. Isto tem consequências interessantes. Enquanto a frase “isto é uma torta” seria literalmente falsa, a frase “Sherlock Holmes é um detetive” – tomada literalmente – é sem sen- tido. Como vimos antes, ao dizer “isto é uma torta” o falante do exemplo está de fato se referindo a uma bola de barro. O demonstrativo “isto” seleciona, neste contexto, a bola de barro. Dado que o barro não é uma torta, o que a frase literalmente diz é falso. Isto não impede, é claro, que a frase seja verdadeira no jogo de faz de conta. Por outro la- do, o nome “Sherlock Holmes” não se refere efetivamente a 177 PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. Sagid Salles qualquer coisa que seja. Lembrando que estamos supondo que a única função dos nomes é referir-se, este nome não tem significado. Sendo assim, a frase “Sherlock Holmes é um detetive” também deve ser destituída de significado. Mas falantes podem simular usar esta frase para expressar uma proposição. 2. TEORIAS DO FAZ DE CONTA: SIMULANDO REFERÊNCIA E PREDICAÇÃO E nada impede que ela seja verdadeira na- quele jogo de faz de conta (criado por Conan Doyle). Do mesmo modo que o faz de conta pode envolver re- ferência simulada, pode envolver atribuição simulada de propriedades. Conforme nota Walton (1990:423), isto não deveria ser muito surpreendente. Uma vez que simulamos fazer referência a alguma coisa através de um termo singu- lar, a tentação de ir além e simular atribuir propriedades pode ser irresistível. Assim, não apenas simulamos nos refe- rirmos pelo nome “Sherlock Holmes”, ainda simulamos atribuir propriedades através do uso (simulado) de predica- dos como “é um detetive”, “é inteligente”, etc. Ao dizer “Sherlock Holmes é um detetive” – fazendo a simulação de referência e a simulação de atribuição de propriedades – es- tamos simulando fazer uma afirmação. Com isto, podemos entender por que teorias do faz de conta são teorias não comprometidas com objetos. Nomes ficcionais como “Sherlock Holmes” colocam a perspectiva da referência direta em dificuldade. Aparentemente estes nomes não têm referente. Sendo assim, de acordo com a perspectiva da referência direta, são destituídos de signifi- cado. Mas, obviamente, nomes ficcionais têm significado. Assim, a perspectiva da referência direta tem de estar erra- da. Uma saída para este problema seria afirmar, seguindo Meinong, que não é verdade que nomes ficcionais não pos- suem referentes. Apenas acontece de eles se referirem a coisas inexistentes. PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 178 ARTIGO ORIGINAL FAZENDO DE CONTA QUE VULCANO NÃO EXISTE Teorias do faz de conta fornecem uma resposta alterna- tiva que não nos compromete com objetos inexistentes. Quem usa o nome “Sherlock Holmes” em um contexto de faz de conta não está usando o nome referencialmente, está simulando usar o nome referencialmente. Do mesmo modo, quem diz “Sherlock Holmes é um detetive” não está fazen- do uma afirmação. Esta frase não expressa realmente qualquer proposição e, consequentemente, não expressa al- go que possa ser absolutamente verdadeiro ou absolutamente falso. A pessoa está simulando fazer uma afirmação e expressar uma proposição, que será verdadeira no jogo de faz de conta criado por Doyle. Com isto, temos um modo de explicar discursos envolvendo nomes ficcio- nais sem apelar a qualquer tipo especial de objeto. Mas pode isto nos ajudar com as existenciais negativas? A primeira coisa a se notar é que, embora nomes ficcionais representem uma classe interessante, existenciais negativas frequentemente envolvem nomes não ficcionais. 2. TEORIAS DO FAZ DE CONTA: SIMULANDO REFERÊNCIA E PREDICAÇÃO Um exemplo é nossa frase 1 (“Vulcano não existe”). Evans, por exemplo, tem uma interessante teoria para explicar discur- sos envolvendo nomes vazios no contexto de existenciais negativas. De acordo com o seu tratamento, para entender o que é dito por uma existencial negativa, falante e ouvinte devem estar engajados (ou preparados para se engajar) em algum jogo de faz de conta como aqueles acima (1982:369). Proferimentos de existenciais negativas singulares envolvem o mesmo tipo de simulação de referência que vimos antes. Mas penso que Schiffer (1988:42) tem razão em ressaltar que ele não fornece qualquer modo claro de lidar com exis- tenciais negativas envolvendo nomes não ficcionais. Estas existenciais são justamente as mais problemáticas para as perspectivas do faz de conta. Neste artigo vou me concen- 179 PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. Sagid Salles trar principalmente sobre estes casos. Na próxima seção, ve- remos que Walton tem uma interessante solução para o problema. 3. WALTON: ALÉM DA MERA SIMULAÇÃO Comecemos a pensar em como poderíamos apelar ao faz de conta para solucionar o problema das existenciais negativas singulares. Uma solução aparente seria alegar que frases como 1 (“Vulcano não existe”) não envolvem referência ou atribuição séria de propriedades. Na verdade, proferimentos de tais frases envolvem a simulação de ambos. Quem profe- re 1 não está realmente fazendo uma afirmação, está simulando fazer uma afirmação. Consequentemente, 1 não é absolutamente verdadeira, nem absolutamente falsa. Mas 1 pode ser verdadeira em um jogo de faz de conta. Repare que esta solução satisfaz os dois critérios apre- sentados na seção 1. Primeiro, os falantes estão realmente usando o nome “Vulcano” ao proferir 1. Apenas ocorre que este uso é um uso simulado. Além disto, “existe” pode con- tinuar sendo encarado como um predicado de primeira ordem, que expressa uma propriedade. A diferença é que falantes não estão realmente atribuindo esta propriedade ao suposto referente de “Vulcano”, eles estão simulando fazer isto. Contudo, se a solução consistisse apenas nisto, seria implausível. Já vimos uma das razões para isto. Aparente- mente proferimentos de 1 não são parte de um jogo de faz de conta, nem são destituídos de sentido, nem apenas ficci- onalmente verdadeiros. 1 é absolutamente verdadeira e representa uma importante descoberta empírica. Qualquer um que queira defender o contrário tem o ônus da prova. PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 180 FAZENDO DE CONTA QUE VULCANO NÃO EXISTE ARTIGO ORIGINAL Kroon (2000:101) aponta ainda uma razão mais séria pela qual o tratamento acima não seria suficiente: ele não resolve o problema das existenciais negativas. O que a pers- pectiva acima faz é apenas jogar o mesmo problema para o reino do faz de conta. Falantes estariam simulando fazer re- ferência a um objeto existente para em seguida simular atribuir a eles a propriedade de não existir. Mas isto faz tan- to sentido quanto falantes simularem que um objeto é quadrado para simular que ele não é quadrado. A motiva- ção para entrar em conversas sobre simulação e faz de conta era justamente evitar a inconsistência, mas agora reencon- tramos a inconsistência em outro lugar. Algo tem de estar errado. Dificilmente falantes estão usando existenciais ne- gativas meramente para simular uma inconsistência deste tipo. A simulação deve servir para algum propósito além deste. A perspectiva de Walton se adequa a isto. 3. WALTON: ALÉM DA MERA SIMULAÇÃO De acordo com ele, quem profere 1 está literalmente dizendo que: 1’) Tentativas referenciais deste tipo não são bem- sucedidas. 1’) Tentativas referenciais deste tipo não são bem- sucedidas. A ideia é esta. Quando falantes dizem “Vulcano não existe” eles estão simulando usar o nome “Vulcano” refe- rencialmente. Mas o propósito de fazer isto neste contexto é indicar um tipo de tentativa referencial. Ao acoplar o pre- dicado “não existe” o falante declara que aquela tentativa não é bem-sucedida. Em outras palavras, usamos existen- ciais negativas para indicar e reprovar tipos de tentativas referenciais. Walton pensa mesmo que 1’ representa o sig- nificado literal de 1. O mesmo deve valer para existenciais positivas. Quem profere uma frase da forma “N existe” está indicando um tipo de tentativa referencial e declarando-a 181 PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. Sagid Salles como bem-sucedida (WALTON, 1990:426; 2000:82). como bem-sucedida (WALTON, 1990:426; 2000:82). A noção de tentativa referencial pode não ser muito cla- ra. É claro que quem diz “Vulcano não existe” está indicando um tipo diferente de tentativa referencial de que quem diz “Sócrates não existe”. Mas deve-se tomar o cuida- do de não pensar que um tipo de nome sempre serve para indicar o mesmo tipo de tentativa referencial. Quem diz “Sócrates existe” (com a intenção de usar o nome do filóso- fo) está indicando um tipo de tentativa, e quem diz “Sócrates existe” (com a intenção de usar o nome do famo- so jogador do Corinthians) está indicando outro tipo de tentativa. Podemos conceder a Walton que o contexto cumpre o papel de ajudar a indicar qual tipo de tentativa está em jogo. O tratamento de Walton tem nítidas vantagens sobre aquele que foi mencionado no início desta seção. Primeiro, ele consegue explicar como existenciais negativas podem ser verdadeiras sem incorrer em contradição. Nenhuma con- tradição segue da verdade de 1’. Segundo, ele consegue fornecer um propósito aceitável para o proferimento de existenciais negativas, que vai além de meramente simular uma inconsistência. Contudo, embora Walton respeite o primeiro critério de adequação, o critério do uso, não respeita o critério da simplicidade (Kroon, 2000:104). De acordo com ele, a pa- lavra “existe” não é um predicado de primeira ordem, mas um meio que temos de expressar aprovação ou desaprova- ção de tentativas referenciais. De fato, penso que o primeiro critério é mais intuitivo que o segundo. 4 Eu apresento a objeção de um modo um pouco diferente. Isto porque a exposição de Richard depende do conceito de modos de apresentação, e penso que isto é uma complicação desneces- sária para o argumento. 3. WALTON: ALÉM DA MERA SIMULAÇÃO Se for ne- cessário recusar um dos dois para resolver o problema das existenciais negativas, que seja o segundo. Mas isto não muda o fato de que um tratamento que respeitasse a ambos PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 182 FAZENDO DE CONTA QUE VULCANO NÃO EXISTE ARTIGO ORIGINAL seria preferível. Conforme veremos na próxima seção, Kro- on tem algo deste tipo. Seja como for, os problemas de Walton vão além de não respeitar o segundo critério. Mark Richard (2000:226) apresenta uma interessante objeção.4 Pense num mundo possível exatamente como o nosso, com a única diferença que ninguém jamais usou qualquer termo referencial para se referir ao planeta Marte. Marte está lá, no mesmo lugar, apenas acontece que ninguém tentou referir-se ao planeta. Intuitivamente, a frase 4) Marte não existe. seria falsa com respeito a este mundo imaginado. No entan- to, de acordo com Walton, a paráfrase de 4 seria: seria falsa com respeito a este mundo imaginado. No entan- to, de acordo com Walton, a paráfrase de 4 seria: 4’) Tentativas referenciais deste tipo [o tipo Marte, diga- mos] não são bem-sucedidas. Mas 4’ é verdadeira com respeito ao mesmo mundo. Dado que 4’ é a paráfrase de 4, 4 (“Marte não existe”) também se- ria verdadeira na perspectiva Waltoniana. Isto leva à estranha conclusão de que a frase “Marte não existe” pode- ria ser verdadeira com respeito a um mundo no qual Marte existe. Este é, no mínimo, um resultado dramático. Walton pode protestar que Richard está jogando em outros termos. De fato, frases existenciais singulares são co- tidianamente usadas para aprovar ou reprovar tentativas referenciais. Contudo, podemos criar jogos não oficiais de faz de conta nos quais isto não é assim. Richard está jogando PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 183 Sagid Salles um jogo destes. Ele está simulando que “Marte existe” ex- pressa uma proposição constituída por um objeto (Marte) e uma propriedade (existência). Está imaginando uma situa- ção na qual a proposição simulada é verdadeira. Neste contexto imaginado, é claro, a negação da proposição simu- lada (4) será falsa. Mas isto é apenas dizer que “Marte não existe” é falsa neste jogo de faz de conta.5 O máximo que Walton concederia a Richard é que “Marte existe” é verdadeira no jogo de faz de conta menci- onado acima. Do mesmo modo, é apenas neste jogo que “Marte não existe” expressa uma proposição falsa. O que Richard tem sucesso em mostrar é que, naquele faz de conta, Marte tem a propriedade de existir. Consequentemente, naquele jogo, 4 é falsa. Nada disto implica que Walton esteja errado quanto a análise do que 4 literalmente significa. Contudo, não penso que esta resposta seja boa. Primei- ro, tudo o que Walton nota é que é possível reinterpretar o exemplo de Richard em uma estrutura de simulação e faz de conta (aliás, será muito difícil achar algum discurso que não possa ser interpretado deste modo). Mas do fato de isto ser possível não se segue que seja plausível. O problema é que não é plausível. O exemplo de Richard não parece em- pregar nenhum uso ficcional ou simulado de “existe”. 5 Walton não responde exatamente a este argumento. Mas penso que esta seria de fato a sua res- posta, com base em Walton (2000:90, nota 70). Sagid Salles Sagid Salles Pode fornecer uma resposta do tipo fornecida ao exemplo de Richard. Tais interpretações são possíveis. Mas duvido que sejam plausíveis. Penso que isto coloca um importante desafio para Walton: encontrar motivação semântica para a sua estratégia de análise. 4) Marte não existe. Walton precisaria de boas razões para defender que algo deste tipo está em jogo, razões que vão além do mero fato de o exemplo ser um problema para a sua perspectiva. Isto nos leva a uma última objeção a Walton. Sua teoria é bem motivada metafisicamente. Afinal, ela possui a quali- dade de (supostamente) explicar as existenciais negativas PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 184 FAZENDO DE CONTA QUE VULCANO NÃO EXISTE ARTIGO ORIGINAL sem, ao mesmo tempo, aceitar algum tipo estranho de enti- dades. Contudo, não é claro se ela é bem motivada semanticamente. Seu objetivo é explicar o que falantes re- almente dizem quando proferem frases existenciais singulares. Mas, intuitivamente, falantes não estão falando sobre tentativas referenciais ao proferir estas frases. A tese de Walton é pouco intuitiva e não é fácil reconhecer que os falantes estejam fazendo o que ela diz que eles estão fazen- do. Se a tese é pouco intuitiva quanto a nossos usos cotidi- anos de existenciais negativas, a coisa é ainda pior quando pensamos em usos não cotidianos. Um filósofo solipsista pode acreditar que só ele existe. Ele está preparado para apontar para qualquer coisa e dizer “isto não existe” ou “aquilo não existe”. Na perspectiva de Walton, ele está re- petidamente desaprovando tipos de tentativas referenciais. Peter Unger é menos otimista do que seria um solipsista quanto ao seu próprio eu. Defende a impressionante tese de que ele próprio não existe. Em certa parte, expõe a sua tese do seguinte modo “A posição desafiadora é: eu não existo, e nem você.” (1979:37, tradução minha). Se levar- mos Walton a sério, Unger está defendendo uma tese sobre tentativas referenciais de certo tipo, uma tese da filosofia da linguagem, e não da metafísica. Teístas e ateístas estão fre- quentemente discutindo sobre a existência de Deus. Frases como “Deus existe” ou “Deus não existe” são comuns nes- tas discussões. Mais uma vez, não parece que eles estão falando sobre tentativas referenciais de certo tipo. Podemos pensar ainda em muitos outros exemplos deste tipo. Walton pode reinterpretar cada um destes casos em uma estrutura de faz de conta. Pode alegar que cada caso envolve a participação em um determinado jogo não oficial. 185 PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 4. KROON: ALÉM DO QUE É LITERALMENTE DITO Kroon concorda com Evans e Walton que termos singula- res são artifícios de referência e concorda com Evans que “existe” expressa uma propriedade de primeira ordem pos- suída por tudo. Além disto, prescreve um tratamento do mesmo tipo do de Walton para existenciais negativas. Exis- tenciais negativas são usadas como meios de indicar e desaprovar tentativas referenciais sérias de certo tipo, assim como existenciais positivas são usadas para indicar e apro- var tipos de tentativas referenciais. Contudo, Kroon pensa que Walton cometeu um erro importante: acreditar que a sua análise fornecia o significado literal das existenciais ne- gativas. De acordo com Kroon frases existenciais singulares têm exatamente o significado literal que elas parecem ter. Co- mecemos pelas existenciais positivas. Frases da forma “N existe” expressam uma proposição constituída por um obje- to e uma propriedade. “Marte existe” expressa a proposição de que Marte existe. Do mesmo modo, a frase “Marte não existe” expressa a proposição de que Marte não existe. Da- do que Marte existe, a última frase expressará uma proposição falsa. Entretanto, vimos que existenciais negativas aparente- mente verdadeiras são problemáticas. Tivemos dificuldades em explicar como “Vulcano não existe” pode expressar uma PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 186 FAZENDO DE CONTA QUE VULCANO NÃO EXISTE ARTIGO ORIGINAL proposição verdadeira. Kroon pensa que, uma vez que ter- mos singulares são artifícios de referência direta e que tudo existe, existenciais negativas singulares jamais serão verda- deiras. Assim, “Vulcano não existe” não pode expressar uma proposição verdadeira. Obviamente, esta frase também não pode expressar uma proposição falsa (pois, neste caso, Vulcano existiria). A solução é que tais frases simplesmente não expressam qualquer proposição que seja. Esta não é uma conclusão surpreendente. Kroon aceita que a única função semântica dos nomes é introduzir um objeto no discurso. “Vulcano” é um nome vazio, de modo que falha em realizar a sua função. Por isto, o nome é desti- tuído de significado. Consequentemente, as frases que o contém também serão destituídas de significado. Isto tem a estranha consequência de que ao proferir 1 (“Vulcano não existe”) as pessoas estão literalmente dizendo nada. Mas é exatamente isto que Kroon quer dizer. Quem profere uma existencial negativa singular sabe que sua frase só poderia ser verdadeira ou falsa se o termo singular da frase tivesse um referente. 4. KROON: ALÉM DO QUE É LITERALMENTE DITO As pessoas também sabem que se o termo tivesse um referente, a frase seria falsa, de modo que encontraríamos novamente uma inconsistência. Deste modo, quem profere 1 não pode estar seriamente usando o termo singular. Ele tem de estar simulando usar o termo e atribuir a propriedade de não existir a seu referente (KROON, 2000:110). Na seção 3 vimos que isto, por si só, não resolve o pro- blema das existenciais negativas. Na verdade, apenas transporta o problema para o escopo da simulação. Estarí- amos supondo que as pessoas estariam simulando usar um nome para referirem-se a algo existente e, em seguida, dizer que este algo não existe. Qual pode ser o propósito de tal 187 PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. Sagid Salles Sagid Salles coisa? Para entender a resposta de Kroon, voltemos para o ca- so das existenciais positivas. Quem profere “Marte existe” está expressando literalmente a proposição de que Marte existe. Mas está expressando não literalmente aprovação de um tipo de tentativa referencial. Podemos usar a frase aci- ma como um meio de expressar não literalmente que tentativas referenciais sérias deste tipo são bem-sucedidas. Com existenciais negativas, a coisa é análoga. Quem profere “Vulcano não existe” não está expressando qualquer propo- sição que seja. Porém, a pessoa está simulando expressar uma proposição com o intuito de expressar não literalmen- te outra proposição: que tentativas referenciais sérias deste tipo não são bem-sucedidas. A principal diferença entre Walton e Kroon é que aqui- lo que o primeiro chamou de significado literal de frases existenciais singulares, o segundo chamaria de significado metafórico ou significado figurado (2000:107). Ignoro compli- cações com usos metafóricos e figurados da linguagem, e concedo a Kroon que tudo corre bem neste ponto. Penso que o tratamento de Kroon tem algumas vanta- gens sobre o de Walton. Primeiro, ele obedece aos dois critérios apresentados na seção 1. Quem profere uma exis- tencial negativa está usando, e não mencionando, o termo singular. Além disto, “existe” realmente expressa uma pro- priedade de primeira ordem. Em segundo lugar, a objeção da motivação semântica tem menos força contra Kroon. Dado que existenciais singulares têm exatamente o signifi- cado literal que parecem ter, a tese de Kroon é semanticamente mais bem motivada. Por fim, a objeção de Richard também tem menos força contra Kroon. Ele não precisa aceitar a consequência indesejável de que 4 (“Marte PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 188 FAZENDO DE CONTA QUE VULCANO NÃO EXISTE ARTIGO ORIGINAL não existe”) poderia ser verdadeira com respeito a um mundo onde Marte existe. Afinal, 4 jamais é verdadeira. O máximo que ele precisaria aceitar é que o que 4 metafori- camente expressa poderia ser verdadeiro com respeito a um mundo onde Marte existe. Mas isto é apenas aceitar que “Marte existe” é consistente com “tentativas referenciais do tipo Marte não são bem-sucedidas”. Contudo, embora a tese de Kroon seja semanticamente mais motivada do que a de Walton, ainda sofre do proble- ma de motivação semântica. De acordo com Kroon, frases como “Vucano não existe” são usadas no escopo de uma simulação para expressar algo metaforicamente. Literalmen- te, 1 não expressa qualquer coisa que seja. coisa? Isto tem o mesmo tipo de consequência que vimos no final da seção anterior. Relembre o exemplo do solipsista que diz “isto não existe”. Não parece que ele está simulando usar a frase apenas para expressar metaforicamente uma tese sobre ten- tativas referenciais. Como antes, os exemplos deste tipo podem ser multiplicados. Na próxima seção, volto a isto em maior detalhe. 5. O PROBLEMA DA MOTIVAÇÃO SEMÂNTICA As teorias do faz de conta são metafisicamente bem moti- vadas, na medida em que não nos comprometem com objetos inexistentes ou coisas do tipo. Além disto, vimos que a versão de Kroon escapa pelo menos das objeções mais imediatas a Evans e Walton. A teoria de Kroon, contudo, também parece carecer em alguma medida de motivação semântica. É preciso motivar a alegação de que existenciais singulares, usadas em contextos aparentemente não ficcio- nais, estão sendo usadas no escopo de faz de conta. PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 189 Sagid Salles Sagid Salles Kroon reduz um pouco a força desta objeção. De acor- do com ele, frases existenciais singulares positivas – quando são verdadeiras – expressam literalmente o que deveriam expressar: que um objeto possui a propriedade de existir. Do mesmo modo, frases existenciais negativas – quando são falsas – expressam literalmente o que parecem expressar. A frase “Dilma não existe” expressa a falsidade de que a Dil- ma não possui a propriedade de existir. Contudo, dado que tudo existe, frases existenciais negativas nunca são verdadei- ras. Assim, “Vulcano não existe” não é verdadeira. Mas esta frase, como sabemos, também não é falsa (pois neste caso Vulcano existiria). Sua saída, então, foi recusar que existên- cias negativas aparentemente verdadeiras expressem proposições. Um falante que profere “Vulcano não existe” está simulando fazer uma afirmação existencial (negativa) para expressar não literalmente que tentativas referenciais de certo tipo falham. O problema é que mesmo a tese mais fraca de Kroon parece semanticamente desmotivada. A princípio, é implau- sível que Peter Uger estivesse usando a frase “eu não existo” para comunicar não literalmente que tentativas referenciais de certo tipo falham, e também é implausível sustentar que cientistas estivessem usando “Vulcano não existe” para ex- pressar não literalmente que tentativas referenciais de certo tipo falham, e assim por diante. Em todos estes contextos, é inicialmente implausível que falantes estejam simulando (fazendo de conta, etc.) usar uma frase que, literalmente, é sem sentido, apenas para comunicar não literalmente uma tese sobre nossas tentativas de referência. Este é o que chamo de problema da motivação semântica: o problema de motivar a afirmação de que falantes estão engajados em um jogo de faz de conta ou alguma espécie de PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 5. O PROBLEMA DA MOTIVAÇÃO SEMÂNTICA 190 FAZENDO DE CONTA QUE VULCANO NÃO EXISTE ARTIGO ORIGINAL atividade de simulação quando a intuição é que eles não es- tejam. As teorias do faz de conta são inicialmente plausíveis para explicar nossos usos da linguagem em contextos cla- ramente ficcionais, mas não é claro qual a motivação semântica para estendê-las para contextos aparentemente não ficcionais. Se a única motivação para aceitarmos as teo- rias do faz de conta é a sua capacidade de resolver puzzles, então tais teorias devem ser rejeitadas. Por mais importante que seja a habilidade destas teorias em resolver puzzles, tais teorias são teorias sobre o que as pessoas de fato fazem com a linguagem (o que elas realmente dizem, o modo como re- almente usam nomes em certos contextos, etc.). É necessário alguma motivação para acreditarmos que nossos falantes estejam realmente fazendo o que estas teorias dizem que eles estão fazendo. Neste caso, é preciso motivação para aceitar que os falantes realmente estejam engajados em ati- vidades de simulação ou faz de conta, quando eles parecem de fato não estar. Não penso que o jogo está perdido para as teorias de faz de conta, pois não sugiro que tal motivação não exista. Meu objetivo é apenas chamar atenção para a sua necessi- dade. Por fim, gostaria de terminar distinguindo o problema global do problema local da motivação para teorias do faz de con- ta. Nosso problema é encontrar uma motivação para acreditar que certa parcela de nossa linguagem está sendo usada no escopo de um jogo de faz de conta. Uma estraté- gia é procurar por uma característica desta parcela que nos permita fazer isto. Talvez haja algo no modo como usamos termos singulares ou então a palavra “existe” que explique a razão de existenciais negativas aparentemente verdadeiras, a despeito das aparências, serem usadas no escopo de um jo- 191 PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. Sagid Salles go de faz de conta. Neste caso, o problema da motivação é um problema local, o problema de encontrar uma caracte- rística de nossos usos de uma parcela de linguagem que nos justifique acreditar que sejam usos em algum sentido não sérios. Esta parece ser a estratégia mais fácil. Desconfio, contudo, que não é bem isto que, por exemplo, Walton tem esperança de encontrar.6 Ele parece pensar que jogos de faz de conta são algo muito mais pre- sentes em nossa linguagem do que pensamos. 6 Veja, por exemplo:” I suspect that make-believe may be crucially involved as well in certain reli- gious prectices, in the role of sports in our culture, in the instituition of morality, in the postulation of “theoretical entities” in science (...)” (Walton 1990:7). 5. O PROBLEMA DA MOTIVAÇÃO SEMÂNTICA E talvez seja mais fácil procurar não por uma característica dos nossos usos de uma parcela de nossa linguagem, mas por uma ca- racterística de nossos usos em geral que justifica afirmações pontuais sobre certas parcelas. Por exemplo, pode ser que haja alguma razão para acreditarmos que o fenômeno do faz de conta é algo tão presente em nossos usos da linguagem que não seja mais surpreendente que existenciais negativas sejam empregadas no escopo de algum tipo de simulação. Neste caso, o que queremos justificar não é apenas que uma parcela pontual de nossos usos são feitos no escopo de faz de conta, mas que o fenômeno do faz de conta é comple- tamente difundido por nossos usos comuns e aparentemente sérios. Uma vez que isto é feito, poderíamos então sustentar que não deveria ser tão surpreendente que isto ocorra no caso das existenciais negativas. Neste caso, es- taríamos interpretando o problema da motivação como um problema global. Eis uma hipótese de como justificar que o faz de conta seja um fenômeno global em nossos usos da linguagem. Eu PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 192 FAZENDO DE CONTA QUE VULCANO NÃO EXISTE ARTIGO ORIGINAL reconheço que o que segue é apenas uma hipótese crua, pa- ra a qual ainda não tenho justificativa adequada, mas serve para tornar claro que tipo de estratégia poderia ser usada. Podemos começar ressaltando que falantes comuns geral- mente não têm garantias infalíveis de que seus usos de termos singulares realmente terão sucesso em se referirem a algo. Dificilmente alguém negaria isto. Agora, o máximo que eles podem fazer é usar os termos singulares como se eles de fato se referissem a algo. Deste modo, todos os nossos usos de termos singulares seriam feitos no escopo de uma simulação, pelo menos no sentido fraco de que estamos usando eles como se de fato se referissem a algo. Quando o termo de fato se refere a um objeto, seremos capazes de usá- lo para expressar proposições singulares sobre o objeto em questão. Quando o termo falha em se referir a um objeto, não temos sucesso em expressar proposições com ele, mas continua sendo verdade que estamos usando-o como se re- ferisse a algo. Dado que fazemos isto o tempo todo, a simulação de referência se torna algo tão natural que sequer passa a ser notada. 5. O PROBLEMA DA MOTIVAÇÃO SEMÂNTICA Por isto ficamos tão surpresos quando nos deparamos com o fato de que certo uso de certo termo singular (como “Vulcano” por exemplo) é simulado. Tão logo notamos que, na verdade, todo uso se dá num escopo de “como se”, percebemos que não há qualquer problema particular com o caso de “Vulcano”. Obviamente isto nos levaria a um conjunto de problemas que requerem solução. Por exemplo, neste caso, qual seria a diferença entre o uso de “Vulcano” e os usos comuns de “Sherlock Holmes”? Se ambos são usados no escopo de uma simulação, o que os distingue? Uma possível resposta seria a seguinte. Nós usa- mos “Vulcano” como se estivéssemos fazendo referência apenas porque não temos garantias infalíveis de que isto re- 193 PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. Sagid Salles almente seja o caso. Mas temos a intenção de que ele refira a um objeto do mundo. Contudo, no caso de “Sherlock Holmes”, nós geralmente usamos sem sequer ter a intenção de nos referirmos a qualquer coisa que seja. Sendo assim, a atividade de faz de conta envolvida no segundo é muito mais evidente e, consequentemente, não gerará surpresa. Talvez algo análogo pudesse ser defendido para nossos usos de predicados e assim por diante. De toda forma, como já foi dito, aqui isto não passa de uma mera hipótese para ex- plicar como uma resposta ao problema global poderia ser procurada. Seja como for, se conseguirmos encontrar uma respos- ta satisfatória ao problema da motivação, as teorias do faz de conta passarão a ser, penso eu, uma estratégia muitíssi- mo plausível para lidar não apenas com existenciais negativas, mas com muitos outros puzzles que não foram tratados aqui. Infelizmente, ainda não conheço uma respos- ta satisfatória a este problema. Abstract: My goal in this paper is to present and analyze some versions of make-believe theories for singular negative existentials. I will quickly present Evans’ perspective and, in greater detail, the perspectives of Kroon and Wal- ton. I will claim that neither Evans nor Walton provide the right account of the phenomenon of singular negative existentials, and that Kroon’s perspec- tive is better than both. However, I will argue that the three theories have the same problem, which I call the problem of semantic motivation. Keywords: singular negative existentials; make-believe theories; the problem of semantic motivation. semantic motivation. REFERÊNCIAS DONNELLAN, Keith. “Speaking of Nothing”. In: The Philosophical Review. Vol. 83, No 1, pp. 3-31. Duke University Press, 1974. PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 194 FAZENDO DE CONTA QUE VULCANO NÃO EXISTE ARTIGO ORIGINAL EVANS, Gareth. The Varieties of Reference. Clarendon Press – Oxford University Press – New York. 2002. EVANS, Gareth. The Varieties of Reference. Clarendon Press – Oxford University Press – New York. 2002. KRIPKE. "Vacuous Names and Fictional Entities". In: Philosophical Troubles (vol. 1). Oxford University Press. pp. 52-74, 2011. KROON, Fred. “Disavowal Through Commitment: Theories of Negatives Existentials”. In: Everett, A. Hofweber, T. (orgs). Empty Names, Foction, and the Puzzles of Non-Existence. CSLI publications. 2000, pp. 95-116. MEINONG, Alexius. “The theory of objects”. In: Chisholm, Roderick M. org. (1960) Realism & the background of phenomenology. Free Press. pp.76-117, 1904. RICHARD, Mark. “Semantic Pretense”. In: Everett, A. Hofweber, T. (orgs): Empty Names, Foction, and the Puzzles of Non-Existence. CSLI publications. 2000, pp. 205-232. RUSSELL, Bertrand. “On Denoting”. In: Analytic Phi- losophy: An Anthology. Martinich, A. P. and Sosa, David, orgs.. University of Texas at Austin: Blackwell. pp.32-41, 2006. SCHIFFER, Stephen. “Review of Gareth Evans, The Varieties of Reference”. Journal of Philosophy 85, no. 1, pp. 33-42, 1988. PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 195 SEARLE, John R. “O Estatuto Lógico do Discurso Ficcional”. In: Expressão e Significado. Trad. Ana Cecília G. A. de Camargo e Ana Luiza Marcondes 195 Sagid Salles PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. Garcia. São Paulo: Martins Fontes, 1995. SAINSBURY, R. M. Fiction and Fictionalism. London, Routledge, 2010. UNGER, Peter. “I Do Not Exist”. In: Philosophical Pa- pers, vol. 2. Oxford University Press, 2006. pp 36-52. WALTON, Kendall. “Existence as Metaphor?”. In: Everett, A. Hofweber, T. (orgs): Empty Names, Foction, and the Puzzles of Non-Existence. CSLI publica- tions. 2000. pp. 69-94. _____. Mimesis as Make-Believe. Cambridge, Havard University Press, 1990. ZALTA, Edward. “The Road Between Pretense The- ory and Abstract Object Theory”. In: Everett, A. Hofweber, T. (orgs): Empty Names, Foction, and the Puzzles of Non-Existence. CSLI publications. 2000, pp. 117-147. _____. Mimesis as Make-Believe. Cambridge, Havard University Press, 1990. ZALTA, Edward. “The Road Between Pretense The- ory and Abstract Object Theory”. In: Everett, A. Hofweber, T. (orgs): Empty Names, Foction, and the Puzzles of Non-Existence. CSLI publications. 2000, pp. 117-147. PHILÓSOPHOS, GOIÂNIA, V.20, N.2, P.171-196, JUL./DEZ. 2015. 196
https://openalex.org/W4293235706	https://insu.hal.science/insu-03748539/document	English	null	Implications of Underground Nuclear Explosion Cavity Evolution for Radioxenon Isotopic Composition	Pure and Applied Geophysics	2,022	cc-by	6,692	To cite this version: Yunwei Sun, Charles R. Carrigan, Eric Pili, Tarabay Antoun. Implications of Underground Nuclear Explosion Cavity Evolution for Radioxenon Isotopic Composition. Pure and Applied Geophysics, 2022, 10.1007/s00024-022-03026-8. insu-03748539 Implications of Underground Nuclear Explosion Cavity Evolution for Radioxenon Isotopic Composition Yunwei Sun, Charles R. Carrigan, Eric Pili, Tarabay Antoun To cite this version: Yunwei Sun, Charles R. Carrigan, Eric Pili, Tarabay Antoun. Implications of Underground Nuclear Explosion Cavity Evolution for Radioxenon Isotopic Composition. Pure and Applied Geophysics, 2022, 10.1007/s00024-022-03026-8. insu-03748539 Distributed under a Creative Commons Attribution 4.0 International License 1 Lawrence Livermore National Laboratory, Livermore, CA 94550, USA. E-mail: sun4@llnl.gov 2 M.H. Chew & Associates, Livermore, CA 94550, USA. 3 CEA, DAM, DIF, 91297 Arpajon, France. 4 Universite´ de Paris, Institut de physique du globe de Paris, CNRS, 75005 Paris, France. HAL Id: insu-03748539 https://insu.hal.science/insu-03748539v1 Submitted on 10 Aug 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Pure Appl. Geophys. 2022 The Author(s) https://doi.org/10.1007/s00024-022-03026-8 YUNWEI SUN,1 CHARLES R. CARRIGAN,2 ERIC PILI,3,4 and TARABAY ANTOUN1 Different half-lives of these xenon iso- topes originating from three different decay networks can produce detectable signals (Saey et al., 2010; Sloan et al., 2016; Bowyer, 2020). The radioactivity of each xenon isotope is characterized by a unique dependence on time as a result of its decay chain and half-lives of precursors reaching peak activity at a distinctive time. In the absence of physical parti- tioning of the radioactive inventory in the post- detonation cavity, seepage, and prompt venting, radioxenon proﬁles will only reﬂect the mechanism of radioactive decay and ingrowth in a closed and well-mixed (i.e., batch-mode) system (De Geer, 2013; Kalinowski & Liao, 2014; Yamba et al., 2016). Xe) have been assessed as indicators of UNEs (Bowyer et al., 1998; Saey & De Geer, 2005; Kalinowski & Pistner, 2006; Kalinowski et al., 2010; Saey et al., 2010; Kalinowski, 2011; Galan et al., 2018). Different half-lives of these xenon iso- topes originating from three different decay networks can produce detectable signals (Saey et al., 2010; Sloan et al., 2016; Bowyer, 2020). The radioactivity of each xenon isotope is characterized by a unique dependence on time as a result of its decay chain and half-lives of precursors reaching peak activity at a distinctive time. In the absence of physical parti- tioning of the radioactive inventory in the post- detonation cavity, seepage, and prompt venting, radioxenon proﬁles will only reﬂect the mechanism of radioactive decay and ingrowth in a closed and well-mixed (i.e., batch-mode) system (De Geer, 2013; Kalinowski & Liao, 2014; Yamba et al., 2016). Keywords: Radioactive decay, transport, underground nuclear explosion, noble gas, xenon isotopic evolution. YUNWEI SUN,1 CHARLES R. CARRIGAN,2 ERIC PILI,3,4 and TARABAY ANTOUN1 YUNWEI SUN,1 CHARLES R. CARRIGAN,2 ERIC PILI,3,4 and TARABAY ANTOUN1 Abstract—Isotopic ratios of radioxenons sampled in the atmosphere or subsurface can be used to verify the occurrence of an underground nuclear explosion (UNE). Differences in the half-lives of radioactive xenon precursors and their decay-chain networks produce different time-dependent concentration proﬁles of xenon isotopes allowing isotopic ratios to be used for tracking UNE histories including estimating the time of detonation. In this study, we explore the potential effects of post-detonation cavity pro- cesses: precipitation of iodine precursors, gas seepage, and prompt venting on radioxenon isotopic evolution which inﬂuences UNE histories. Simpliﬁed analytical models and closed-form solutions yielding a potentially idealized radioactive decay/ingrowth chain in a closed and well-mixed system typically have limited application by not including the partitioning of the radionuclide inventory between a gas phase and rock melt created by the detonation and by ignoring gas transport from the cavity to host rock or ground sur- face. In reality, either subsurface transport or prompt release that is principally responsible for gas signatures violates the closed-sys- tem (or batch-mode) assumption. A closed-form solution representing time-dependent source-term activities is extended by considering the cavity partitioning process, slow seepage, and/or prompt release of gases from the cavity and applied to realistic systems. (Saey & De Geer, 2005; Carrigan et al., 2016). Advances in the technology for measuring radio- xenon concentrations have enhanced the detection of both UNE signatures and the global background of radioxenons produced by civilian facilities including nuclear reactors and medical isotope production plants (Le Petit et al., 2015; Achim et al., 2016; Gueibe et al., 2017; Haas et al., 2017; Hoffman & Berg, 2018). Isotopic ratios of radioxenons (e.g., ½131m 133m 133 135Xe) have been assessed as indicators (Saey & De Geer, 2005; Carrigan et al., 2016). Advances in the technology for measuring radio- xenon concentrations have enhanced the detection of both UNE signatures and the global background of radioxenons produced by civilian facilities including nuclear reactors and medical isotope production plants (Le Petit et al., 2015; Achim et al., 2016; Gueibe et al., 2017; Haas et al., 2017; Hoffman & Berg, 2018). Isotopic ratios of radioxenons (e.g., ½131m 133m 133 135Xe) have been assessed as indicators of UNEs (Bowyer et al., 1998; Saey & De Geer, 2005; Kalinowski & Pistner, 2006; Kalinowski et al., 2010; Saey et al., 2010; Kalinowski, 2011; Galan et al., 2018). IM release number: LLNL-JRNL-828896. Implications of Underground Nuclear Explosion Cavity Evolution for Radioxenon Isotopic Composition YUNWEI SUN,1 CHARLES R. CARRIGAN,2 ERIC PILI,3,4 and TARABAY ANTOUN1 YUNWEI SUN,1 CHARLES R. CARRIGAN,2 ERIC PILI,3,4 and TARABAY ANTOUN1 2. Model and Solution Development In this section, we propose ordinary differential equations (ODEs) of the multi-compartment system coupled with radionuclide decay/ingrowth networks and source-term activities, and derive closed-form solutions for given network structures (Fig. 2, see also Bourret et al., 2021). The study domain is composed of multiple compartments (gas-ﬁlled cav- ity, melt puddle, and host rock) connected by inter- compartment mass exchange (precursor rainout, seepage from gas-ﬁlled cavity to host rock, and/or prompt venting from the cavity to ground surface). Figure 1 Vertical cross-section of conceptual model with rainout (R) from the cavity (white) to melt puddle (red), seepage (S) from the cavity to host rock (yellow), and prompt release (V) from the cavity to ground surface (in a user-deﬁned time period). The back diffusion from melt puddle to cavity is neglected Figure 1 Vertical cross-section of conceptual model with rainout (R) from the cavity (white) to melt puddle (red), seepage (S) from the cavity to host rock (yellow), and prompt release (V) from the cavity to ground surface (in a user-deﬁned time period). The back diffusion from melt puddle to cavity is neglected The mass change of all radionuclides in each decay chain in the multi-compartment system described in Fig. 1 can be expressed as context of our cavity-melt partitioning model (Car- rigan et al., 2020; Sun et al., 2021), the detonation system following a UNE is described by several interacting subsystems or compartments (e.g., cavity, rock-melt puddle, and host rock). Each compartment may exchange radionuclides with other compart- ments. Although the slow seepage from the cavity to host rock was considered by Sun et al. (2021) in the analytical solution of generalized systems, prompt venting during a speciﬁed period of time has not been included. In the fully coupled numerical model of multiphase transport and radionuclide decay and ingrowth (Carrigan et al., 2016; Bourret et al., 2021), the transport of xenon isotopes produced from chain reactions in the cavity was accurately described. 1. Introduction Monitoring for underground nuclear explosions (UNEs) uses atmospheric detection of radionuclide gases released by either seepage or prompt venting at the ground surface of the test site following a UNE A realistic UNE system, in general, may be composed of a cavity and melt puddle (Fig. 1) with possible post-detonation partitioning of radionuclides from the gas-ﬁlled cavity into the underlying puddle of molten rock (R), gas seepage into the fractured zone of containment (S), and/or prompt venting of larger volumes of cavity gas (V) either of which is potentially capable of producing detectable xenon signatures at ground surface or downwind. In the Figure 1 Vertical cross-section of conceptual model with rainout (R) from Y. Sun et al. Pure Appl. Geophys. Y. Sun et al. Y. Sun et al. Figure 1 Vertical cross-section of conceptual model with rainout (R) from the cavity (white) to melt puddle (red), seepage (S) from the cavity to host rock (yellow), and prompt release (V) from the cavity to ground surface (in a user-deﬁned time period). The back diffusion from melt puddle to cavity is neglected Y. Sun et al. cavity directly (Carrigan et al., 2021). Since the prompt venting alters mass balance in the cavity, a full-scale mass exchange needs to be considered among all compartments. In this paper, we derived a closed-form solution to a full-scale model consider- ing time-dependent mass exchanges among interactive compartments: (1) thermally induced condensation (rainout) of refractory iodine precursors from cavity to rock melt, (2) seepage of cavity gases into a fractured containment zone, and (3) prompt venting of cavity gases to ground surface. The back diffusion from melt puddle to cavity is not considered because of its low gas ﬂux compared to other exchange processes. 2. Model and Solution Development However, the signature of promptly vented xenons was approximated using the xenon evolution in the d dt cc cp ch cv 2 6664 3 7775 ¼ A R S Q O O O R A O O S O A O Q O O A 2 6664 3 7775 cc cp ch cv 2 6664 3 7775; ð1Þ ð1Þ where cc, cp, ch, and cv are the mass vectors in cavity, puddle, host rock, and prompt release, respectively. A is a matrix of the ﬁrst-order decay and ingrowth rates, R is the rainout-rate matrix from cavity to puddle, S is the seepage-rate matrix, and Q is the prompt venting matrix. O denotes n n zero matrices and n is the number of radionuclides in the decay chain. (a) (b) (c) Implications of Underground Nuclear Explosion Cavity Implications of Underground Nuclear Explosion Cavity (a) p g p y (a) (b) (b) (c) Figure 2 di i d /i h k f d h i 131 b 133 d 135 N b d b hi f (c) g Radioactive xenon decay/ingrowth networks of decay chains a 131, b 133, and c 135. Numbers on arrows denote branching factor (%). Red and green icons represent radioactive and stable xenon isotopes while pink and yellow icons denote precipitating and possible precipitating precursors. The decay networks are based on England and Rider (1994) g Radioactive xenon decay/ingrowth networks of decay chains a 131, b 133, and c 135. Numbers on arrows denote branching factor (%). Red and green icons represent radioactive and stable xenon isotopes while pink and yellow icons denote precipitating and possible precipitating precursors. The decay networks are based on England and Rider (1994) Y. Sun et al. Pure Appl. Geophys. 2.2. Precipitation of Precursors The post-detonation precipitation of the refractory radioactive inventory involves a time-dependent mass transfer from the vapor-ﬁlled cavity to the rock melt or puddle at the bottom of the cavity. This is a complex problem that is dependent on several parameters (e.g., cavity pressure, temperature, yield, cavity-gas composition, etc.) and for the purposes of this paper only a somewhat general and simpliﬁed model is considered here. It is assumed that during the rapid cooling of the post-detonation vapor phase as described in Sect. 2.1, the vaporized iodine precursors (In, Sn, Sb, Te-m, and Te) condense out, mixing with the melt phase in the puddle compart- ment (Pili et al., 2017; Carrigan et al., 2020). Since no signiﬁcant short-term diffusion mechanism has been identiﬁed from the puddle to cavity (Carrigan et al., 2020), any back diffusion from the puddle to the cavity gas is neglected. The rainout is expressed as T ¼ T0 þ 8:95 105 q3:156 R Y 1 3 4:576 q0:411 ; ð2Þ ð2Þ where T [C] is the temperature, T0 [C] is the initial temperature prior to detonation, R [m] is the radial distance from the working point (in a spherical coordinate system), Y [kt] is the energy-equivalent yield, and q [g cm3] is the bulk density of the rock. A simple Newtonian cooling is assumed (Olsen, 1967) for calculating the rainout time as t ¼ 1 b ln Tm T0 Tc T0 ; b ¼ ln 2 tT=2 ð3Þ ð3Þ where Tm [C] is the post-shot temperature in the cavity, Tc [C] is the condensation temperature, b is the temperature decay factor, and tT=2 is the half-time of cavity temperature. Using (2) and assuming a cavity radius of 20 m, the cavity temperature can be calculated as shown in Fig. 3a for an uncompacted soil (1.2 g cm3) and two denser rock types with three different rock densities. The corresponding where Tm [C] is the post-shot temperature in the cavity, Tc [C] is the condensation temperature, b is the temperature decay factor, and tT=2 is the half-time of cavity temperature. Using (2) and assuming a cavity radius of 20 m, the cavity temperature can be calculated as shown in Fig. 3a for an uncompacted soil (1.2 g cm3) and two denser rock types with three different rock densities. 2.1. Cavity Temperature rainout times of In, Sn, Sb, and Te-m/Te are shown in Fig. 3b. The residual heat of a UNE is translated into a temperature distribution in the cavity and surrounding area. The post-shot temperature distribution can be estimated with a Hugoniot phenomenological equa- tion of state model, as described by Butkovich (1974): 2.5. Solution Development Equation (1) is arranged as a lower triangular matrix S ¼ diag ð 0 0 0 0 0 0 zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ}\|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{ precursors s1 z}\|{ 131mXe s2 z}\|{ 131Xe Þ; dc dt ¼ A c; c ¼ ½cc; cp; ch; cv ð11Þ ð11Þ ð7Þ where s1 and s2 are seepage rates of 131mXe and 131Xe. where A is lumped matrix diagonalized as where A is lumped matrix diagonalized as A ¼ SKS1 ð12Þ ð12Þ where K is an eigenvalue matrix of A, S is the matrix whose columns are the eigenvectors of A, and S1 is the inverse matrix of S. The analytical formulation of S and S1 is described in Sun et al. (2012, 2015, and 2021). 2.2. Precipitation of Precursors The corresponding as d dt cc cp ¼ R O O R cc cc ; ð4Þ ð4Þ where R is a diagonal rainout-rate matrix. For (a) (b) Figure 3 a Cavity temperature versus yield number in three different geologic conditions. b Rainout schedule of iodine precursors under Newtonian cooling (b) (a) Figure 3 a Cavity temperature versus yield number in three different geologic conditions. b Rainout schedule of iodine precursors under Newtonian cooling Figure 3 a Cavity temperature versus yield number in three different geologic conditions. b Rainout schedule of iodine precursors under Newtonian cooling Implications of Underground Nuclear Explosion Cavity Implications of Underground Nuclear Explosion Cavity example, the rainout-rate matrix of decay chain 131 (Fig. 2a) can be speciﬁed as occ ot ¼ Pðt0; t1Þ v dcc dx ¼ Pðt0; t1Þ v L ðcc 0Þ ¼ Pðt0; t1Þ q cc ð8Þ where v [m s1] is the velocity of venting ﬂow, L [m] is the venting distance, q [s1] is the mass loss rate due to prompt venting, and Pðt0; t1Þ [–] is a boxcar function deﬁned as ð5Þ where Ti; i ¼ 1; 2; 3; 4, are the assumed conden- sation temperatures of In, Sn, Sb, and Te-m/Te, respectively, ri; i ¼ 1; . . .; 5, are rainout rates, and HðTiÞ is the Heaviside step function with HðTiÞ ¼ 0; 8 T [ Ti and HðTiÞ ¼ 1; 8 T Ti. Pðt0; t1Þ ¼ 0; t\t0 1; t0 t t1 0; t [ t1: 8 > < > : The mass change due to the prompt venting is described in matrix format as The mass change due to the prompt venting is described in matrix format as 2.3. Seepage of Isotopic Xenon d dt cc cv ¼ Q O O Q cc cc : ð9Þ ð9Þ It is also assumed that xenon isotopes produced in the cavity transport away to the host rock, and the mass loss from the cavity, due to excess pressure, is linearly proportional to the mass in the cavity, which is consistent with the ideal gas law. The mass change is expressed as For decay chain 131, Q in Eq. (9) is speciﬁed as Q ¼ diag ð 0 0 0 0 0 0 zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ}\|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{ precursors Pðt0; t1Þ q zﬄﬄﬄﬄﬄﬄﬄ}\|ﬄﬄﬄﬄﬄﬄﬄ{ 131mXe Pðt0; t1Þ q zﬄﬄﬄﬄﬄﬄﬄ}\|ﬄﬄﬄﬄﬄﬄﬄ{ 131Xe Þ; ð10Þ ð10Þ d dt cc ch ¼ S O O S cc cc ; ð6Þ where t0 and t1 [s] are the starting and ending time of prompt venting. ð6Þ where S is the diagonal matrix representing the seepage rates of xenon isotopes from cavity to host rock. For decay chain 131, S in Eq. (6) is speciﬁed as 2.4. Prompt Venting of Isotopic Xenon Prompt release (venting) driven by pressure gradient (between the cavity and ground surface) is described by one-dimensional advection and expressed in the form of ﬁrst-order reaction Deﬁning a ¼ S1c, Eq. (11) is expressed in terms of a as da dt ¼ K a ð13Þ ð13Þ with a transformed initial condition a0 ¼ S1c0 Table 1 Deterministic models with multiple physical processes Y. Sun et al. Pure Appl. Geophys. Pure Appl. Geophys. Y. Sun et al. Deterministic models with multiple physical processes where c0 is the vector of initial concentrations. Each ODE in Eq. (13) is independent of the others, but with the ﬁrst-order decay rate k. In other words, the coupled ODEs (Eq. 11) in terms of c by ingrowth, rainout, seepage, and prompt venting, are decom- posed into N ODEs with the same formulation, xenon signatures. Assuming zero initial inventory in the puddle for 1 kt U235 ﬁssion, four models are conceptualized as listed in Table 1 for demonstrating the effect of source-term processes on xenon inven- tories and isotopic ratios of activities. A reference model (#0) representing the idealized case of isotopic evolution in a cavity determined entirely by the radioactive decay chain (Kalinowski et al., 2010; Kalinowski, 2011; Yamba et al., 2016) is used to compare models #1, 2, 3 for demonstrating the indi- vidual effect of rainout, seepage, and prompt venting, respectively. Model #4 includes all four processes (Table 1) dai dt ¼ ki ai; 8i ¼ 1; 2; . . .; N; ð14Þ ð14Þ where ai is the transformed concentration of ci and the solution of ai is ai ¼ a0 i expðkitÞ; 8i ¼ 1; 2; . . .; N: ð15Þ ð15Þ Using c ¼ S a, the solution of c is Using c ¼ S a, the solution of c is cðtÞ ¼ S expðk1tÞ 0 0 0 expðk2tÞ 0 .. . .. . .. . .. . 0 0 expðkNtÞ 2 66664 3 77775 S1 c0; ð16Þ 3.1. Evolution of Xenon Isotopes Assuming the cavity temperature decaying from 3000 to 20 C with temperature half-life of 600 [s] and a prompt venting between 1 105 [s] (1.16 [d]) and 1.864 105 [s] (2.16 [d]), the xenon inventories of Model #4 in the cavity and promptly vented gas were simulated as shown in Fig. 4. ð16Þ The closed-form solution can be veriﬁed for abso- lutely sequential reactions by comparing the Bateman equation (Bateman, 1910) and for an integrated sys- tem (with all source-term activities) by comparing a numerical solution using ode113s in MATLAB (MathWorks, 2000). The closed-form solution can be veriﬁed for abso- lutely sequential reactions by comparing the Bateman equation (Bateman, 1910) and for an integrated sys- tem (with all source-term activities) by comparing a numerical solution using ode113s in MATLAB (MathWorks, 2000). As shown in Fig. 4a–c, the xenon inventories in the cavity are clearly reduced due to the prompt venting between 105 and 1.864 105 [s]. 135Xe in vented gas peaks during the venting period due to its short half-life, while other three xenons peak after the one-day venting (Fig. 4d). A late-time venting may not contain detectable signature 135Xe because of its short half-life. The inventory proﬁles shown in Fig. 4 can be used to calculate isotopic ratios of xenon radioactivities. Fig. 5a shows the Multi-Isotope Ratio Chart (MIRC, the correlation of 133mXe/131mXe and 135Xe/133Xe) in the cavity and host rock with individual and combined source activities. While 3. Results and Analyses In this section, we simulate the evolution of xenon isotopes in the cavity, melt puddle, host-rock, and promptly vented gas, and xenon isotopic ratios for demonstrating the impact of source-term activities on Implications of Underground Nuclear Explosion Cavity (a) (b) (d) (c) Figure 4 Inventories of xenon isotopes in cavity and promptly vented gas. a 131mXe and 131Xe. b 133mXe and 133Xe. c 135mXe and 135Xe. d 131mXe, 133mXe, 133Xe, and 135Xe in promptly vented gas (a) (c) Figure 4 Inventories of xenon isotopes in cavity and promptly vented gas. a 131mXe and 131Xe. b 133mXe and 133Xe. c 135mXe and 135Xe. d 131mXe, 133mXe, 133Xe, and 135Xe in promptly vented gas g pes in cavity and promptly vented gas. a 131mXe and 131Xe. b 133mXe and 133Xe. c 135mXe and 135Xe. d 131mXe, 133mXe, 133Xe, and 135Xe in promptly vented gas Inventories of xenon isotopes in cavity and promptly vented gas. a 131mXe and 131Xe. b 133mXe and 133Xe. c 135m 133mXe, 133Xe, and 135Xe in promptly vented gas Test Site, with a known detonation time (Ringbom et al., 2014), may indicate the precursor precipitation occurred in a leaky system. Three Japanese data sets that are located below the ER curve (blue curve) and close to the 131mXe/133Xe ratio in host rock (green curve) reﬂect a possible seepage from the cavity to host rock since those samples were taken off site. Both 131mXe/133Xe ratios in cavity (red curve) and host rock (green curve) behave similarly after 0.2 d in this example. If those samples (circles) were taken from the cavity directly, the red curve might be used to interpret the early-time precipitation. Two Russian data points (green squares) located above the ER curve may indicate a possible venting from the cavity. Test Site, with a known detonation time (Ringbom et al., 2014), may indicate the precursor precipitation occurred in a leaky system. Three Japanese data sets that are located below the ER curve (blue curve) and close to the 131mXe/133Xe ratio in host rock (green curve) reﬂect a possible seepage from the cavity to host rock since those samples were taken off site. Both 131mXe/133Xe ratios in cavity (red curve) and host rock (green curve) behave similarly after 0.2 d in this example. If those samples (circles) were taken from the cavity directly, the red curve might be used to interpret the early-time precipitation. 4. Discussion and Conclusion For predicting time-dependent isotopic evolution, the idealized standard model (ER) assuming a batch- mode closed cavity may not be appropriate when iodine precursors precipitate from the cavity to the melt puddle, xenon isotopes transport to host rock, or prompt venting occurs. We extended our closed-form solution to a multi-compartment system coupled with complex radioactive decay/ingrowth networks, tem- perature-dependent precursor precipitation, cavity gas seepage, and speciﬁed prompt venting. The standard model (#0 in Table 1) serves as a reference with deviations potentially indicating precipitation of refractories, xenon seepage, and/or prompt venting have occurred and if the cavity is closed. For 3.2. Effect of Rainout on Xenon Activity Ratios and produces 131mXe/133Xe ratio higher than the curve subject to idealized batch-mode conditions. Similarly to seepage effect, the time of prompt venting (Model #3) deviates the MIRC (Fig. 7c) and 131mXe/133Xe ratio (Fig. 7d) curves away from the standard ER curves after the prompt venting starts. Although a different venting rate q alters xenon inventories in the cavity, it does not change the MIRC and 131mXe/133Xe ratio. The time scale of seepage and venting is referred to OTA (1989). In absence of seepage and prompt venting, the rainout effect on both MIRC and 131mXe/133Xe ratio in the cavity is simulated using Model #1 and shown in Fig. 6a, b for three different rainout rates [1 104, 1 103, 1 102] [s1] under Newtonian cooling condition from 3000 to 20 C with temper- ature half-life of 600 [s]. As shown in Fig. 6a, a fast rainout moves the correlation to the right away from the ER curve (batch-mode closed without rainout). Similarly, the fast rainout results in a low ratio of 131mXe/133Xe (Fig. 6b). In addition to the rainout-rate effect, the temperature proﬁle also affects MIRC and 131mXe/133Xe ratio. Fig. 6c, d show the MIRC and 131mXe/133Xe ratio for tT=2 ¼ [300 600 1200] [s] and r ¼1 103 [s1]. All possible rainout scenarios (for given r and tT=2) move both MIRC and 131mXe/133Xe ratio to the right side of their ER curves. 3. Results and Analyses Two Russian data points (green squares) located above the ER curve may indicate a possible venting from the cavity. rainout shifts the England and Rider curve (blue) to the right (see also Carrigan et al., 2020), seepage (cyan) and prompt venting (dashed magenta) move the curve back to the left on the plot. The time- dependent ratio of 131mXe to 133Xe (in the cavity) shown in Fig. 5b indicates that rainout brings the England and Rider (ER) curve (Model #0, blue) down toward the x-axis while both seepage and prompt venting lift the ratio value above the blue curve under the ER condition. The comparison between observed data and Model #0 curve reveals if the system is closed with rainout or open with possible seepage or prompt venting. The observed data of 131mXe/133Xe ratio from atmospheric samples associated with the February 2013 declared DPRK UNE at the Punggye-ri Nuclear (a) (b) Figure 5 a Correlation of xenon isotopic ratios between 133mXe/131mXe and 135Xe/133Xe, and b ratio of 131mXe/133Xe versus time in the cavity. The vertical dashed lines in both a and b indicate the starting time of prompt venting. ‘(c)’ and ‘(h)’ in ﬁgure legends indicate model results in cavity and host rock, respectively Y. Sun et al. Pure Appl. Geophys. (a) Y. Sun et al. Y. Sun et al. Pure Appl. Geophys. (b) Figure 5 a Correlation of xenon isotopic ratios between 133mXe/131mXe and 135Xe/133Xe, and b ratio of 131mXe/133Xe versus time in the cavity. The vertical dashed lines in both a and b indicate the starting time of prompt venting. ‘(c)’ and ‘(h)’ in ﬁgure legends indicate model results in cavity and host rock, respectively 3.3. Effect of Seepage and Prompt Venting on Xenon Activity Ratios When s is assumed to be [1 106, 1 105, 1 104] [s1] without considering rainout and prompt venting, the MIRC and 131mXe/133Xe ratio in the cavity are simulated using Model #2 as shown in Fig. 7a, b. A high seepage rate moves the MIRC correlation away from the ER curve towards the left Implications of Underground Nuclear Explosion Cavity (a) (b) (d) (c) Figure 6 a Effect of rainout rate on MIRC with tT=2 = 600 [s]. b Ratio of 131mXe/133Xe versus time with different rainout rates with tT=2 = 600 [s]. c Effect of temperature half-life on MIRC with r ¼ 1 103 [s1]. d Ratio of 131mXe/133Xe versus time with different temperature half-lives (Fig. 3b) with r ¼ 1 103 [s1] Implications of Underground Nuclear Explosion Cavity (b) (a) (d) (c) Figure 6 a Effect of rainout rate on MIRC with tT=2 = 600 [s]. b Ratio of 131mXe/133Xe versus time with different rainout rates with tT=2 = 600 [s]. c Effect of temperature half-life on MIRC with r ¼ 1 103 [s1]. d Ratio of 131mXe/133Xe versus time with different temperature half-lives (Fig. 3b) with r ¼ 1 103 [s1] example, data points of cavity samples located above the ER curve in 131mXe/133Xe(t) (Fig. 5b) indicate possible seepage or prompt venting while those data points of the cavity samples below the ER curve demonstrate an early-time precipitation of iodine precursors. Only early-time processes (i.e., precipi- tation, seepage, and prompt venting) under thermal conditions are considered in our model and analytical solution. Late-time driving force of gas transport (e.g., barometric pumping) in fractured rock is not considered. A prompt venting that can be physically deﬁned by operational activities (e.g., starting and ending time), uncertain hydrodynamic and geological conditions (e.g., depth of burial, rock properties, venting pathways) is described by venting rate q with a boxcar function (Pðt0; t1Þ in Eq. 8). Although the Newtonian cooling is used in this paper for describing the fast cooling processes (from 3000 to 988 C), the true temperature proﬁle either from an onsite moni- toring or hydrodynamic modeling can be inputted to calculate precursor precipitation schedule. Then, the difference between observed xenon activity ratios and those simulated using ER model may be possibly used to calculate yield number inversely and recover historic source-term activities. 3.3. Effect of Seepage and Prompt Venting on Xenon Activity Ratios If the cavity temper- ature remains above 988 C for a long time (e.g., tT=2 [ 100 [d]), the precursors of iodine will have enough time to decay away for complete xenon (a) (b) (c) (d) Figure 7 a Effect of seepage on MIRC. b Effect of seepage on 131mXe/133Xe ratio. c Effect of venting time on MIRC. d Effect of venting time on 131mXe/133Xe ratio. Venting duration is 1 day and venting rate is 1 102 [s1] Y. Sun et al. Pure Appl. Geophys. (a) Y. Sun et al. Y. Sun et al. Y. Sun et al. Pure Appl. Geophys. (b) (d) (c) Figure 7 a Effect of seepage on MIRC. b Effect of seepage on 131mXe/133Xe ratio. c Effect of venting time on MIRC. d Effect of venting time on 131mXe/133Xe ratio. Venting duration is 1 day and venting rate is 1 102 [s1] g a Effect of seepage on MIRC. b Effect of seepage on 131mXe/133Xe ratio. c Effect of venting time on MIRC. d 131mXe/133Xe ratio. Venting duration is 1 day and venting rate is 1 102 [s1] radioxenon, which will have the effect of shifting the MIRC toward the ER ingrowth curve and away from the rainout curve. Future analytical models will attempt to quantify this effect of iodine loss during the period of rapid venting. production in the cavity and the effect of rainout is eliminated. Then, the analytical solution with r ’ 0 applies for seepage and prompt venting only. The generalized analytical solution can be used as a handy and robust tool for conducting sensitivity analyses and verifying high-ﬁdelity computer codes with a low computational cost. Acknowledgements In this paper, we have only considered precipita- tion of the refractory precursors of iodine. We have assumed most or all iodine remains in the cavity gas phase and does not precipitate into the underlying melt or onto hot cavity surfaces. Such an assumption is probably justiﬁable for gas seepage rather than for rapid venting. If rapid venting occurs, it is expected that isotopes of iodine may be released with We thank anonymous reviewers for their constructive reviews and helpful comments leading to an improved manuscript. This research was funded by the National Nuclear Security Administration, Defense Nuclear Nonproliferation Research and Development (NNSA DNN R&D), US Department Implications of Underground Nuclear Explosion Cavity of Energy and performed under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under Contract number DE- AC52-07NA27344. 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https://openalex.org/W2398007338	http://www.zurnalai.vu.lt/vertimo-studijos/article/download/10000/7846	English	null	EVALUATING MACHINE TRANSLATION QUALITY: A CASE STUDY OF A TRANSLATION OF A VERBATIM TRANSCRIPTION FROM SLOVAK INTO GERMAN	Vertimo studijos	2,017	cc-by	6,330	ISSN 2029-7033. VERTIMO STUDIJOS. 2015. 8 ISSN 2029-7033. VERTIMO STUDIJOS. 2015. 8 EVALUATING MACHINE TRANSLATION QUALITY: A CASE STUDY OF A TRANSLATION OF A VERBATIM TRANSCRIPTION FROM SLOVAK INTO GERMAN Jozef Štefčík Department of Translation Studies Faculty of Arts Constantine the Philosopher University in Nitra, Slovakia jstefcik@ukf.sk Jozef Štefčík The paper addresses the issue of using online statistical machine translation tools for the transla tion of specific text types and the problems of their translatability when using automated transla tion systems. An attempt is made to analyze and evaluate the machine translation of a verbatim transcription from Slovak into German. WHY IS MACHINE TRANSLATION BECOMING MORE IMPORTANT? Over the last decade, machine translation (MT) has become an important research topic in both the academic sphere and commercial sector. Translation is essential for international trade, not only because of the need to translate commercial contracts and legislation, but also for the use of instructions and manuals for imported products. Another aspect of the commercial interest in MT involves the cost of human translation and translation services, particularly in view of the fact that translation is highly demanding work that requires an exceptional degree of professionalism and considerable amount of time. A translator can usually translate no more than 9–10 pages a day (depending on the subject and complexity), which sometimes leads to delays, for example, in marketing a new product. This is also a major issue for EU institutions owing to the need to translate legislation into the 24 official languages of the European Union, along with numerous other documents. The cost and speed of translation are major factors in determining the social, political and economic importance of MT in many other areas of human activity. For these reasons, IT specialists and researchers are continuing to develop MT from common online systems (Google Translate, Bing Translate, etc.) through to the sophisticated systems designed for the specific needs of companies (e.g. DGT uses the Moses system). There is also a philosophical dimension to MT in the endeavour to 139 Jozef Štefčík automate human thinking and linguistic production. “From an academic perspective, MT is interesting because it allows the application and testing various hypotheses in linguistics, computer science and artificial intelligence” (Munková 2013). Thus, MT has achieved a considerable advantage in speed and quality in recent years. Its usage is changing the status of professional translators and creating a new role for them as pre-editors or post-editors of MT. The very notion of translation is changing. Currently, however, MT is used with caution where a high degree of quality is required, but it is also a recognized fact that certain text types allow MT to be more successfully applied than others. 2) In großen Organisationen, die sich mit einer großen Anzahl von Kunden ______, ist es wichtig für das reibungslose Funktionieren der verschiedenen Abteilungen und Geschäftsprozesse, dass die neuesten Informationen über die Kunden zur Verfügung steht.“ TEXT ANALYSIS AND TEXT TYPES FOR THE PURPOSES OF MT Most text types are more or less hybrid in form. Translators must, therefore, decide whether the relevant source text can be machine-translated or not. Trained and qualified translators have an advantage over untrained translators because they can perform macro-and micro-stylistic translation-relevant text analysis faster and more easily, which enables them to overcome time constraints. They can quickly decide which texts or text segments can be translated by people or by MT, or in which segments the re- edited text would require only minimal changes. Some text types have proven to be relatively reliable when translated by MT: technical, legal, marketing and management, tourism and catering, manuals, instructions, EU directives, regulations, insurance contracts and the like. For this reason we believe it would be most useful to analyse the following text types in the most frequent language pairs and how MT renders them: manuals, reference books, scientific reports, records, certificates, balance sheets and other financial statements. iii Here is a brief example of a machine-translated text into English: 1) In large organizations dealing with large numbers of customers it are essential for the effective operation of various departments and business processes did the latest customer information is available”. (In: “Managing Customer data, 2007 Global Industries”) 1) In large organizations dealing with large numbers of customers it are essential for the effective operation of various departments and business processes did the latest customer information is available”. (In: “Managing Customer data, 2007 Global Industries”) The translation contains mistakes, but the content is understandable, and a translator can easily post-edit such a text. The German variant of the MT of the same text via Google Translate (analytical language) is as follows: 2) In großen Organisationen, die sich mit einer großen Anzahl von Kunden ______, ist es wichtig für das reibungslose Funktionieren der verschiedenen Abteilungen und Geschäftsprozesse, dass die neuesten Informationen über die Kunden zur Verfügung steht.“ 140 Evaluating Machine Translation Quality: A Case Study A few words are missing in this translation: the verb to deal with and the adjective available are not translated at all (… von Kunden beschäftigen (…) zur Verfügung stehen accordingly), but after post-editing the text would be understandable. TEXT ANALYSIS AND TEXT TYPES FOR THE PURPOSES OF MT One might expect even better results if, prior to translation, each text were subjected to a text analysis based on the following points (Byrne 2012, 90): 1) topic; 2) text category; 3) text function; 4) target audience; 5) purpose of the text (how will the text be used); 6) distinguishing features. 7) potential problems in translation Translation-relevant text analysis gives us more information about the difficulties to be expected when translating and the external tools that can help translators to produce better quality within a limited timeframe. After textual macro-analysis, the translator might consider microanalysis, which can be based on several steps. Arnold (1994) proposed three steps in the process of MT:h 1) Preparation of input (pre-editing): intra-linguistic transfer. The translator makes the source text “translatable” by simplifying it on a linguistic level; 1) Preparation of input (pre-editing): intra-linguistic transfer. The translator makes the source text “translatable” by simplifying it on a linguistic level; 2) Translation using a translation system – an inter-linguistic transfer. This is a typical translation process, which consists of three common steps: analysis, transfer, synthesis. 3) Revision of translations – an intra-linguistic transfer. In this phase, the post- editing, revising and proofreading of the translated text (“raw translation”) are important. These steps of pre-editing and post-editing are shown in Table 1 (intra-language translation) and Table 2 (inter-language translation) below. Table 1. A sample of pre-editing of the text for MT (Kit, Pan, Webster 2002, 44). Source text Pre-Editing of the Source text Let the water run hot at the sink and then pull the connector from the recess in the back of the dishwasher. Upon the completion of the above task, lift the connector to the faucet by pressing down the thumb release. 1. Turn on the faucet at the sink until the water runs hot. 2. Pull the connector from the recess in the back of the dishwasher. 3. Press down on the thumb release and lift the connector onto the faucet. Table 1. A sample of pre-editing of the text for MT (Kit, Pan, Webster 2002, 44). 141 Jozef Štefčík Table 2. A sample of post-editing of the same text after MT translation into German. Target text – Translation into German Target text of the pre-edited version Lassen Sie das Wasser laufen heiß in der Wanne und dann ziehen Sie den Stecker aus der Aussparung in der Rückseite der Spülmaschine. TEXT ANALYSIS AND TEXT TYPES FOR THE PURPOSES OF MT Nach Abschluss der oben genannten Aufgabe, heben Sie den Anschluss an den Wasserhahn durch Drücken des Daumen Release. 1. Drehen Sie den Wasserhahn an der Spüle, bis das Wasser heiß ist. 2. Ziehen Sie den Stecker aus der Aussparung in der Rückseite der Spülmaschine. 3. Drücken Sie auf den Daumen Release und heben Sie den Stecker auf den Wasserhahn. Table 2. A sample of post-editing of the same text after MT translation into German. It is clear that the pre-edited text provides a better output. It could be edited much faster than the text on the left, which has a more complicated and longer sentence structure. TRANSLATION OF A SPECIFIC TEXT TYPE: WRITTEN RECORDS Records belong to a commonly used text type. Written records (e.g. transcripts, minutes, protocols, and all kinds of written documents containing factual material) document reported speech from events that have already taken place (with the exception of a memory record) and rely on descriptions and observations. There are different types of written records: • Based on results (streamlined, structured summaries that keep essential and non-essential information apart), • Based on results (streamlined, structured summaries that keep essential and non-essential information apart), • Based on history (chronological, most realistic representation of the time sequence, the essential and the non-essential equally documented), • Based on history (chronological, most realistic representation of the time sequence, the essential and the non-essential equally documented), • Based on purpose (retrieved from memory or from recordings). Each written record should be accurate (precise language), objective (no personal opinions), non-judgmental (no observations or judgments), and positive (FLT). The source text of our written record selected for analysis was stylistically dense and contained administrative, narrative and technical features; the goal was to describe what was discussed in the communication act objectively and in detail. The analysed source text of the record had several functions – it reported, instructed, and advocated at the same time. The text contained many verbs and subordinate clauses. The text also had many time references, names and technical terms that referred to specific areas and operations. The main problems in translating any written record is the information within the text itself, represented by second or third parties, which may prove difficult to decode. The authors of the records tend to use originally reformulated phrases from 142 Evaluating Machine Translation Quality: A Case Study of a Translation of a Verbatim Transcription from Slovak to German speeches that were recorded on audio and transcribed without the necessary attention to context. Since external translators often lack the relevant context, they are mostly unable to get the message across. We therefore want to determine the extent to which the studied hybrid text can generate linguistically appropriate output when prepared for revision by using the online system of statistical MT, Google Translate. EVALUATING MT The evaluation of MT plays a key role in the field of MT and there have certainly been many attempts to evaluate it (Hutchins and Somers 1992, Arnold et al. 1994, Callison-Burch et al. 2006, to name but a few). In order to make evaluation more efficient, experts have begun to think about automatic evaluation methods without human intervention. There have been projected series of automated quality assessment metrics to achieve the effectiveness in the evaluation process. Automatic evaluation metrics were used for additional human evaluation, while providing high efficiency and consistency at a relatively low cost. Most of these are based on measuring similarities between automated translations and referential–human–translation. Automatic evaluation metrics can be based on statistical principles (n-grams or editing distance) or on deep linguistic structures (morphological, syntactic or semantic information). The evaluation of MT or of MT systems is an essential area of research not only in an attempt to determine the effectiveness of existing MT systems, but also to optimize their performance. Progress in MT relies on the quality of newly developed MT systems, the goal of their evaluation being to demonstrate a greater effectiveness than that of existing systems. However, here we stumble on the question of translation quality (texts generated by MT systems), and more exact methods of quality evaluation criteria. Is it possible to claim that this particular translation is the only correct translation of the original and there is no other correct translation? How to evaluate the quality of two translations that are not identical but both represent the original? Or two translations that are only partially correct? It is a very difficult task which is affected by several factors. It primarily depends on the recipient (for whom is the evaluation of MT performed?) and its further use (for what purpose is the evaluation of MT used?). Style in translation can be crucial in some applications and irrelevant in others. Therefore we avoid using the term “good” translation, but rather “appropriate” or “adequate” translation. The evaluation of MT can be approached in two ways: Glass Box evaluation or Black Box evaluation. Glass Box measures the quality of the evaluation system based on system characteristics. It focuses on the linguistic coverage of the system and the theory used in natural language processing. This type of evaluation would be relevant for scientists and developers. TRANSLATION QUALITY ASSESSMENT There have been many discussions over the last decade about the evaluation of translation quality, or Translation Quality Assessment (hereinafter referred to as TQA), which is equally important for both the professional quality control of content and for study programmes that prepare would-be translators for the profession. Although there is a widely acknowledged need to establish the general criteria for assessing the quality of translations or to come up with a definition of what might be considered “good, satisfactory or accepted” translation, there is still no universal definition of translation quality or even generally accepted methods of evaluation. Although there are national and international standards of translation (ATA, Sical, etc.), they are not widely accepted as objective criteria for assessing the quality of translation. Basically, when assessing the quality of a translated text, we tend to stick to the following criteria: • linguistic correctness,i • fidelity to the source text, • readability of the target text, • equivalence, • transfer of the meaning (appropriateness, shifts…) (Ackaert et al. 2013) • transfer of the meaning (appropriateness, shifts…) (Ackaert et al. 2013). These general criteria may be categorized into several subgroups or attributed to the following translation-errors, according to which professional translators and translation instructors may assess translated texts: omissions, negative shifts of meaning, register, punctuation, spelling, grammar, style and vocabulary (cf. also Vilar et al. 2006).h These are the basic aspects to consider in evaluating translations. However, translations cannot be restricted to the learning environment, or to one purpose or target group. We may generally claim that translation quality should be perceived from the point of view of its recipients, their needs, and their knowledge of the subject matter, etc. This could be regarded as a final text approach based on human or manual evaluation. On the other hand, the evaluation of MT requires a different approach, since the output (translated text) cannot be regarded as a final text in need of revision. The evaluation of MT can be done manually or with the help of certain software, i.e. it may be performed automatically. 143 Jozef Štefčík EVALUATING MT Scientists would need it in terms of confirming or rejecting hypotheses. Developers attempt to figure out if the MT system works the same way it was projected to in order to determine its limits. Black Box measures the 144 Evaluating Machine Translation Quality: A Case Study of a Translation of a Verbatim Transcription from Slovak to German Evaluating Machine Translation Quality: A Case Study of a Translation of a Verbatim Transcription from Slovak to German quality evaluation system based on the generated translation. This type of evaluation is intended for recipients and translators. Translators need to know whether the use of the system will improve their productivity from the point of view of quantity and post-editing. The recipient is interested in the cost, speed and readability of the translation. This is why we also focus on the Black Box evaluation in our study, which uses internal (intrinsic) and external (extrinsic) methods to evaluate the accuracy and applicability of MT. Internal methods (evaluation scales, trial order, error analysis, etc.) subjectively assess the quality of MT based on the comparison (hypothesis) with a referential translation which is considered to be the “gold standard”. Evaluators subjectively evaluate the main characteristics of reliable translation quality, such as the adequacy and fluency of MT text. External methods, called the task-oriented methods (post-editing or reading with comprehension) are focused on efficiency and text usability with regard to a specific task. Automatic internal metrics do not require human intervention. It represents a significant breakthrough in the field of MT in terms of the quality of automatic evaluation and MT system optimization. Automatic metrics (accuracy, coverage, WER, PER, BLEU and others), which determine the quality of translation or errors through comparison, calculate the similarity between hypothesis and reference translation (and a given set of reference translations) and provide a relatively rapid feedback on the quality of the system as well as the newly- created text generated by the MT system. With the introduction of IT-technologies as a tool for computer-assisted translation memories a new era of translation came into existence. At present, translatology is considered an interdisciplinary science working in conjunction with other disciplines. The idea of interdisciplinary science is based on the hypothesis that a natural language can use a variety of symbols; it can be fully analysed, controlled and mathematically encoded. 1. F-measure (Precision and Recall)h These are the easiest automatic evaluation metrics and are often used in natural language processing. They are based on a word congruency hypothesis with the words in the referential translation, regardless of the word position in a sentence. They have a mutually opposite relationship, which means that increasing accuracy scores may lead to the reduction of the coverage score and vice versa. A disadvantage of accuracy metrics is where the hypothesis falls short in terms of the number of words, but acquires a high accuracy score (but low coverage). The reverse also applies: we can get a hypothesis containing all possible words, which increases the likelihood that some of these words will also appear in the referential translation, but the hypothesis is too long, with a high score coverage, but low accuracy score. The question is how to solve this problem. We do not want to generate a sentence (hypothesis) that would include misspelled words, but we do not want to have any omissions as well. Therefore, experts from the field of MT come up with the F-measure, also known as the F-score. It is actually the harmonic mean of the two metrics (accuracy and coverage). EVALUATING MT With MT, this primary hypothesis of interdisciplinary studies was extended following the results of experimentation with natural language processing. This is based on the fact that language is so rich and complex that it cannot be completely analysed and split into a set of rules that can be subsequently encoded as computer program algorithms. There have been many suggestions of how to measure quality, some focusing on target specific syntactic constructions, others assessing various sentences as a whole on the N-point scale or on automated translations with a reference. However, these methods have been mainly tested on major languages (English and other world languages). There have been very few attempts to focus on inflectional languages such 145 Jozef Štefčík as Slovak, with its specific morphological richness. In the present paper we focus on the evaluation of MT in the language pair Slovak (Source) vs. German (Target). In our evaluation we use the following metrics: 2. WER (Word-error rate) WER metrics or error word rate was first used in the evaluation of statistical MT. It belongs to the first generation metrics of automatic MT evaluation systems. WER rate was taken from the field of speech recognition and is based on the edit distance taking into account the word order. The edit distance is represented by the Levenshtein distance, which is defined as a minimum number of single-character edits (insertion, removal and substitution) needed to achieve a conformity of two sequences (sentences). 3. PER (Position-independent Error Rate)h 3. PER (Position-independent Error Rate)h This is occasionally used in the evaluation of MT. It is similar to metrics coverage by using the same denominator – the length of the referential translation or the number of words in the reference. As the name suggests, it is a certain degree of error rate. It does not measure congruence, but the mismatch. It takes into account defective and superfluous words which must be removed in long translations. 146 Evaluating Machine Translation Quality: A Case Study of a Translation of a Verbatim Transcription from Slovak to German Here are some examples from the source text: Here are some examples from the source text: 3) „Priestorové pokrytie považuje za dostatočné...“ (literal translation as „räumliche Deckung“ – this phrase does not exist either in the source text or in the target text) 4) ak sa urobia opatrenia v jednej časti nie je možné to uzavrieť tým, že tam to 3) „Priestorové pokrytie považuje za dostatočné...“ (literal translation as „räumliche Deckung“ – this phrase does not exist either in the source text or in the target text) 4) „….ak sa urobia opatrenia v jednej časti, nie je možné to uzavrieť tým, že tam to končí...“(unclear wording, too many demonstrative pronouns without factual reference) 5) „... treba mať k dispozícii zoznam chránených druhov a živočíchov a voči nim vykonávať prieskum“ (stylistically unclear – to make an investigation against sb.) 5) „... treba mať k dispozícii zoznam chránených druhov a živočíchov a voči nim vykonávať prieskum“ (stylistically unclear – to make an investigation against sb.) 6) „...Údaje majú zhŕňať celoročný aspekt…“ (the nominal expression is redundant) 7) „....v otázke migrácie živočíchov nie je dôležité okrem smernice na ochranu vtáctva zohľadniť aj existenciu Alpsko-karpatského koridoru pre vysokú zver...“ (the phrase in bold is superfluous and disturbs the cohesion, coherence, and thus the meaning of the whole statement) 7) „....v otázke migrácie živočíchov nie je dôležité okrem smernice na ochranu vtáctva zohľadniť aj existenciu Alpsko-karpatského koridoru pre vysokú zver...“ (the phrase in bold is superfluous and disturbs the cohesion, coherence, and thus the meaning of the whole statement) 8) „...1x/rok je možné podať žiadosť o zmenu projektu a ešte stále je priestor na zareagovanie...“ (a vague formulation – an opportunity for a response) 9) „…Otázka premietnutia do finančných otázok je zatiaľ neznáma.“ (stylistically unclear and vague) Suggestion: Since neither machine nor translator is capable of translating unclearly formulated sentences into the target language without background knowledge, the original texts should be adapted (pre-edited) before translation. Similarly, it appears essential to instruct the source text writers about how the texts should be written. The source text should be as unambiguous and concise as possible. 2. Variable output quality (MT and referential translation) from a linguistic perspective. In the first phase we translated the target text (translation product) without using Google Translate (human translation with the native German editor). Based on the referential translation we evaluated the statistical MT (Google translation) of the written record by measuring its quality. Case study: Translation of a record from Slovak into German by using the online system of MT In our experiment we used an automated evaluation with metrics of a written record (“verbatim transcript of a meeting”) which was translated from the Slovak language into German without pre-editing. It was a 12-page text documenting a working meeting within the framework of an EU-project involving cross-border regional cooperation between Slovakia and Austria (RECOM). There were two approaches used during the translation process: MT with the “statistical system of MT” and the classical computer-assisted translation with electronic dictionaries. There were two reasons for choosing the online system of MT in our case study: 1. Easy access for any user working online. 2. Online system of statistical MT is the only system that can translate from a large number of language pairs (even from and to synthetic analytic languages). 2. Online system of statistical MT is the only system that can translate from a large number of language pairs (even from and to synthetic analytic languages). The translation (referential translation – RT) in our case study has been revised by a German native speaker. Afterwards, both outputs were compared by a software program MT evaluator (this program was developed in close cooperation between the Institute for Computer Science and the Institute of Translation studies at Constantine the Philosopher University in Nitra). In the qualitative analysis of the translated text using statistical MT, we have taken two important criteria into consideration: 1. F-measure – transfer of the lexically most appropriate words and phrases (adequate, faithful reproduction). 2. WER – syntactical word order appropriateness (linguistic correctness and readability). We have also partially dealt with some shortcomings in the original text which contributed to some mistranslations. The following are problem areas that we have encountered: We have also partially dealt with some shortcomings in the original text which contributed to some mistranslations. The following are problem areas that we have encountered: 1. A lack of input quality from a linguistic perspective. The first difficulty for the translator was some unintelligible text segments, formulations, or sentences in the source text. It was a literal transcript of the audio-discourse that included implicit or unclearly formulated information. The incomprehensibility 147 Jozef Štefčík occurred because of artificial ad-hoc language that was very difficult to understand in the source text without background-knowledge, which is why it was not easy to transfer the text into the target language without consulting the producer of the text. Here are some examples from the source text: The following table shows the percentage results in the congruence between the referential translation and the statistical online MT (Google Translate) from the selected 22 text segments. 148 Evaluating Machine Translation Quality: A Case Study of a Translation of a Verbatim Transcription from Slovak to German Table 3. BLEU 1, 2, 3, 4 – the number of consecutive correct word combinations (1 word phrase, 2 phrases, etc.) (in %) Table 3. BLEU 1, 2, 3, 4 – the number of consecutive correct word combinations (1 word phrase, 2 phrases, etc.) (in %) Precision Recall F-measure PER WER BLEU 1 BLEU 2 BLEU 3 BLEU 4 33,33 50 40 50,5 100 33,33 12,5 0 0 33,33 25 28,57 75 75 25 18,75 10,71 0 58,33 50 53,84 50 50 50 31,17 17,14 9,52 21,43 20 20,69 80 86,67 20 7,18 0 0 71,43 55,56 62,5 44,44 55,56 55,56 25,93 0 0 52 37,14 43,33 62,86 77,14 37,14 8,93 0 0 25 27,27 26,09 72,82 90,91 25 9,09 0 0 41,46 37,78 39,53 62,22 82,22 40 6,83 2,34 0 25 33,33 28,57 67 106,67 35 5,26 0 0 44,44 30,77 36,36 69,23 73,08 34,62 16,29 12,98 9,23 21,43 19,35 20,34 80,65 93,55 22,58 3,35 0 0 25 25 25 75 83,33 33,33 0 0 0 33,33 25 28,57 75 75 25 6,82 0 0 16,67 16,67 16,67 83,33 91,67 16,67 0 0 0 44,44 38,71 41,38 61,29 70,97 38,71 13,4 0 0 20 14,29 16,67 85,71 100 14,29 0 0 0 22,86 27,59 25 72,62 110,34 25,71 0 0 0 33,33 37,5 35,29 62,62 75 33,33 25 14,29 0 29,41 25 27,03 75 95 25 0 0 0 27,78 22,73 25 77,27 86,36 22,73 4,81 0 0 33,33 36,36 34,78 63,73 109,09 33,33 18,18 10 0 41,67 29,41 34,48 70,59 76,47 29,41 6,42 0 0 755 684,46 709,69 1516,88 1864,03 675,74 219,91 67,46 18,75 34,31818182 31,11182 32,25864 68,94909 84,72864 30,71545 9,995909 3,066364 0,852273 Analysis of the best and worst MT results according to the in the referential translation (RT) and MT (MT) In the following lines we tried to pick out the 3 best and the 3 lowest results of MT from the 22 measured segments by using the metrics of F-measure (an average of precision with coverage). 149 Jozef Štefčík ment) Ansprache des Bü ohenau an der Ma d sentence – G anged its form in nwesenden das Pro ted by its synon llen means to in er, two different n the MT) wh anhand von Unte T: Gefolgt von einer Rede des Burgermeisters Hohenau an der March p F. Two words are missing in the translated sentence – Gemeinde – village, and dann – then. The main verb folgen-folgte has changed its form into a participle without changing the whole meaning. 11) Referential translation: Herr Pr. hat den Anwesenden das Projekt BBMH vorgestellt. MT: P Pr. fuhrte das vorliegende Projekt BBMH. 11) Referential translation: Herr Pr. hat den Anwesenden das Projekt BBMH vorgestellt. MT: P Pr. fuhrte das vorliegende Projekt BBMH. 11) Referential translation: Herr Pr. hat den Anwesenden das Projekt BBMH vorgestellt. MT: P Pr. fuhrte das vorliegende Projekt BBMH. In this short sentence the verb was substituted by its synonym (vorstellen – führen), which causes a slight misunderstanding. Vorstellen means to introduce, führen means “to lead” or “to show someone round”. Moreover, two different tenses are used (Perfekt in the referential translation and Präteritum in the MT) which are not stylistically compatible. 12) Referential translation: Das Ziel ist es, anhand von Untersuchungen eine Studie zu erarbeiten und eine geeignete Variante für die Lösung der ganzjährigen Erschließung von Zufahrtswegen zu der Brücke über den Grenzübergang Moravský Sv Ján Hohenau. MT: Das Ziel der Umfrage ist es eine Untersuchung durchzufuhren und empfehlen eine geeignete Alternative zur Bereitstellung Ganzjahrig Zufahrten zur Brucke Grenzubergang mahrischen Vol John Hohenau. MT: Das Ziel der Umfrage ist es eine Untersuchung durchzufuhren und empfehlen eine geeignete Alternative zur Bereitstellung Ganzjahrig Zufahrten zur Brucke Grenzubergang mahrischen Vol John Hohenau. The MT generated one additional element (Umfrage- survey) that causes a shift in meaning. From the lexical perspective there is confusion of the words Lösung (solution) and Bereitstellung (provision). The adjectives were written in capital letters and the geographical names transferred into the target language, which is not acceptable. On the other hand, the MT system generated synonyms which do not alter the contextual meaning: the synonyms durchführen – erarbeiten (carry out research), Lösung – Alternative. The omitted prepositions – über den Grenzübergang (through the checkpoint) – disrupt the cohesionof the text. The best results of F-measure (3. 5. 6. segment) The best results of F-measure (3. 5. 6. segment) 10) Referential translation: Dann folgte die Ansprache des Bürgermeisters der Gemeinde Hohenau an der March, Herrn F. 10) Referential translation: Dann folgte die Ansprache des Bürgermeisters der Gemeinde Hohenau an der March, Herrn F. f l d d h d h f Hohenau an der March, Herrn F. MT: Gefolgt von einer Rede des Burgermeisters Hohenau an der March p F. MT: Gefolgt von einer Rede des Burgermeisters Hohenau an der March p F. The worst results of F-measure (11.14.16. segment) 13) Referential translation: Es geht um die Termine, wann man die Untersuchungsergebnisse, Fokussierung, Bestimmung des Wasserspiegels und Empfehlungen aus der Sicht des Ökosystems u. ä. bekommen kann; Diese werden von der österreichischen Seite sichergestellt. 13) Referential translation: Es geht um die Termine, wann man die Untersuchungsergebnisse, Fokussierung, Bestimmung des Wasserspiegels und Empfehlungen aus der Sicht des Ökosystems u. ä. bekommen kann; Diese werden von der österreichischen Seite sichergestellt. 13) Referential translation: Es geht um die Termine, wann man die Untersuchungsergebnisse, Fokussierung, Bestimmung des Wasserspiegels und Empfehlungen aus der Sicht des Ökosystems u. ä. bekommen kann; Diese werden von der österreichischen Seite sichergestellt. MT: Zu den Terminen wenn Sie die Ergebnisse einer Umfrage Fokus Ermittlung der maximalen Wasserstande und Empfehlungen in Bezug auf das Ökosystem erhalten und dergleichen die bietet AT Seite. 150 Evaluating Machine Translation Quality: A Case Study of a Translation of a Verbatim Transcription from Slovak to German This example sentence of MT demonstrates its potential for nonsense since the sentence elements do not maintain cohesion, perhaps due to incomprehensible or complex sentence structure in the Slovak source text. The machine-translated sentence reproduced only some words, such as Ergebnisse, Empfehlungen and Ökosystem, however without any logical linking. The word Umfrage is redundant and does not make any sense in the given context. 14) Referential translation: Er hat die beauftragten Firmen aufgefordert, den Stand ihrer Arbeit zu präsentieren. 14) Referential translation: Er hat die beauftragten Firmen aufgefordert, den Stand ihrer Arbeit zu präsentieren. p MT: Er nannte das Unternehmen verantwortlich fur die Prasentation der Fortgang der Arbeiten. p MT: Er nannte das Unternehmen verantwortlich fur die Prasentation der Fortgang der Arbeiten. In this sample sentence, in spite of its brevity, we can see the erroneous transfer of all lexical elements – auffordern vs. nennen, the adjective verantwortlich (responsible) is redundant and the verb präsentieren has been changed into a noun (Präsentation). Fortgang der Arbeiten could be regarded as lexically relevant, but in an incorrect morphological form. The synonyms Firmen and Unternehmen could be acceptable but they have been translated in the singular, even though in the referential text the plural is used. 15) Referential translation: Das ganze Gebiet hält er für ausreichend. MT: Raumliche Abdeckung als ausreichend angesehen. In this short sentence only the second part is correct. The first part stands for a non-existent lexical phrase which was caused by artificial source language formulation that needed additional explanation. CONCLUSIONS The aim of this article was to test the evaluation of MT for a specific text type – the written record. The analysis of the machine-translated text has demonstrated that written records are extremely complicated text types that are almost impossible to translate using automated translation systems. The main reason can be seen in the complex register and in the selected language style that uses various means of expression. Secondly, it is also important to take the sentence structure into consideration because machine-translated texts contain long and complex sentence structures. In addition, it should be noted that the lexis in automated translations provides diffuse equivalents which can be attributed to the various specialized areas and hybrid text functions of a written record. We have also found that the quality of the input plays an important role. This is why other types of written records should be tested. The results could be beneficial to both professional and amateur translators and enable them to organize and manage their work more effectively. The aim of this article was to test the evaluation of MT for a specific text type – the written record. The analysis of the machine-translated text has demonstrated that written records are extremely complicated text types that are almost impossible to translate using automated translation systems. The main reason can be seen in the complex register and in the selected language style that uses various means of expression. 151 Jozef Štefčík The final results have proved that MT is not suitable for certain text types because there are many anomalies at all linguistic levels between the referential translation (human translation without the intervention of computer-assisted translating) and MT. A particular disadvantage of the Google MT system for the Slovak-German language pair is that there are not many parallel texts for these languages included in the system as the majority of the text corpora are in English. This also applies to German technical texts that use numerous English terms, as well as borrowings from English. Our study confirmed that written reports (as well as other text types) should be pre-edited and re-written into shorter sentence segments. It is therefore necessary to carry out further research into MT of pre-edited records between various language pairs. 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Proceedings of LREC. 697–702. 152 152 Evaluating Machine Translation Quality: A Case Study of a Translation of a Verbatim Transcription from Slovak to German Evaluating Machine Translation Quality: A Case Study of a Translation of a Verbatim Transcription from Slovak to German Evaluating Machine Translation Quality: A Case Study of a Translation of a Verbatim Transcription from Slovak to German MAŠININIO VERTIMO KOKYBĖS VERTINIMAS: TRANSKRIBUOTO ŽODINIO DISKURSO VERTIMO IŠ SLOVAKŲ KALBOS Į VOKIEČIŲ KALBĄ ATVEJO ANALIZĖ Jozef Štefčík S a n t r a u k a Šiame straipsnyje aptariama automatinio vertimo kokybės vertinimo problema remiantis žodinio teksto vertimo iš slovakų kalbos į vokiečių kalbą pavyzdžiu. Autorius atliko eksperimentą, kurio tikslas – pa tikrinti susitikimų, posėdžių ir kitokių komunikacinių situacijų, per kurias žodžiu pasakomas turinys įrašomas į laikmenas (pvz., naudojant diktofoną ar vaizdo kamerą), o vėliau transkribuojamas kaip doku mentas (pvz., posėdžio protokolas), vertimo į kitą kalbą (konkrečiu atveju – vokiečių) kokybę. Vertinimui pasirinkti tikslumo, leksinės ir sintaksinės atitikties ir kiti kriterijai pagal Ackaerto ir kitų kartu su juo dirbusių mokslininkų (Ackaert et al. 2013) pasiūlytą metodiką. Vertimas atliktas naudojant statistiniu principu veikiančią automatinio vertimo sistemą Google Translate, vėliau lygintas su profesionalaus ver tėjo darbu. Atliktas tyrimas patvirtino, kad kol kas kai kurių tekstų automatinis vertimas neįmanomas dėl daugelio neišspręstų problemų įvairiuose sistemos lygmenyse ir kad norint produktyviai panaudo ti automatinio vertimo galimybes verčiant transkribuotus žodinio diskurso tekstus (kaip, beje, ir kito kio tipo rašytinius tekstus) tekstas turi būti tinkamai parengtas, t. y. atliktas jo išankstinis redagavimas (pre-editing), suskaidant tekstą į trumpesnius segmentus, kurie sistemoje būtų lengviau atpažįstami. Tin kamai parengus tekstą vertimo rezultatai buvo gerokai geresni. 153
https://openalex.org/W2139561552	https://eprints.gla.ac.uk/101890/1/101890.pdf	English	null	Ignored Disease or Diagnostic Dustbin? Sudden Infant Death Syndrome in the British Context	Social history of medicine	2,015	cc-by	14,842	Social History of Medicine page 1 of 22 Social History of Medicine Advance Access published March 2, 2015 Social History of Medicine page 1 of 22 © The Author 2015. Published by Oxford University Press on behalf of the Society for the Social History of Medicine. doi:10.1093/shm/hkv003 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1J. G. Scadding, ‘Principles of Definition in Medicine with Special Reference to Chronic Bronchitis and Emphyse- ma’, The Lancet, 1959, 273, 323–5. Centre for the History of Medicine, University of Glasgow, Lilybank House, Bute Gardens, Glasgow, G12 8RT, Scot- land, UK. Email: Angus.Ferguson@glasgow.ac.uk Angus Ferguson is Lord Kelvin Adam Smith Fellow in Social Sciences at the University of Glasgow, where he is also an Associate Academic in the Institute of Health and Wellbeing. His current research focuses on the history of medical confidentiality and privacy. In 2013, Ashgate published his book Should a Doctor Tell? The Evolution of Medical Con- fidentiality in Britain as part of its Medical Law and Ethics series. Centre for the History of Medicine, University of Glasgow, Lilybank House, Bute Gardens, Glasgow, G12 8RT, Scot- land, UK. Email: Angus.Ferguson@glasgow.ac.uk Angus Ferguson is Lord Kelvin Adam Smith Fellow in Social Sciences at the University of Glasgow, where he is also an Associate Academic in the Institute of Health and Wellbeing. His current research focuses on the history of medical confidentiality and privacy. In 2013, Ashgate published his book Should a Doctor Tell? The Evolution of Medical Con- fidentiality in Britain as part of its Medical Law and Ethics series. s Ferguson, A. H. (2015) Ignored disease or diagnostic dustbin? Sudden infant death syndrome in the British context. Social History of Medicine. Copyright © 2015 The Author This work is made available under the Creative Commons Attribution License (CC BY 4.0) Version: Published http://eprints.gla.ac.uk/101890 Deposited on: 06 March 2015 Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk Ferguson, A. H. (2015) Ignored disease or diagnostic dustbin? Sudden infant death syndrome in the British context. Social History of Medicine. Copyright © 2015 The Author Deposited on: 06 March 2015 Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk 1J. G. Scadding, ‘Principles of Definition in Medicine with Special Reference to Chronic Bronchitis and Emphyse- ma’, The Lancet, 1959, 273, 323–5. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author 2015. Published by Oxford University Press on behalf of the Society for the Social History of Medicine. doi:10.1093/shm/hkv003 Ignored Disease or Diagnostic Dustbin? Sudden Infant Death Syndrome in the British Context Angus H. Ferguson* Summary. Sudden Infant Death Syndrome (SIDS) was defined in 1969 and incorporated into the Inter- nationalClassification ofDiseasesa decadelater.To advocates ofSIDSasadiagnosis,medicalinterestin sudden infant death was long overdue. However, the definition of SIDS lacked positive diagnostic cri- teria, provoking some to view it as a ‘diagnostic dustbin’ for the disposal of problematic cases where cause of death was unclear. This paper examines the development of medical interest in sudden infant death in Britain during the middle decades of the twentieth century. It highlights the importance of recognising the historicity of SIDS as a diagnosis facilitated by changes in law and medicine over the courseofthenineteenthandtwentiethcenturies.ItsuggeststhatSIDSprovidesadefinitivecasestudyof themedicalisationoflifeanddeath,andauniqueexampleofanofficiallyrecogniseddiseasethathadno symptoms, signs, pathology or patients. at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from Keywords: SIDS; cot death; diagnosis; medicalisation; symptoms; frameworks of disease The concept of a disease is thus an abstraction from the reality of phenomena observed in patients, useful because it permits of thinking, speaking, and writing in generalisations.1 Sudden Infant Death Syndrome (SIDS) was defined and adopted as a diagnosis during the second international conference on the causes of sudden death in infants, held in East- sound, Washington in 1969.2 The inaugural conference, designed to review recent work and recommend future research priorities, had taken place in Seattle in 1963. Abraham Bergman, an American paediatrician and central figure in promoting medical interest in sudden infant death, noted that at the time of the first conference ‘the biggest unknown was whether the bulk of infants dying suddenly and unexpectedly were victims of a distinct Ireland and Czechoslovakia. Abraham B. Bergman, J. Bruce Beckwith and C. George Ray, Sudden Infant Death Syndrome. Proceedings of the Second Inter- national Conference on Causes of Sudden Death in Infants (Seattle: University of Washington Press, 1970). 1J. G. Scadding, ‘Principles of Definition in Medicine with Special Reference to Chronic Bronchitis and Emphyse- ma’, The Lancet, 1959, 273, 323–5. 2ThoughpredominantlyattendedbyAmericans,therewas a European presence through specialists from Northern Page 2 of 22 Angus H. Ferguson disease entity or were dying coincidentally from a number of known diseases’.3 By 1969, according to Bergman, the picture was becoming a great deal clearer.4 In 1978, the ninth revision of the International Classification of Diseases added SIDS to the list of officially recognised diseases.5 The attention SIDS has received in recent decades is often portrayed as a stark contrast to ignorance of it in earlier periods. 5From 1978, ICD9 [The International Classification of Dis- eases, 9th Revision]includedthe category798—Sudden Infant Death Syndrome. Previously cases had been included under category 795: Sudden Death (Cause Unknown). For a summary of this see Sylvia R. Limerick, ‘Sudden Infant Death in Historical Perspective’, Journal of Clinical Pathology, 1992, 45 (Supplement), 3–6. 6Bergman, The ‘Discovery’, xi. 7Bergman et al., Sudden Infant Death Syndrome, 18. 8Dorland’s Medical Dictionary, 21st edn (London: W.B. Saunders Co., 1968) defined Symptom as ‘an organic or physiologic manifestation of disease which a patient is usually aware of and frequently complains of’. Thesamework defined Sign as‘anobservablephys- ical phenomenon so frequently associated with a given condition as to be considered indicative of its presence’. Similarly, the 15th edition of Hutchison’s Clinical Methods (London: Bailliere, Tidall & Cassell, 1968) dis- tinguished between the interrogation of the patient’s history, including symptoms, and the physical examin- ation of the patient, including the search for signs. 9John L. Emery, ‘Is Sudden Infant Death Syndrome a Diagnosis?’, British Medical Journal, 1989, 299, 1240. 10Ibid. 3Abraham B. Bergman, The ‘Discovery’ of Sudden Infant Death Syndrome. Lessons in the Practice of Political Medicine (New York: Praeger, 1986), 8. Bergman was Director of out-patient services at the Children’s Ortho- pedic Hospital and Medical Center and Associate Pro- fessor of Pediatrics and Preventive Medicine at the University of Washington, Seattle. Together with his colleague Bruce Beckwith, a pathologist, he co-chaired the1969meetingonCausesofSuddenDeathinInfants held at the University of Washington. 3Abraham B. Bergman, The ‘Discovery’ of Sudden Infant Death Syndrome. Lessons in the Practice of Political Medicine (New York: Praeger, 1986), 8. Bergman was Director of out-patient services at the Children’s Ortho- pedic Hospital and Medical Center and Associate Pro- fessor of Pediatrics and Preventive Medicine at the University of Washington, Seattle. Together with his colleague Bruce Beckwith, a pathologist, he co-chaired the1969meetingonCausesofSuddenDeathinInfants held at the University of Washington. 9John L. Emery, ‘Is Sudden Infant Death Syndrome a Diagnosis?’, British Medical Journal, 1989, 299, 1240. 10Ibid. Ignored Disease or Diagnostic Dustbin? Sudden Infant Death Syndrome in the British Context In recounting his experience of championing SIDS as a disease requiring focused research by medical science, Bergman asserted that ‘despite the magnitude of the problem, until the mid 1970s SIDS must have been the most ignored disease in history.’6 However, SIDS has often proven to be controversial— not least because it is a diagnosis of exclusion. The 1969 conference defined SIDS as ‘the sudden death of any infant or young child, which is unexpected by history, and in which a thorough post-mortem examination fails to demonstrate an adequate cause for death’.7 In other words, cases were not identified using positive criteria, such as an agreed pattern of symptoms, signs or explanatory pathology, but by the absence of evidence for an alterna- tive cause of death.8 Consequently, SIDS was something of an anomaly in medicine: an of- ficially recognised disease with no symptoms, no signs, no explanatory pathology and no patients. John Emery, Professor of Paediatric Pathology in Sheffield, noted that the recom- mendation that all such cases should be registered as Sudden Infant Death Syndrome was rapidly adopted internationally. He pointed to five key reasons for this: at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from It enabled doctors to tell parents that their child had died of natural causes and that no one could have prevented it. It excused all concerned from any defect in care, diagnosis and treatment. Pathologists welcomed the diagnosis: the less they found, the more certain they could be. Because the syndrome was of unknown origin health authorities had no basis for prevention. And finally, this diagnosis facilitated the development of parent support groups and the raising of money for research.9 However, the absence of positive diagnostic criteria for SIDS was a source of concern. Emery’s article queried whether SIDS was a real disease or a ‘diagnostic dustbin’ for the dis- posal of cases in which investigation failed to reveal an obvious cause of death.10 Sudden Infant Death Syndrome in the British Context Page 3 of 22 This paper explores Bergman’s assertion that SIDS was an ignored disease, and Emery’s characterisation of it as a diagnostic dustbin, through the analysis of medical interest in sudden infant death in Britain during the years prior to the definition and recognition of SIDS in 1969. While sudden infant death became a focus of international medical research, there are good reasons to focus on Britain. 12Medicalisation is used to mean the adoption of a medical model of understanding for phenomena pre- viously understood within a different framework. 11Karl Hufbauer, ‘Federal Funding and Sudden Infant Death Research, 1945–1980’, Social Studies of Science, 1986, 16, 61–78; Limerick, ‘Sudden infant death in historical perspective’, 5; Bergman, The ‘Discovery’. Ignored Disease or Diagnostic Dustbin? Sudden Infant Death Syndrome in the British Context In America the promotion of SIDS as a legitimate diagnosis was important in securing federal funding for research.11 In Britain, by contrast, both the Ministry of Health and the Medical Research Council (MRC) were active in funding research, combining investigation of possible clinical, pathological and social causes, prior to the definition of SIDS. Indeed, as detailed in later sections of the paper, by the mid-1960s the MRC was struggling to persuade researchers to apply for available funding. Therefore,while only partof the biggerpicture of international workonSIDS, Britain presents an important case study of the lead up to the definition and recognition of SIDS. at Periodicals Dept on March 6, 201 http://shm.oxfordjournals.org/ Downloaded from at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from The analysis contextualises the medicalisation of sudden infant deaths using the histori- ography of death registration, infant mortality and relevant changes in law and medicine in the nineteenth and early twentieth centuries, highlighting the importance of recognising the historicity of SIDS.12 Having discussed why medical interest came to focus on SIDS in the mid-twentieth century, the paper examines the early co-ordinated studies of sudden infant death in Britain, investigating the extent to which their findings contributed to the discovery of a new disease. The final section of the paper examines SIDS in relation to the social construction of disease and the frameworks of understanding used by historians of medicine. It concludes that, while the lack of positive diagnostic criteria entails that SIDS does not fit into traditional frameworks of disease, the diagnosis had power and signifi- cance. This was especially so for the parents and families of victims for whom a medical ex- planation, even one devoid of diagnostic, prophylactic or therapeutic substance, helped to assuage guilt and legal suspicion and facilitated the establishment of interdisciplinary support networks. at Periodicals Dept on March 6, 2015 http://shm.oxfordjournals.org/ m 13See Edward Higgs, Life, Death and Statistics (Hatfield: Local Population Studies, 2004) on England and Wales; and Anne Cameron, ‘The Establishment of Civil Registration in Scotland’, The Historical Journal, 2007, 50, 377–95 on Scotland. See, forexample, LudmillaJordanova, ‘TheSocialCon- struction of Medical Knowledge’, Social History of Medicine, 1995, 8, pp. 361–81, 367. See, forexample, LudmillaJordanova, ‘TheSocialCon- struction of Medical Knowledge’, Social History of Medicine, 1995, 8, pp. 361–81, 367. 13See Edward Higgs, Life, Death and Statistics (Hatfield: Local Population Studies, 2004) on England and Wales; and Anne Cameron, ‘The Establishment of Civil Registration in Scotland’, The Historical Journal, 2007, 50, 377–95 on Scotland. See, forexample, LudmillaJordanova, ‘TheSocialCon- struction of Medical Knowledge’, Social History of Medicine, 1995, 8, pp. 361–81, 367. 13See Edward Higgs, Life, Death and Statistics (Hatfield: Local Population Studies, 2004) on England and Wales; and Anne Cameron, ‘The Establishment of Civil Registration in Scotland’, The Historical Journal, 2007, 50, 377–95 on Scotland. 17R. I. Woods, P. A. Watterson and J. H. Woodward, ‘The Causes of Rapid Infant Mortality Decline in England and Wales, 1861–1921. Part I’ Population Studies, 1988, 42, 343–66. 15The Births and Deaths Registration Act, 1836 intro- duced civil registration in England and Wales; and the Registration of Births, Deaths and Marriages (Scotland) Act, 1854 introduced civil registration in Scotland. Ignored Disease? Civil registration of vital statistics was instituted in nineteenth-century Britain through legisla- tionrequiringtherecordingofbirths,marriagesanddeathsinEnglandandWalesin1836and Scotland in 1854.13 Although the initial drive behind civil registration centred on facilitating the transfer of property to legitimate heirs, over time the use to which registration was put, and its relevance to medicine, grew. From the late nineteenth century, the dual emphasis onensuringthatcertificationofdeathwascarriedoutbydoctorsandthatitincludedaspecific cause, framed in clinico-pathological terms, was vital to the rise of medical interest in SIDS. WhilemanyanalysesofSIDSbeginbypointingtoevidenceofsuddeninfantdeathsdating back to biblical times, there are reasons why coordinated medical interest focused on the Page 4 of 22 Angus H. Ferguson issue in the middle decades of the twentieth century.14 In the UK, prior to the advent of civil registration of vital statistics, infants did not attract much medical or legal concern.15 There- fore sudden infant deaths, specifically, were not ignored, rather they were a subset of a broader group of deaths that received little attention. From 1877 onwards the annual report of the Registrar General included figures for infant mortality.16 This highlighted a problem of persistently high infant mortality, in contrast to a pattern of annually falling rates of mortality in the general population.17 As David Armstrong notes ‘the creation of a specific mortality rate for infants at this time suggests both the emergence of a social awareness of these young deaths and, more importantly, the social recognition of the infant as a discrete entity.’18 Official recognition of infants as a distinct group for analysis had significant legal implica- tions. Recording the birth and death of infants was an important step towards legislation safeguarding such vulnerable lives. gy 22See articles and letters to The Times on 27 August 1864, 8; 23 December 1904, 5; 3 January 1905, 13; 15 February 1908, 9; 3 April 1908, 11; 18 April 1908, 5. 16This included deaths under the age of one. David Arm- strong,‘TheInventionofInfantMortality’,Sociology of Health and Illness, 1986, 8, 211–32, 212. 21Elizabeth deG. R. Hansen, ‘“Overlaying” in 19th-century England: Infant Mortality or Infanti- cide?’, Human Ecology, 1979, 7, 333–52. 23George C. Alter and Anne G. Carmichael, ‘Classifying the Dead: Toward a History of the Registration of Cause of Death’, Journal of the History of Medicine and Allied Sciences, 1999, 54, 114–32. 18Armstrong, ‘The Invention of Infant Mortality’, 212. 19 f f h 20Children Act, 1908, 8 Edw. 7 Ch. 67 ss.13. 2 14Examples of SIDS literature citing the biblical account of overlaying include: Bergman, The ‘Discovery’, xi; Limerick, ‘Sudden Infant Death in Historical Perspec- tive’, 3; John L. Emery and E. M. Crowley, ‘Clinical His- tories of Infants Reported to Coroner as Cases of Sudden Unexpected Death’, British Medical Journal, 1956, 2, 1518–21, 1518; The Department of Health, Report of the Chief Medical Officer’s Expert Group on The Sleeping Position of Infants and Cot Death (London: HMSO, 1993), 3. 14Examples of SIDS literature citing the biblical account of overlaying include: Bergman, The ‘Discovery’, xi; Limerick, ‘Sudden Infant Death in Historical Perspec- tive’, 3; John L. Emery and E. M. Crowley, ‘Clinical His- tories of Infants Reported to Coroner as Cases of Sudden Unexpected Death’, British Medical Journal, 1956, 2, 1518–21, 1518; The Department of Health, Report of the Chief Medical Officer’s Expert Group on The Sleeping Position of Infants and Cot Death (London: HMSO, 1993), 3. 19Infant Life Protection Act, 1897, 60 & 61 Vict. Ch. 57. ss.8. 18Armstrong, The Invention of Infant Mortality , 212. 19Infant Life Protection Act, 1897, 60 & 61 Vict. Ch. 57. ss.8. 18Armstrong, ‘The Invention of Infant Mortality’, 212. 19Infant Life Protection Act, 1897, 60 & 61 Vict. Ch. 57. ss.8. 19Infant Life Protection Act, 1897, 60 & 61 Vict. Ch. 57. ss.8. Ignored Disease? Evidence of an increasing concern about the fate of infants is found in the Infant Life Protection Act, 1897, which required that suspicious infant deaths be notified to district coroners, or, in Scotland, to the procurator fiscal, so that an inquest to establish the cause of death could be carried out.19 Under the terms of the Children Act, 1908, the overlaying of a child by an adult under the influence of drink was made a criminal act of negligence.20 Such legislation demonstrated a perceived need to protect infants from parental neglect or malevolence.21 As the predominant explanation given for unexpected infant deaths, ‘overlaying’ both reflected and perpetuated con- temporary concerns about the working, drinking and sleeping habits of the working class population.22 at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from AnneCarmichaelandGeorgeAlterindicatethat,atitsoutset,registrationofvitalstatistics was motivated by the interest of statisticians in recording population trends and patterns rather than by medical theory or practice.23 However, as doctors became more involved in the process of registering cause of death, a tension arose between the interests of doctors and statisticians. The emergent statisticians of the eighteenth and early nineteenth centuries were inter- ested in statistical classification—that is, in grouping individual entities in such a way as Sudden Infant Death Syndrome in the British Context Page 5 of 22 to derive general principles governing deaths. The medical men were interested in no- menclature, in assessing distinctions with a view to understanding multiple causes of morbid phenomena. In other words, one group were lumpers, the other splitters.24 Both agendas had implications for SIDS. As already noted,the statisticians’ categorisation of registration data highlighted infant mortality as a specific area of concern, which was a factorinsparkinglegalandmedicalinterest.Paediatricsbecameestablishedasadistinctspe- cialism during the early decades of the twentieth century, with subdivisions focusing atten- tion on perinatal, neonatal and postneonatal conditions.25 This period produced increased medical interest in stillbirths, with legislation requiring registration of stillbirths in England and Wales from 1927, and in Scotland—with the additional requirement that a specific cause be given—a decade later.26 at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from On one level, there are similarities between the rise of medical interest in stillbirth and sudden infant death. Cases of either often attracted legal rather than medical attention, with suspicion of abortion or infanticide driving investigation. 26Nicky Hart, ‘Infant Mortality: Gender and Stillbirth in Reproductive Mortality before the Twentieth Century’, Population Studies, 1998, 52, 215–29; Gayle Davis, ‘Stillbirth Registration and Perceptions of Infant Death, 1900–60: the Scottish Case in Nation- al Context’, Economic History Review, 2009, 62, 629–54. 27Deaths occurring between 28 weeks gestation and one year old. See Hart, ‘Infant Mortality’, 215–16. 28Nirupa Dattani and Nicola Cooper, ‘Trends in Cot Deaths’, Health Statistics Quarterly, 2000, 5, 10–16. 29Hart, ‘Infant Mortality’, 219–20. 30On the introduction of ultrasound, see Malcolm Nicol- son and John E. E. Fleming, Imaging & Imagining the Fetus.TheDevelopment of Obstetric Ultrasound (Balti- more: The Johns Hopkins University Press, 2013). Gayle Davis, ‘Stillbirth Registration and Perceptions of Infant Death, 1900–60: the Scottish Case in Nation- al Context’, Economic History Review, 2009, 62, 629–54. Ibid., 121. 25Ontheestablishmentofpaediatrics asaspecialism,see David Armstrong, Political Anatomy of the Body. Making Knowledge in Britain in the Twentieth Century (Cambridge: Cambridge University Press, 1983), 55. Perinatal covered the period from 28 weeks gestation until the end of the first week of life, and therefore included stillbirths; neonatal re- ferred to the first 4 weeks of life after birth; pPostneo- natal referred to the period between 4 weeks and 1 year old. 24Ibid., 121. 25 31Bergman, The ‘Discovery’, 10. 37Tim Devis and Cleo Rooney, ‘Death Certification and the Epidemiologist’, in Health Statistics Quarterly, 1999, 1, 21–33; Anne Hardy, ‘“Death is the Cure of All Diseases”: Using the General Register Office Cause of Death Statistics for 1837–1920’, Social History of Medicine, 1994, 7, 472–92; Gunter B. Risse, ‘Cause of Death as a Historical Problem’, Con- tinuity and Change, 1997, 12, 175–88; K. Codell Carter, ‘Causes of Disease and Causes of Death’, Con- tinuity and Change, 1997, 12, 189–98. Ignored Disease? Similarly, once medical atten- tion came to focus on recording and examining cases in more detail, both stillbirths and sudden infant deaths were found to be much more common than previously thought. In the middle decades of the twentieth century, stillbirths were recognised as the single largest cause of reproductive mortality.27 Early studies in the 1950s suggested that suddeninfantdeathsmightaccountforaround20percentofpostneonatalinfantmortality, and changes in registration practices entailed that it rose to account for nearly half of post- neonatal infant mortality in the 1970s and 1980s.28 However, the differences between stillbirth and SIDS are significant in terms of their re- spective medicalisation. Setting aside cases of abortion or infanticide, medical explanations of stillbirth focused on intrauterine conditions, particularly the age, health and nutrition of the mother.29 The mother, as much as the fetus, was the patient—arguably more so until ultrasound enabled the medical gaze to focus on the placenta in the 1960s—and the search for causal explanations considered both.30 By contrast, SIDS typically affected the postneonatal infant population, with the number of cases peaking around the age of 2–4 months, when the viability and separate existence of the infant was established. As Bergman noted, ‘SIDS does not claim the tiniest, most fragile, immature victims. It is not a phenomenon of the newborn period.’31 Reflecting this, early studies on sudden infant death generally defined cases for examination in a way that separated sudden infant Page 6 of 22 Angus H. Ferguson death from stillbirth. For example, the UK Ministry of Health’s study in the 1950s specified the age range of interest as ‘from two weeks to two years’ old.32 Other international studies focused onthe period 2–11 months.33 While recent researchhas soughtto establish a link between obstetric conditions and particular cases of sudden infant death, earlier medical research often linked the latter with examples of sudden death in childhood or adulthood.34 As discussed below, early attempts to identify, diagnose and avert potential cases of sudden infant death had devastating consequences for healthy infants. Alter and Carmichael note that doctors’ growing involvement in death registration ‘created a set of relationships between physicians and patients that was not linked to medical care and did not necessarily reinforce the physician’s role as comforter or healer’.35 This was an important step towards SIDS, an exclusively retrospective and non- therapeutic diagnosis, becoming the focus of medical interest. 38In Scotland the matter was referred to the procurator fiscal for investigation. The role of procurator fiscal in Scotland combines the duties of coroner and public prosecutor in England. 33See for example, Preben Geertinger, ‘Sudden Unex- pected Death in Infancy. With Special Reference to the Parathyroids’, Pediatrics, 1967, 39, 43–48. 37Tim Devis and Cleo Rooney, ‘Death Certification and the Epidemiologist’, in Health Statistics Quarterly, 1999, 1, 21–33; Anne Hardy, ‘“Death is the Cure of All Diseases”: Using the General Register Office Cause of Death Statistics for 1837–1920’, Social History of Medicine, 1994, 7, 472–92; Gunter B. Risse, ‘Cause of Death as a Historical Problem’, Con- tinuity and Change, 1997, 12, 175–88; K. Codell Carter, ‘Causes of Disease and Causes of Death’, Con- tinuity and Change, 1997, 12, 189–98. 32A. Leslie Banks, ‘An Enquiry into Sudden Death in Infancy’, Monthly Bulletin of the Ministry of Health, 1958, 17, 182–91. 34Recent research includes Gordon C. S. Smith, Angela M. Wood, Jill P. Pell, Ian R. White, J. A. Crossley and Richard Dobbie, ‘Second-Trimester Maternal Serum Levels of Alpha-Fetoprotein and the Subsequent Risk of Sudden Infant Death Syndrome’, New England Journal of Medicine, 2004, 351, 978–86; Gordon C. S. Smith, Jill P. Pell and Richard Dobbie, ‘Risk of Sudden Infant Death Syndrome and Week of Gesta- tion of Term Birth’, Pediatrics, 2003, 111, 1367–71; Gordon C. S. Smith and Ian R. White, ‘Predicting the Risk for Sudden Infant Death Syndrome from Obstetric Characteristics: A Retrospective Cohort Study of 505 011 Live Births’, Pediatrics, 2006, 117, 60–6. For an earlier study, see for example, Ann Dally, ‘Status Lym- phaticus:SuddenDeathinChildren from “Visitationof God”toCotDeath’,MedicalHistory,1997,41,70–85. 35Alter and Carmichael, ‘Classifying the Dead’, 130. 36Armstrong, ‘The Invention of Infant Mortality’, 211. 39Ian A. Burney, Bodies of Evidence (London: The Johns Hopkins University Press, 2000). In Scotland specialists in forensic medicine were attached to the ancient uni- versities and developed international reputations as experts in this field. See M. Anne Crowther and Brenda White, On Soul and Conscience: The Medical Expert and Crime. 150 Years of Forensic Medicine in Glasgow (Aberdeen: Aberdeen University Press, 1988). Ignored Disease? Moreover, as Armstrong notes, registration of vital statistics led to greater specificity in analysis of death: ‘the notion of a pathological cause of death in the form of a disease was introduced to replace so-called “natural” death’.36 Doctors came under ever greater pressure to frame the specific cause of death in clinico-pathological terms in all cases.37 at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from In the absence of an obvious cause, sudden infant deaths were better suited to a system which simply recorded the fact of death. With growing emphasis on providing a specific clinico-pathological cause, one of two courses could be followed. The death might be regis- tered under an alternate heading—such as accidental suffocation or respiratory disease. This practice entailed that the scale of sudden infant death was not recognised until studies began to examinethe problem in moredepth inthe middledecades of the twentieth century. Alternatively, the case could be referred to a coroner for further investigation.38 While coroners were often legally trained, their investigation might draw on the expertise of a doctor specialising in forensic medicine or a pathologist’s autopsy report.39 However, 39Ian A. Burney, Bodies of Evidence (London: The Johns Hopkins University Press, 2000). In Scotland specialists in forensic medicine were attached to the ancient uni- versities and developed international reputations as experts in this field. See M. Anne Crowther and Brenda White, On Soul and Conscience: The Medical Expert and Crime. 150 Years of Forensic Medicine in Glasgow (Aberdeen: Aberdeen University Press, 1988). Sudden Infant Death Syndrome in the British Context Page 7 of 22 this wasrare incasesof suddendeath ininfants. OneLondoncoroner suggestedthatcounty councils objected to the expense involved.40 Another pointed to procedural reasons: Itmaybethatacoronerwillactonthestrengthofthedoctor’sopinionastothecauseof deathwithoutnecessarilyhavingtheconfirmationofapost-mortemexamination.Prior to 1927 this was usually the case, as it was not then possible for a coroner to order a post-mortem examination without holding an inquest; therefore the medical opinion as to the cause of death, based on circumstantial evidence and external evidence only, was usually accepted.41 The Coroners (Amendment) Act, 1926, introduced scope for a coroner to request an autopsy without automatically provoking an inquest, provided the death was found to result from natural causes. This gave pathologists unprecedented opportunity to examine the accidental suffocation hypothesis frequently recorded as the cause of sudden infant death. 40The Times 18 April 1908, 5. 41W. H. Davison, ‘Accidental Infant Suffocation’, British Medical Journal, 1945, 2, 251. 42Ibid., 251; Douglas Swinscow, ‘So-called Accidental Mechanical Suffocation of Infants’, British Medical Journal, 1951, 2, 1004–7. 43E. Emrys-Roberts, ‘Status Lymphaticus’, Journal of Pathology and Bacteriology, 1913, 18, 513–26; Matthew Young and Hubert Turnbull, ‘An Analysis 44Ann Dally, ‘Status Lymphaticus: Sudden Death in Chil- dren from“Visitation of God” to Cot Death’. of the Data Collected by the Status Lymphaticus Inves- tigationCommittee’, JournalofPathologyandBacteri- ology, 1931, 34, 213–58; T. H. B. Bedford, ‘The Pathology of Sudden Death: A Review of 198 Cases “Brought in Dead”’, Journal of Pathology and Bacteri- ology, 1933, 36, 333–47. of the Data Collected by the Status Lymphaticus Inves- tigationCommittee’, JournalofPathologyandBacteri- ology, 1931, 34, 213–58; T. H. B. Bedford, ‘The Pathology of Sudden Death: A Review of 198 Cases “Brought in Dead”’, Journal of Pathology and Bacteri- ology, 1933, 36, 333–47. 47A. Leslie Banks, ‘An Enquiry into Sudden Death in Infancy’, Monthly Bulletin of the Ministry of Health and the Public Health Laboratory Service, 1958, 17, 182–91. 49Banks, ‘An Inquiry into Sudden Death’, 183. In a dis- cussion of Sudden Death in Infancy at the first BMA Annual Clinical Meeting held in Southampton 4–7 De- cember 1958, Dr Gairdner, consultant pediatrician at Addenbrooke’s Hospital and member of the Ministry 48Banks had a combination of medical and legal qualifi- cations. The Department of Human Ecology at Cam- bridge had recently emerged out of a growing interest in social medicine, epidemiology and health statistics. A. Leslie Banks, ‘The Department of Human Ecology and the Medical Officer of Health’, Public Health, 1950, 63, 211–12; A. Leslie Banks, ‘The De- partment of Human Ecology, Cambridge’, Public Health, 1954, 67, 199–200. 46Bedson was appointed to the Goldsmith Chair of Bac- teriology, London University in 1934. A significant pro- portion of his research focused on viruses as causal factors of disease. In 1951, Camps was pathologist at the Chelmsford and Essex hospital. He subsequently became Reader (1954) and then Professor (1963) of Forensic Medicine at the London Hospital Medical School. Ignored Disease? Dally’s research details specific medical interest in sudden infant death during the first half of the twentieth century, highlightingthe considerablerisksinvolvedinattempting toidentify acausalmech- anismanddevelopprophylacticinterventions.WhileanMRCcommitteereportin1931con- cluded that there was no such thing as Status Lymphaticus, the theory continued to find support into the 1940s.45 at Periodicals Dept on March 6, 201 http://shm.oxfordjournals.org/ Downloaded from In summary, by the mid-twentieth century there was increased medico-legal focus on infants, and on identifying and recording the specific causes of infant mortality, framed in clinico-pathological terms. Changes to the Coroners Act in 1926 provided greater scope for investigation of cases where the specific cause of death was unclear, allowing patholo- gists to test the evidence for existing theories regarding ‘overlaying’ and accidental suffoca- tionasthecausesofsuddeninfantdeath.Buttheearlydecadesofthetwentiethcenturyalso highlighted the risks of prophylactic medical intervention. Against this backdrop, the Min- istry of Health began to support research into the aetiology of sudden infant deaths in the 1950s. at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from at Periodicals Dept on March 6, 2015 http://shm.oxfordjournals.org/ om 48Banks had a combination of medical and legal qualifi- cations. The Department of Human Ecology at Cam- bridge had recently emerged out of a growing interest in social medicine, epidemiology and health statistics. A. Leslie Banks, ‘The Department of Human Ecology and the Medical Officer of Health’, Public Health, 1950, 63, 211–12; A. Leslie Banks, ‘The De- partment of Human Ecology, Cambridge’, Public Health, 1954, 67, 199–200. 45Ibid. 81. Ignored Disease? Subsequently, local studies reported findings from increased numbers of post- mortem examinations raising doubts about the validity of the overlaying, or accidental suf- focation, hypothesis.42 These doubts, brought to the attention of the Ministry of Health in the1950s,weretheproximatecauseofthe coordinatedstudiesonsuddeninfantdeaththat took place in the 1950s and 1960s. at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from Evidently,itisimportanttounderstandtheriseofmedicalinterestinsuddeninfantdeaths, and the historicity of SIDS as a diagnosis, in relation to developments in the registration of vital statistics, medical law and paediatrics in the late nineteenth and early twentieth centur- ies. While these factors combined to produce a context conducive to the medicalisation of sudden infant deaths by the 1960s, there had been earlier attempts to provide medical explanation and intervention for sudden infant deaths.43 Ann Dally’s work on Status Lym- phaticus reveals that up until the 1950s, British medical students were taught that an enlarged thymus gland could indicate susceptibility to sudden death in infants, children and adults. In some versions of the theory, the enlarged gland itself was the direct cause of death—inhibiting circulation and respiration inthe victim. By other accounts, an enlarged thymus was indicative of a constitutional weakness that predisposed the individual to unex- pected sudden death from apparently trivial causes, such as the administration of inocula- tions in childhood or chloroform during surgery.44 Uncertainty regarding the function of the thymus gland contributed to medical advice that, in cases where it was enlarged, it should be removed. However, there was no estab- lished norm regarding the size of the thymus. As Dally details, the decision to establish a norm using the findings of autopsies on children who had died in hospital had disastrous consequences, as it was subsequently revealed that this cohort presented with atypically small glands as a consequence of the role of the thymus in combating infection. Normal Page 8 of 22 Angus H. Ferguson glands were categorised as dangerously large—an error compounded by the negative effectsofadvicetosurgicallyremovethethymus,oruseirradiationtoreduceitssize.Surgical interventions had high mortality rates, and irradiation was subsequently linked to high rates of cancer. An inaccurate understanding of the aetiology entailed that measures intended to be prophylactic caused significant iatrogenic harm to healthy individuals. of Health’s Steering Committee, suggested the figure was 1,600 deaths for England and Wales. British Medical Journal, 1958, 2, 1467. Interestingly, this was similar to the reported figure for overlaying half a century earlier: The Times, 18 April 1908, 5. 57Emery and Crowley, ‘Clinical Histories of Infants’, 1518; G. Thurston, ‘Sudden Infant Deaths’, British Medical Journal, 1957, 1, 107; E. Duthie and R. Goodbody, ‘Sudden Infant Deaths’, British Medical Journal, 1957, 1, 285; Editorial, ‘Sudden Death in Infancy’, British Medical Journal, 1957, 1, 1411. Ministry of Health and MRC Studies y In1951,ProfessorSamuelBedson,renownedbacteriologist,andDrFrancisE.Camps,foren- sicpathologist,wroteamemorandumtotheMinistryofHealth.46Theysuggestedthatthere was growing evidence that some cases of sudden death of infants attributed to accidental mechanicalsuffocationmightbeduetoothercauses,suchasacuteinfection.47TheMinistry of Health invited Leslie Banks, Professor of Human Ecology at Cambridge, to chair a steering committee to investigate further.48 In particular, the enquiry was targeted towards deter- mining whether the deaths often attributed to accidental suffocation were actually a result of bacterial or viral infections, or the inhalation of food or vomit. A preliminary study confirmed the weakness of the accidental suffocation hypothesis, and also suggested that the problem was larger than previously acknowledged—1,400 cases annually in England and Wales, equating to over 20 per cent of postneonatal infant mortality.49 Sudden Infant Death Syndrome in the British Context Page 9 of 22 Banks’ committee conducted the enquiry under three broad headings, examining micro- biological, pathological and social factors. The investigation was concentrated in Cam- bridgeshire, where previous investigation had taken place, and the London County Council area.50 On receiving notification of the sudden death of a child, aged between two weeks and two years old and living in one of the study areas, coroners were to report the findings of their investigation on a form designed to facilitate the study. Part one of the form recorded post-mortem findings, including results of histological and bacteriologic- al tests.51 The second section recorded social data and was completed by a health visitor assigned to investigate the background and circumstances of death. This covered prior signs of illness or symptoms, the location and position in which the body was found and any other findings relevant to evaluating suffocation as the likely cause of death. at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from In 1958, the interim report of the committee identified trends in the 81 cases investi- gated.52 Sudden death was most common in the first year, affected boys more than girls and peaked in winter. The report underlined the need for more detailed pathological inves- tigation. While naked eye pathological appearances might suggest mechanical asphyxia, some thought histological findings indicated acute respiratory infection.53 However, few deaths could be ascribed to bacterial infection and early results appeared to exclude this as an important factor.54 Consequently, bacteriological analysis of cases was discontinued in London. 56Banks, ‘An Inquiry into Sudden Death’, 189. The issue of enquiring into the case history and context follow- ing a sudden death was controversial at the time. Dif- ferent results might be obtained depending on a variety of variables, including who asked the questions and when. 51The type and extent of tests to be included was the source of discussion amongst the steering committee members and the pathologists in each site. 50Peterborough was initially included, but was removed due to low numbers of relevant cases. Cambridgeshire relatedtotheCityofCambridgeandthosepartswithin a ten-mile radius. The London data came from the metropolitan boroughs within the Coroners’ districts of Sir Bentley Purchase and W. R. H. Heddy: Hamp- stead, Marylebone, St. Pancras, Finsbury, Holborn, Islington, Hackney, Stoke Newington, Deptford, Greenwich, Woolwich and Lewisham. 55Ministry of Health, Enquiry into Sudden Death in Infancy (London: HMSO, 1965), 7. 5264 in London and 17 in Cambridge. 53Banks, ‘An Inquiry into Sudden Death’, 187. 54Ibid., 187. 5264 in London and 17 in Cambridge. 53Banks, ‘An Inquiry into Sudden Death’, 187. 63Undertaking research at University College Hospital in London, Gunther became a leading expert on infant feeding; Robert Royston Amos Coombs, was a re- searcher in immunology at the University of Cam- bridge, where he became Reader in Immunology (1963–66) and Quick Professor in Immunology (1966–88). Ministry of Health and MRC Studies It continued in Cambridge, although a further 50 cases also produced negative findings.55 Similarly, evidence of a virus was isolated in only two of the 50 London cases in which testing had been undertaken. In three other cases the presence of a virus could not be ruled out. Analysis of the social factors cast further doubt on the overlaying/accidental suffocation hypothesis. Only nine cases were recorded as occurring when the infant was in bed with a parent and there was little evidence for other causes of accidental suffocation. The previous standard of care for victims was recorded as good, and while in many cases parents recol- lected a history of minor symptoms, such as a slight cold or some vomiting of feeds, ‘these were almost all of an apparently trivial kind’.56 As was noted at the time, such recol- lection after death could be ascribed to the obvious desire to find any sort of foothold for investigation and explanation.57 While the interim report noted that the study had not yet identified the cause of sudden death, after an MRC review of the interim findings it was Page 10 of 22 Angus H. Ferguson agreed that the work should continue. Although recognising that the social investigation was important, the report concluded that the intensification of virus investigation was the crux.58 agreed that the work should continue. Although recognising that the social investigation was important, the report concluded that the intensification of virus investigation was the crux.58 The MRC review had been undertaken by four referees. Sir Wilson Jameson, Sir Wilfrid Sheldon and Dr Alan Carruth Stevenson were in favour of continuing the work.59 By con- trast, Sir Dugald Baird was unimpressed by the interim report and doubted that there was sufficient value in continuing the research.60 Such doubts were not reflected in the reply sent to the Ministry of Health: Theconcensus(sic)ofopinionofthoserefereeswehaveconsultedisthatthereisaneed for studies of this kind, and in view of the promising start that has been made it would seemappropriateforProfessorBanksandhiscolleagues,shouldtheysowish,tosubmit concrete proposals for future work for consideration by the Board.61 at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from This offer of funding from the MRC Clinical Research Board (CRB) was accepted in order to add immunology to the lines of investigation. 58Banks, ‘An Enquiry into Sudden Death’, 191. 59Jamesonwasabarrister-at-law and had held anumber of teaching positions focused on public health in London. He served as Chief Medical Officer to the Min- istry of Health and Ministry of Education 1940–50; Sheldon was physician-pediatrician to the Queen 1952–71 and a consulting physician and pediatrician at King’s College Hospital and the Hospital for Sick Children, Great Ormond Street; having gained clinical experience in hospitals in both Glasgow and London, and served in the Royal Army Medical Corps, Steven- son was Professor of Social and Preventive Medicine at Queen’s University Belfast 1948–58, before becom- ing Reader in Public Health at London University and Director of the MRC Population Genetics Unit and Lecturer in Human Genetics at Oxford University 1958–74. 62W. E. Parish, A. M. Barrett and R. R. A. Coombs, ‘Inhal- ation of Cow’s Milk by Sensitized Guinea Pigs in the Conscious and Anaesthetized State’, Immunology, 1960, 3, 307–24; Parish, Barrett, Coombs, M. Gunther and F. E. Camps, ‘Hypersensitivity to Milk and Sudden Death in Infancy’, The Lancet, 1960, 276, 1106–10. 61MRC memo 10 December 1963. National Archives, Kew (hereafter: NA) FD7/715. 60Baird was Regius Professor of Midwifery at the Univer- sity of Aberdeen 1937–65, and Honorary Director of the MRC Obstetric Medicine Research Unit. 60Baird was Regius Professor of Midwifery at the Univer- sity of Aberdeen 1937–65, and Honorary Director of the MRC Obstetric Medicine Research Unit. Ministry of Health and MRC Studies In 1960, a team of researchers from Cam- bridge and London published research demonstrating that inhalation of cow’s milk could produce a severe immunological reaction leading to sudden death in sensitised guinea pigs.62 They believed this might provide an explanation of cases of sudden death involving artificially fed infants and MRC funding was used to explore the hypothesis. Mavis Gunther had been investigating the effects of bottle feeding on infants, and, combining this with Robin Coombs’ work in immunology, a collaborative project was devised to assess whether an overwhelming immunological reaction might result from regurgitated milk getting into the respiratory tract or lungs of the infant.63 The significance of extending the Steering Committee’s study in this direction was twofold. First, immunological work became the major focus of British research into sudden infant deaths. Secondly, Coombs became a critic of the Ministry of Health Steering Committee, and a catalyst for the promo- tion of MRC research. Prior to departing for Washington to attend the first international symposium on the causes of sudden death in infants, in July 1963 Coombs wrote a letter urging the MRC to Sudden Infant Death Syndrome in the British Context Page 11 of 22 set up a small working party or conference to discuss cot deaths.64 The letter was partially motivated by the fact that his recent work with Parish was coming to an end. He felt that an independent evaluation of the research was necessary before developing it further with Gunther, who had a funding application pending at the MRC. Although their experi- mental findings had yet to produce convincing evidence in support of the anaphylactic shock hypothesis, there were still promising leads. However, ‘the next move is a rather dif- ficult one, and all depends on how much credence one puts on the hypothesis in the light of all our experimental evidence.’65 Evidentlytheywerestrugglingtoprovethatamodifiedanaphylacticreaction,triggeredby the regurgitation of cows’ milk into the respiratory tract or lungs of a sensitised sleeping infant, was a likely cause of cot death. While there was some evidence for this hypothesis in results of their tests in anaesthetised guinea pigs, it was no more than circumstantial. Nonetheless, even without solid proof that anaphylaxis resulting from hypersensitivity to cows’ milk was definitely the cause of some cases of cot death, they might still assist in re- ducing the risk of such deaths. 64Coombs to Bunje 12 July 1963. NA FD7/715. 65Ibid. 66Ministry of Health, Enquiry, 17. 67MRC Note of meeting with Coombs (on 4 October 1963) dated 10 October 1963. NA FD7/715. 68The report acknowledged that the greater length and detail of the questionnaire proved problematic and met with some resistance. Pasupathy, Jill P. Pell, Michael Fleming and Gordon C. S. Smith, ‘Trends in Socioeconomic Inequalities in Risk of Sudden Infant Death Syndrome, Other Causes of Infant Mortality, and Stillbirth in Scotland: Population Based Study’, British Medical Journal, 16 March 2012, 344–58. Page 12 of 22 Angus H. Ferguson children of the same age and sex, and drawn from the same residential areas, as cases of sudden death. Findings from each strand of investigation were used to place cases into one of two cat- egories: ‘explained’ or ‘unexplained’. The former included cases in which either a definite or apossiblecauseofdeathhadbeenidentified.69The‘unexplained’categorywasreservedfor caseswheredetailedinvestigationresultedinnocausebeingidentified.Outofatotalof130 cases in which the history and macroscopic investigations were backed up with detailed histological post-mortem investigations, 93 were classified as ‘unexplained’. Both the bac- teriological and viral investigations echoed the negative findings of the interim report. The analysis of social circumstances drew on reports of 152 cases, of which 108 remained unex- plained.Therewasincreasedrecollectionofsymptomsofrespiratoryillnessfromtheparents of sudden death cases when compared to the control group—68 per cent compared to 31 per cent.Echoingtheinterim findings, thesewereoften verytrivial—snuffles oraslighthead cold—and had not given cause for anxiety at the time. The final report acknowledged the likelihood of bias, suggesting that at least some of the difference could be attributed to the fact that parents of victims ‘seeking to find some explanation for the tragedy, are likely to recall any little symptom, whereas the mother of a healthy lively control child might easily forget that the baby had a slight cold ten days previously.’70 A short period of breastfeeding and early bottle feeding; the use of a soft pillow; illegitimacy; being born to a young mother; low household income; low social class; and poorer quality housing were all identified as factors leading to an increased risk of sudden infant death. Clearly then, while cases of sudden infant death were found across the socioeconomic spectrum, the risk factors identified for sudden infant death echoed earlier concerns surrounding over- laying—highlighting poor families, living inbadhousing, andillegitimacy as majorfactors.71 at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from Theimmunologicalresearchhadfocusedonestablishingevidencethathypersensitivity to cows’milkmightprovokefatalanaphylacticshock.Studiesoflungtissuewerelargelyincon- clusive. Similarly, evidence of cows’ milk in the lungs of 25 out of 60 ‘unexplained’ cases was noted,althoughthisfindingwascontextualisedbythefactthatitwasalsopresentin8outof 25 ‘explained’ cases—suggesting that it might be a characteristic, rather than a trigger, of terminal events. Perhaps most significantly, serum antibody titres to cows’ milk protein were on average higher for ‘unexplained’ cases than for normal infants of the same age. Combined with the fact that ‘unexplained’ cases were more likely to be bottle-fed, this aspect seemed worthy of further investigation. Pasupathy, Jill P. Pell, Michael Fleming and Gordon C. S. Smith, ‘Trends in Socioeconomic Inequalities in Risk of Sudden Infant Death Syndrome, Other Causes of Infant Mortality, and Stillbirth in Scotland: Population Based Study’, British Medical Journal, 16 March 2012, 344–58. 69The former included heart disease or tracheobronchi- tis; the latter included slight inflammatory changes in the respiratory tract or bacteriological evidence of acute infection. 70 69The former included heart disease or tracheobronchi- tis; the latter included slight inflammatory changes in the respiratory tract or bacteriological evidence of acute infection. 70Ministry of Health, Enquiry, 11. 71For more recent discussion of socioeconomic status and SIDS, see Angela M. Wood, Dharmintra 71For more recent discussion of socioeconomic status and SIDS, see Angela M. Wood, Dharmintra 70Ministry of Health, Enquiry, 11. Ministry of Health and MRC Studies Their strategy focused on infant feeding, in particular the risks associated with the use of dried milk powders specially treated to render the soluble proteins insoluble in the stomach. If ways could be found to reduce the likelihood of infants regurgitating artificial feeds, then the risk of cot death resulting from anaphylactic shock might be correspondingly reduced regardless of the fact that the specific mechanism or aetiology had not been experimentally proven. at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from The MRC extended Gunther’s funding and this research later informed the recommenda- tions on breastfeeding and research on artificial feeds arising out of the Steering Commit- tee’s final report.66 On his return from Washington, Coombs arranged a meeting to discuss the possibility of an MRC conference. Evidently he was not inspired by the current work of the Steering Committee. He suggested that it was ‘a committee of amateurs’ in need of expert guidance and opinions. As it had not met for two years, he regarded it as ‘moribund’ and indicated that its chairman was aware, and approved, of his request that the MRC set up another body.67 Negotiations towards arranging a small conference took place over subsequent months. While there was Ministry of Health support for an MRC sponsored meeting, this was delayed until publication of the final report of the Steering Committee. The Steering Committee issued its final findings in autumn 1964. Following the recom- mendation of its interim report, exhaustive virological examination had been undertaken on specimens from a further 51 cases but results remained negative. Consequently, this aspectofthe workwassuspendedin1961inordertoconcentrateonanalysisofsociological and immunological factors. Investigation of social factors was refined, omitting questions that prior analysis had shown to be irrelevant and adding more detailed questions about bedding, the clinical history of cases and the use of medicines.68 It was also extended to in- corporate control group data, undertaking the same questionnaire with parents of healthy Page 12 of 22 Angus H. Ferguson Page 12 of 22 Angus H. Ferguson Although a large number of deaths remained unexplained after detailed investigation, the report concluded by highlighting what appeared to be statistically significant risk factors: early bottle-feeding, the use of a soft pillow, and, at least in some cases, recent in- fection. Therefore, the report recommended that certain precautions be taken. If a pillow was used for sleeping, it should be hard rather than soft. All babies should be exclusively Sudden Infant Death Syndrome in the British Context Page 13 of 22 breastfed for the first two weeks of life. In order to minimise any risk of fatal anaphylactic shock resulting from inhalation of cow’s milk, a means of treating cows’ milk should be found to ensure that all the proteins coagulated in the infant’s stomach, thereby minimising the risk of regurgitated food proteins provoking an overwhelming immunological response.72 Having taken the Steering Group’s investigations as far as he felt possible, Banks sug- gested to Sir George Godber that the MRC should determine if further investigation was ne- cessary.73 Godber and Sir Harold Himsworth decided that any future meeting should be confined to consideration of further research on the anaphylactic shock hypothesis.74 An MRC working party conference was convened, under the Chairmanship of Professor Arnold Ashley Miles on 16 June 1965.75 Coombs, who had originally pushed for the meeting, was asked to present a paper on the anaphylactic hypothesis as a starting point for the discussion. Echoing his previous comments to the MRC in 1963, Coombs’ presenta- tion noted the strengths and weaknesses of attempts to produce firm evidence for the theory. In the ensuing debate, the participants expressed reservations, pointing to the cir- cumstantial,unconvincingnatureoftheevidenceproducedtosupportthehypothesis,high- lighting in particular the problem of using anaesthesia to mimic sleep conditions in guinea pigs. at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from at Periodicals Dept on March 6, 2015 http://shm.oxfordjournals.org/ m In summing up, Miles felt that no clear programme of further investigation had emerged from the meeting. 75Ashley Miles was Professor of Experimental Pathology, University of London and Director of the Lister Institute of Preventive Medicine, London, 1952–71. In 1945, he co-edited (with G. S. Wilson) Topley and Wilson’s Prin- ciples of Bacteriology, Virology and Immunity. Atten- dees of the June meeting included: Professor A. A. Miles, Dr R. R. A. Coombs, Dr M. J. R. Dawkins, Dr D. M. T. Gairdner, Dr L. E. Glynn, Dr Mavis Gunther, Professor D. V. Hubble, Dr J. H. Humphrey, Dr E. M. Ring, Professor H. O. Schild, Dr D.R.Stanworth,DrY.AuzinsandDrB.E.C.Hopwood. 76Minutes of the Conference on Cot Deaths, London, 16 June 1965. NA FD7/715. 77Officenoteontheconferenceoncotdeaths,6October 1965. NA FD7/715. 78Coombs’s grant comprised £900 for scientific assist- ance and £200 expenses for chemical coupling of red cell antibodies with protein allergens. MRC grant com- mencing 1 October 1965. NA FD7/715. On the reti- cence of other researchers, see MRC note, 18 March 1966. NA FD7/715. 72Ministry of Health, Enquiry, 17. 73Godber was Chief Medical Officer, Ministry of Health. 74Himsworth served as Secretary of the MRC 1949–68 and Deputy Chairman 1967–8. Godber and Hims- worth’s decision is documented in Hopwood to Miles, 9 March 1965. NA FD7/715. 75Ashley Miles was Professor of Experimental Pathology, University of London and Director of the Lister Institute of Preventive Medicine, London, 1952–71. In 1945, he co-edited (with G. S. Wilson) Topley and Wilson’s Prin- ciples of Bacteriology, Virology and Immunity. Atten- dees of the June meeting included: Professor A. A. Miles, Dr R. R. A. Coombs, Dr M. J. R. Dawkins, Dr D. M. T. Gairdner, Dr L. E. Glynn, Dr Mavis 74Himsworth served as Secretary of the MRC 1949–68 and Deputy Chairman 1967–8. Godber and Hims- worth’s decision is documented in Hopwood to Miles, 9 March 1965. NA FD7/715. 78Coombs’s grant comprised £900 for scientific assist- ance and £200 expenses for chemical coupling of red cell antibodies with protein allergens. MRC grant com- mencing 1 October 1965. NA FD7/715. On the reti- cence of other researchers, see MRC note, 18 March 1966. NA FD7/715. 72Ministry of Health, Enquiry, 17. 73Godber was Chief Medical Officer, Ministry of Health. Medicine; and Dr J. A. Dudgeon of the Department of Microbiology at the Great Ormond Street Hospital for Sick Children. MRC correspondence in NA FD23/ 1882: Dudgeon to Levy, 29 April 1966; Allison to Levy, 29 April 1966; Godfrey to Miles, 3 May 1966; Levy to Dudgeon, 5 May 1966; Levy to Allison, 5 May 1966. Page 12 of 22 Angus H. Ferguson There was consensus of opinion that, despite equivocal evidence for the anaphylaxis hypothesis, some further work should be undertaken—a conclusion driven more by a general interest in further research on infant immunology than any clear belief that it was likely to reveal the aetiology of unexplained infant deaths.76 The recommenda- tion passed on to the MRC was that, if its CRB was in favour of pursuing research, a working partyshouldbesetuptoexaminethevalidityandfeasibilityoffurtherworkonthecows’milk protein hypersensitivity hypothesis.77 In fact, the CRB advised that such a working party was unlikely to serve any useful purpose. More generally, there was little evidence of desire to undertake significant further research on this aspect. Coombs received a small grant, but when the MRC approached other researchers there was reluctance to develop work on the anaphylaxis hypothesis.78 Even Gunther, whose research had been supported by ongoing grants from the MRC, declined the opportunity to apply for funding to develop a Page 14 of 22 Angus H. Ferguson new project. She cited the need to concentrate on writing a book on infant feeding which had been set aside whilst working on cot death research: new project. She cited the need to concentrate on writing a book on infant feeding which had been set aside whilst working on cot death research: theendingofmy grantshouldbeseenquiteapartfromtheneedforastructureofsome sort to consider further work on cot death. The events have been like a game of chess with a sequence of moves. The Council took over formally from the Ministry’s Steering Committee and then by limiting all future consideration to aspects of milk allergy and then finding no means of tackling milk allergy, have cornered the subject into absolute immobility.79 at Periodicals Dept on March 6, 201 http://shm.oxfordjournals.org/ Downloaded from Gunther urged the MRC to set up a new committee to consider various lines of enquiry, in- cluding the virology of intrauterine and neonatal infections; further study of the peculiarities oftheimmunestateinthefirstsixmonthsoflife;moredetailedpost-mortemhistologyofcot death; and further statistical analysis of the registration of cases. She enclosed a copy of an article she had recently written, which postulated that exposure to viruses, such as rubella,couldresultinintrauterineorneonatalinfectionandthedevelopmentofantibodies. 83Marie A. Valdes-Dapena, ‘Progress in Sudden Infant Death Research, 1963–69’, in Bergman et al., Sudden Infant Death Syndrome, 3. Faulkner to Gunther, 18 April 1966. NA FD23/1882. 82Opinions were received from Dr Anthony Allison from the Department of Virology and Bacteriology at the National Institute for Medical Research; Professor Sir Ashley Miles of The Lister Institute of Preventive 80The article was subsequently published: Mavis Gunther, ‘Cot Deaths: Anaphylactic Reaction After Intrauterine Infection as Another Potential Cause’, The Lancet, 1966, 287, 912–14. Gunther to Faulkner, 10 March 1966. NA FD23/1882. 80The article was subsequently published: Mavis Gunther, ‘Cot Deaths: Anaphylactic Reaction After Intrauterine Infection as Another Potential Cause’, The Lancet, 1966, 287, 912–14. Page 12 of 22 Angus H. Ferguson As an alternative to the artificial feeding hypothesis, Gunther suggested that a severe antibody-antigen reaction may be another possible cause of anaphylactic shock resulting in cot death.80 at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from Replyingthefollowingmonth,theMRCindicatedthatanewcommitteewasunlikely.The letter stressed that they had not taken over investigation of sudden infant deaths from the Ministry of Health, but rather, in discussion they had come to the view that further examin- ation of the hypersensitivity hypothesis would be a productive next step. However, they had been unable to stimulate interest in undertaking new work in the field.81 Nonetheless, the letter expressed a desire to encourage research on the virology of intrauterine and neonatal infections. To this end, the MRC gauged expert opinion on the merits of Gunther’s idea. The respondents were in favour of investigations into the role of viruses in cot death, in light of recent advances in knowledge of respiratory viral infections, although they recognised a po- tential difficulty in obtaining suitable material for study. Dudgeon, who already had MRC funding for research on rubella, suggested he could follow this up as an aspect of his current work. He indicated that his colleague, Dr Soothill, an immunologist, might also be interested in undertaking some investigation.82 81Faulkner to Gunther, 18 April 1966. NA FD23/1882. 79Gunther to Faulkner, 10 March 1966. NA FD23/1882. 79Gunther to Faulkner, 10 March 1966. NA FD23/1882. 79Gunther to Faulkner, 10 March 1966. NA FD23/1882. 80The article was subsequently published: Mavis Gunther, ‘Cot Deaths: Anaphylactic Reaction After Intrauterine Infection as Another Potential Cause’, The Lancet, 1966, 287, 912–14. 81Faulkner to Gunther, 18 April 1966. NA FD23/1882. 82Opinions were received from Dr Anthony Allison from the Department of Virology and Bacteriology at the National Institute for Medical Research; Professor Sir Ashley Miles of The Lister Institute of Preventive The Second International Conference, 1969 The Second International Conference, 1969 ‘The first step towards the solution of any problem is the recognition of the existence of that problem’ was Marie Valdes-Dapena’s observation whilst summarizing progress between the first and second international conferences on sudden infant death.83 The conference Sudden Infant Death Syndrome in the British Context Page 15 of 22 in 1963 had recognised the existence of the problem and since then work had begun on finding a solution, and, drawing on her earlier published review of the world literature on sudden infant deaths, Valdes-Dapena summarised the findings of recent research.84 This echoed both the range of hypotheses examined in Britain and also the negative results of attempts to establish a causal explanation. Whilst acknowledging British support for the anaphylactic shock hypothesis linked with hypersensitivity to cow’s milk, Valdes-Dapena noted that attempts to evidence the theory had been unsuccessful.85 She also highlighted Gunther’s paper on intrauterine infection as a possible sourcefor a hypersensitivity reaction. While noting that the theory had merit, the fact that studies had not consistently isolated evidence of viral infection was a ‘major stumbling block’.86 Valdes-Dapena’s presentation was followed by a discussion of terminology, led by Bergman and Beckwith, which resulted in the decision to use Sudden Infant Death Syndrome.87Thenewdiagnostictermwasdeemedtohave‘theimportantvirtueofcommu- nicating to the medical profession the concept that this is, in fact, a distinctive clinico- pathological entity’ which made it preferable to the potentially misleading ‘cot’ or ‘crib’ death.88 However, despite strong rhetoric emphasising the strides taken towards confirm- ing SIDS as a ‘real disease’, studies had yet to identify a clear aetiology, or even signs that could be used to identify cases at risk prior to death.89 SIDS was a disease without known symptoms, signs, explanatory pathology or patients. This highlights a further factor in the historicity of SIDS: the medicalisation of such deaths required an ontological model of disease. at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from at Periodicals Dept on March 6, 2015 http://shm.oxfordjournals.org/ m A Disease without Symptoms, Signs, Pathology or Patients In his introductory chapter to Framing Disease, Charles Rosenberg highlights that the phys- ical manifestation and perception of symptoms is the starting point of defining disease.90 However, the importance of symptoms in the diagnostic process has changed over time. 91John V. Pickstone, ‘Ways of Knowing: Towards a His- torical Sociology of Science, Technology and Medi- cine’, British Journal of History of Science, 1993, 26, 433–58; Pickstone, Ways of Knowing. A New History of Science, Technology and Medicine (Manchester: Manchester University Press, 2000). 88For the long quote in this sentence, see Ibid., 14. While often associated with periods when infants were asleep, not all cases of sudden infant death occurred when the infant was horizontal or in bed—cases had been recorded where the infant had been awake and was being carried around or was in a cart, see 90Charles E. Rosenberg, ‘Introduction. Framing Disease: Illness, Society, and History’, in Charles E. Rosenberg and Janet Golden, eds, Framing Disease. Studies in Cultural History (New Brunswick: Rutgers University Press, 1992), xiii–xxvi, xvi. 87‘Discussion of Terminology and Definition of the Sudden Infant Death Syndrome’, in Bergman et al., Sudden Infant Death Syndrome, 14–22. 84Marie A. Valdes-Dapena, ‘Sudden and Unexpected Death in Infancy: A Review of the World Literature 1954–66’, Paediatrics, 1967, 39, 123–38. 85Ibid., 133. 86Ibid ibid., 15; Valdes-Dapena, ‘Sudden and Unexpected Death in Infancy’, 123. 89 89For the confirmation of SIDS as a ‘real disease’, see Bergman et al., Sudden Infant Death Syndrome, ix. ibid., 15; Valdes-Dapena, ‘Sudden and Unexpected Death in Infancy’, 123. 89For the confirmation of SIDS as a ‘real disease’, see Bergman et al., Sudden Infant Death Syndrome, ix. 90 Press, 1992), xiii–xxvi, xvi Individual Sickness’, in A. C. Crombie, ed., Scientific Change (London: Heinemann, 1961), 629–58. The Second International Conference, 1969 The patient’s narrative case history, prominent in eighteenth-century medicine, was grad- uallyabsorbedintomodelsofdiseasebasedonstandardisedunderstandingsofpathological anatomy, supplemented by germ theory and the growing importance of laboratory based analysis.91 Technology increasingly facilitated the diagnostic process, with implications both for the diagnosis of disease before symptoms became manifest to seemingly healthy individuals, and for those unable to effectively communicate their own case history. Page 16 of 22 Angus H. Ferguson Increasingly,thepatient’sawarenessandexperienceofdiseaseandthescientificallydefined existence of disease could be separated. AsRosenbergnotes,the‘modernhistoryofdiagnosisisinextricablyrelatedtodiseasespe- cificity, to the notion that diseases can and should be thought of as entities existing outside the unique manifestations of illness inparticular menand women’.92 This formulation is one example of an idea expressed by a number of historians of medicine relating to a shift from a physiological concept of disease to an ontological concept of disease.93 The former under- stood disease as a general constitutional imbalance, particular to, and largely inseparable from, its manifestation in individual patients. The latter understood disease as a localised and standardised entity which could be more easily theorised and analysed apart from its presenceinspecificpatients.Theontologicalideaofdiseaseasspecificentitywasconnected with the rise of scientific and laboratory analysis as the basis of diagnostic medicine over the course of the nineteenth and twentieth centuries.94 at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from Writing in the 1970s, when SIDS was becoming established as an internationally recog- nised diagnosis, Stanley Reiser argued that medicine increasingly prioritised the importance of objective evidence provided by scientific laboratory tests and technological investigation of the body, over patients’ subjective accounts of their symptoms or doctors’ unaided obser- vations.95 Aside from the obvious point that infants were unable to describe their case history, victims of sudden unexpected death were, by definition, unlikely to have been active patients providing subjective evidence of illness to attract medical attention prior to death. A SIDS diagnosis could only be retrospectively applied to the deceased following autopsy. The growth of the ontological concept of disease allowed for analysis in isolation from patient experience. This gave greater scope for the medicalisation of sudden infant deaths as SIDS, a disease diagnosis, in the second half of the twentieth century. However, it was still a jump to get from a more amenable context for the recognition of sudden infant death as a medical problem, to the acceptance of SIDS as an official diagnosis. 94See David Harley, ‘Rhetoric and the Social Construc- tion of Sickness and Healing’, Social History of Medi- cine, 1999, 12, 407–35, 418. 95Stanley Joel Reiser, Medicine and the Reign of Technol- ogy (Cambridge: Cambridge University Press, 1978), ix. 96Georges Canguilhem, The Normal and the Pathologic- al (Zone Books, New York, 1989), 92–3; Mary Tiles, ‘The Normal and the Pathological: The Concept of a Scientific Medicine’, The British Journal for the Philoso- phy of Science, 1993, 44, 729–42, 741. 92Charles E. Rosenberg, ‘The Tyranny of Diagnosis: Spe- cific Entities and Individual Experience’, The Millbank Quarterly, 2002, 80, 237–60, 237. 93Eric J. Cassell, ‘Ideas in Conflict: The Rise and Fall (and Rise and Fall) of New Views of Disease’, Daedalus. JournaloftheAmericanAcademyofArtsandSciences, 1986, 115, 19–42; Owsei Temkin, ‘The Dependence of Medicine Upon Basic Scientific Thought’, in C. M. Brooks and P. F. Cranefield, eds, The Historical Development of Physiological Thought (New York: Hafner Publishing, 1959), 5–21; Owsei Temkin, ‘The Scientific Approach to Disease: Specific Entity and 92Charles E. Rosenberg, ‘The Tyranny of Diagnosis: Spe- cific Entities and Individual Experience’, The Millbank Quarterly, 2002, 80, 237–60, 237. 93Eric J. Cassell, ‘Ideas in Conflict: The Rise and Fall (and Rise and Fall) of New Views of Disease’, Daedalus. JournaloftheAmericanAcademyofArtsandSciences, 1986, 115, 19–42; Owsei Temkin, ‘The Dependence of Medicine Upon Basic Scientific Thought’, in C. M. Brooks and P. F. Cranefield, eds, The Historical Development of Physiological Thought (New York: Hafner Publishing, 1959), 5–21; Owsei Temkin, ‘The Scientific Approach to Disease: Specific Entity and 92Charles E. Rosenberg, ‘The Tyranny of Diagnosis: Spe- cific Entities and Individual Experience’, The Millbank Quarterly, 2002, 80, 237–60, 237. The Second International Conference, 1969 For, as Canguilhem notes, the development of a standardised model of disease, potentially allowing it to be diagnosed even before symptoms have become manifest to the patient, is typically a result of the recording and investigation of previous cases.96 With SIDS, the absence of symptoms, signs or explanatory pathology in previous cases made it difficult to develop a standardised model that could be used to diagnose potential cases of SIDS in living infants. An alternative option was to use epidemiology to seek an objective understanding of the disease derived from analysis of population level statistics rather than the symptoms or signs Sudden Infant Death Syndrome in the British Context Page 17 of 22 Fig. 1 Infant mortality rates in England and Wales, 1957–1968 Source: Based on the figures contained in Table 1.2 in the Department of Health and Social Security, Confidential Enquiry into Postneonatal Deaths 1964–1966 (London: HMSO, 1970), 4. at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from ig. 1 Infant mortality rates in England and Wales, 1957–1968 Fig. 1 Infant mortality rates in England and Wales, 1957 1968 Source: Based on the figures contained in Table 1.2 in the Department of Health and Social Security Confidential Enquiry into Postneonatal Deaths 1964–1966 (London: HMSO, 1970), 4. g y g Source: Based on the figures contained in Table 1.2 in the Department of Health and Social Security, Confidential Enquiry into Postneonatal Deaths 1964–1966 (London: HMSO, 1970), 4. from individual patients. As Figure 1 demonstrates, in the decade prior to the definition of SIDS, overall infant mortality was decreasing, primarily as a result of falling neonatal mortal- ity. Although numerically smaller, postneonatal mortality was relatively static over the period.97 from individual patients. As Figure 1 demonstrates, in the decade prior to the definition of SIDS, overall infant mortality was decreasing, primarily as a result of falling neonatal mortal- ity. Although numerically smaller, postneonatal mortality was relatively static over the period.97 As the Department of Health and Social Security’s enquiry made clear, the comparative trendsmadepostneonatalmortalityasignificantcauseforconcern,provokingfurther inves- tigation.98Theenquiryhighlightedthat37percentofpostneonatalmortalitywasattributed to ‘cot death’, and later studies drew attention to such deaths as the largest individual cause of mortality in this group.99 The increasing prominence of sudden infant deaths attracted medical attention—a point noted in Emery’s writings on the subject.100 However, when considered at the level of population statistics, these deaths posed pro- blems for epidemiologists. 97c.5,000 deaths annually. 98DepartmentofHealthandSocialSecurity,Confidential Enquiry into Postneonatal Deaths 1964–1966 (London: HMSO, 1970). 99Fit for the Future. Report of the Committee on Child Health Services (House of Commons Parliamentary Papers 1976–77. Cmnd. 6684, 65). 100Emery and Crowley, ‘Clinical Histories’, 1518; Emery, ‘Is Sudden Infant Death Syndrome a Diagnosis?’, 101Medically trained, Froggatt was Professor in the De- partment of Social and Preventive Medicine at Queen’s University, Belfast; see Peter Froggatt, ‘The Contribution of Epidemiology to the Study of Sudden Infant Death Syndrome’, in Bergman et al., Sudden Infant Death Syndrome, 25–31. 1240; John L. Emery, ‘Classifying and Recording Un- expected Deaths of Infants’, Journal of Clinical Path- ology, 1973, 26, 386. 1240; John L. Emery, ‘Classifying and Recording Un- expected Deaths of Infants’, Journal of Clinical Path- ology, 1973, 26, 386. 101Medically trained, Froggatt was Professor in the De- partment of Social and Preventive Medicine at Queen’s University, Belfast; see Peter Froggatt, ‘The Contribution of Epidemiology to the Study of Sudden Infant Death Syndrome’, in Bergman et al., Sudden Infant Death Syndrome, 25–31. 100Emery and Crowley, ‘Clinical Histories’, 1518; Emery, ‘Is Sudden Infant Death Syndrome a Diagnosis?’, 1240; John L. Emery, ‘Classifying and Recording Un- expected Deaths of Infants’, Journal of Clinical Path- ology, 1973, 26, 386. The Second International Conference, 1969 Peter Froggatt identified three difficulties: definition, post- mortem diagnosis and controls.101 Death registration forms used categories from the Inter- national Classification of Diseases which provided a universal set of commonly understood diagnoses intended to produce uniform and comparable statistics across regions and coun- tries. However, there were grey areas in classifications and inconsistency in registration Page 18 of 22 Angus H. Ferguson practice.102 Suddeninfantdeathsmightberegisteredunderavarietyofheadings,reflecting differences in local understanding and approach, and depending on whether a full case history had been taken and/or an autopsy carried out. Echoing findings of earlier studies, Froggatt noted that the registered cause of death could be influenced by the socioeconomic circumstances of cases. Suspicion of neglect or foul play in cases from poorer backgrounds could lead to a registration of ‘accidental suffocation’ or ‘cause unknown’. In more affluent areas, doctors might register a clear cause, even in the absence of evidence, in order to ease the upset and avert any stigma of suspicion. Cases of sudden infant death shared character- isticswithrespiratorydeaths—apointevidencedbythesubsequentdiagnostictransferfrom respiratory infection to SIDS in the 1970s and 1980s—and the former were often registered as the latter despite the absence of any evidence of respiratory infection. Inconsistent regis- tration inhibited the development of longitudinal trends in sudden infant death, affecting comparisons of incidence across geographical regions. practice.102 Suddeninfantdeathsmightberegisteredunderavarietyofheadings,reflecting differences in local understanding and approach, and depending on whether a full case history had been taken and/or an autopsy carried out. Echoing findings of earlier studies, Froggatt noted that the registered cause of death could be influenced by the socioeconomic circumstances of cases. Suspicion of neglect or foul play in cases from poorer backgrounds could lead to a registration of ‘accidental suffocation’ or ‘cause unknown’. In more affluent areas, doctors might register a clear cause, even in the absence of evidence, in order to ease the upset and avert any stigma of suspicion. Cases of sudden infant death shared character- isticswithrespiratorydeaths—apointevidencedbythesubsequentdiagnostictransferfrom respiratory infection to SIDS in the 1970s and 1980s—and the former were often registered as the latter despite the absence of any evidence of respiratory infection. Inconsistent regis- tration inhibited the development of longitudinal trends in sudden infant death, affecting comparisons of incidence across geographical regions. Sleep” Cot Death and Infant Care’ (unpublished PhD thesis, University of Cambridge, 1995). 102For more on the historiography of cause of death, see Risse, ‘Cause of Death as a Historical Problem’; Codell Carter, ‘Causes of Disease and Cause of Death’; Hardy, ‘Death is the Cure of All Diseases’. 103John L. Emery, ‘Certification of Cot Deaths’, British Medical Journal, 1972, 5841, 669. 104Nirupa Dattani and Nicola Cooper, ‘Trends in Cot Deaths’, Health Statistics Quarterly, 2000, 5, 10–16. 105Thisperiodincorporatestherevisedmedicaladviceon infantsleeping positionin1991whichledtothe‘Back to Sleep’ campaign. See Christine Hiley, ‘“Back to 106SUDI = Sudden Unexpected Death in Infancy. On ter- minology, see, for example, Sylvia R. Limerick and Chris J. Bacon, ‘Terminology Used by Pathologists in Reporting on Sudden Infant Deaths’, Journal of Clin- ical Pathology, 2004, 57, 309–11; Stephen J. Gould, Martin A. Weber and Neil J. Sebire, ‘Variation and Un- certaintiesintheClassificationofSuddenUnexpected Infant Deaths Among Paediatric Pathologists in the UK: Findings of a National Delphi study’, Journal of Clinical Pathology, 2010, 63, 796–99. 103John L. Emery, ‘Certification of Cot Deaths’, British Medical Journal, 1972, 5841, 669. 102For more on the historiography of cause of death, see Risse, ‘Cause of Death as a Historical Problem’; Codell Carter, ‘Causes of Disease and Cause of Death’; Hardy, ‘Death is the Cure of All Diseases’. 112Mark’s father worked in an investment house in New York and evidently Mark’s parents were able to afford to press the matter, setting up a foundation to lobby and fund scientific research. The Second International Conference, 1969 illustrated the difficulty of using animal models.108 at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from Emery’s publications highlight the importance of statistics to the rise of medical interest in SIDS, but without symptoms, signs or pathology, it is obvious why he suggested SIDS appeared to be a diagnostic dustbin. However, the validity of this provocative label is de- pendent on regarding diagnoses purely in terms of symptoms, signs and pathology. Rosen- berg notes that the act of diagnosis not only structures medical practice but also confers social approval on particular sickness roles and legitimises bureaucratic relationships.109 Conceptualising particular characteristics as a disease both reflects and shapes social atti- tudes. As Elizabeth Fee puts it, ‘medicine itself is not neutral but carries both liberating and repressive functions’.110 A medical diagnosis can influence society’s judgement of indi- viduals in positive or negative ways. In cases of SIDS, the diagnosis provided a medical model of understanding that presented the first step towards an explanation, helping to counter legal suspicions of intentional harm that often surrounded the parents of SIDS victims. Emery’s publications highlight the importance of statistics to the rise of medical interest in SIDS, but without symptoms, signs or pathology, it is obvious why he suggested SIDS appeared to be a diagnostic dustbin. However, the validity of this provocative label is de- pendent on regarding diagnoses purely in terms of symptoms, signs and pathology. Rosen- berg notes that the act of diagnosis not only structures medical practice but also confers social approval on particular sickness roles and legitimises bureaucratic relationships.109 Conceptualising particular characteristics as a disease both reflects and shapes social atti- tudes. As Elizabeth Fee puts it, ‘medicine itself is not neutral but carries both liberating and repressive functions’.110 A medical diagnosis can influence society’s judgement of indi- viduals in positive or negative ways. In cases of SIDS, the diagnosis provided a medical model of understanding that presented the first step towards an explanation, helping to counter legal suspicions of intentional harm that often surrounded the parents of SIDS victims. Many publications open with personal accounts of cases, paying tribute to the role that the families of SIDS victims have played in promoting medical interest and research.111 Bergman notes the death of Mark Addison Roe in Connecticut in 1958 was a catalyst for sci- entific research into SIDS in the USA. J. L. Culbertson, H. F. Krous and R. D. Bendell, Sudden Infant Death Syndrome. Medical Aspects and Psychological Management (London: Edward Arnold, 1989); Francis E. Camps and Robert G. Carpenter, Sudden and Unexpected Deaths in Infancy (Cot Deaths). Report of the Proceedings of the Sir Samuel Bedson Symposium held at Adden- brooke’s Hospital, Cambridge (Bristol: John Wright and Sons, 1972). 107Froggatt, ‘The Contribution of Epidemiology’, 26. 108Ann Dally, ‘Status Lymphaticus’, 70–85. 109Rosenberg ‘The Tyranny of Diagnosis’, 239. 110Elizabeth Fee, ‘Henry E. Sigerist: From the Social Pro- duction of Disease to Medical Management and Sci- entific Socialism’, The Milbank Quarterly, 1989, 67, 127–50, 129. There is a significant literature on this aspect of medicine, from critiques of medical author- ity in the works of Foucault and Illich, to analyses of medicalisation of death through technological pro- longation of life, to studies of growing numbers of cases of medically unexplained symptoms. The med- icalisation of SIDS represents a case of liberation. 111Bernard Knight, Sudden Death in Infancy. The ‘Cot Death’ Syndrome (London: Faber and Faber, 1983); J. L. Culbertson, H. F. Krous and R. D. Bendell, Sudden Infant Death Syndrome. Medical Aspects and Psychological Management (London: Edward Arnold, 1989); Francis E. Camps and Robert G. Carpenter, Sudden and Unexpected Deaths in Infancy (Cot Deaths). Report of the Proceedings of the Sir Samuel Bedson Symposium held at Adden- brooke’s Hospital, Cambridge (Bristol: John Wright and Sons, 1972). The Second International Conference, 1969 at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from Arguably, SIDS was adopted as a diagnosis because it would facilitate better registration statistics.Givenhisreservationsaboutthelackofpositivediagnosticcriteria,Emeryfavoured SIDSbeingenteredassecondaryinformationafterwhateverthepathologistconsideredwas the most likely primary cause.103 Certainly, increased medical awareness of the issue impacted on registration figures. Between 1971 and 1988 the postneonatal mortality rate in England and Wales dropped from 5.9 to 4.1 per 1,000 live births, while the recorded rate of SIDSincreased from0.3 to2.0 per 1,000live births—reflecting anincrease from5 per cent to nearly 49 per cent of overall postneonatal mortality.104 The increase in SIDS mirrored a corresponding decline in deaths from respiratory conditions, indicating diagnostic trans- fer. Between 1988 and 1992, there was sharp decline in all forms of postneonatal mortality, including SIDS.105 Rates were stable between 1992 and 1997, before transfer away from SIDS to other causes of death. Recent studies indicate that, while the term ‘SIDS’ is still used, pathologists register cases under a variety of terms—SIDS; SUDI; ‘unascertained’— reflecting local preferences and carrying nuances of interpretation.106 Froggatt’s second and third problems were the fact that SIDS was solely a post-mortem diagnosis and it was difficult to identify suitable controls for comparative study. This limited the range of potential investigations and made it difficult to determine whether risk factors identified for the SIDS group were characteristic of SIDS in particular. The resultant study population will be biased toward factors known to be associated with infant mortality in general—that is, in all probability, toward bigger and poorer Sudden Infant Death Syndrome in the British Context Page 19 of 22 families, a preponderance of males and of winter deaths, a lower birth weight, and so on.Wenote,ofcourse,thatthesearetheveryfactorsincriminatedinsuddenunexpect- ed death.107 families, a preponderance of males and of winter deaths, a lower birth weight, and so on.Wenote,ofcourse,thatthesearetheveryfactorsincriminatedinsuddenunexpect- ed death.107 While epidemiological research highlighted sudden infant deaths as an area of concern, this was only a tentative step towards identification of a disease. Progress required the develop- ment of causal explanations demonstrating the mechanism(s) by which the risk factors resulted in sudden death, followed by experimental testing of the hypotheses. However, as Dally’s account of medical interest in Status Lymphaticus underlines, there were signifi- cant risks in attempting this using healthy infants; and the work of Coombs et al. 111Bernard Knight, Sudden Death in Infancy. The ‘Cot Death’ Syndrome (London: Faber and Faber, 1983); and Sons, 1972). The Second International Conference, 1969 Having been pronounced healthy at a check-up two weeks beforehand, Mark’s sudden death left his parents seeking an explanation. Unable to identify a suitable foundation or research project, in 1962 the Roes decided to set one up using the substantial insurance policy Mark’s grandfather had taken out at his birth.112 Thus, for Bergman, in America the change in medicine’s attitude to SIDS came about ‘not through any actions of the high priests of science and medicine, nor through Page 20 of 22 Angus H. Ferguson theinitiativesofgovernmentpolicymakers.Changewaswroughtbyasmallbandofparents who had lost babies to SIDS, aided by a few good-hearted physicians by means of an orga- nised political campaign.’113 In Britain, the Foundation for the Study of Infant Deaths was established in 1971 at a sym- posium, held in Cambridge, which arose out of a grandmother’s attempts to find an explan- ation for her infant grandson’s unexpected death.114 During this meeting, Professor Wedgwood, of the department of pediatrics in Seattle, presented some work of his collea- gues, Bergman and Beckwith, which made clear their awareness of the significance of pro- viding a medical diagnosis: The result of the tragedy is not only the loss of life of an infant, but also the disruption of the family as a personal and social unit. The incident has profound psychological and social concomitants. Thus recognition and understanding both of the biology and the sociology of the syndrome, and hopefully in the future the prevention of the deaths has importance that goes far beyond the immediate death of the single infant—it reflects on the whole well-being of a family. Only by recognition and under- standing of both these facts—the occurrence of the syndrome and the prevalence of the family reactions—can the physician assist properly in his chosen role. As scientists we may be more comfortable in discussing and studying the biological process; but we are negligent if we disregard the psychological and social implications of this disease.115 at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from Wheretypicallyamedicaldiagnosiswasofgreatsignificancetothepatientdiagnosed,inthe case of SIDS there were no patients. Rather, the diagnosis was only of value to others— paediatricians, pathologists, epidemiologists, medical researchers and, above all, to the parents and relatives of victims. It deflected some of the legal suspicion falling on parents and provided the beginnings of an explanation for the loss of their child. 113Bergman, The ‘Discovery’, xi. 114TheFoundation wasrelaunchedastheLullabyTrustin April 2013, see <http://www.lullabytrust.org.uk/ history-of-the-charity> (accessed 26 January 2015). Echoing the case of Mark Addison Roe, the grand- mother of a child who had died suddenly and unex- pectedly donated money which was used to sponsor the Sir Samuel Bedson Symposium at Adden- brooke’s Hospital Cambridge. Details of the confer- ence, chaired by the Countess of Limerick, were subsequently published: Camps and Carpenter, Sudden and Unexpected Deaths in Infancy. This text dates the meeting as having taken place on 17 and 18 April 1970. 115R. J. Wedgwood,‘Reviewof the U.S.A. Experience’, in Camps and Carpenter, eds, Sudden and Unexpected Deaths in Infancy, 23. Wedgwood was Professor and Chairman of the Department of Pediatrics at the Uni- versity of Washington, Seattle. 116Since its foundation in 1971, the Foundation for the Study of Infant Deaths / Lullaby trust has invested over £11 million in research <http://www.lullabytrust. org.uk/ourwork> (accessed 26 January 2015). The Scottish Cot Death Trust receives around 86 per cent of its income from fundraising events and public donations <http://www.scottishcotdeathtrust. org/about/how-are-we-funded.php> (accessed 26 January 2015). of Killing Children’, in The Independent, 12 April 2005; N. Fleming, ‘Nine Out of Ten Cot Deaths “Occur Naturally”’, in Daily Telegraph, 31 December 2004; ‘Gene Breakthrough that Could Free Murder Mother: Solicitor’s Babies May Have Been Cot Death Victims After All’, in The Daily Mail, 16 July 2001. 117Emery was aware of the importance of family welfare in cases where an infant died suddenly and unexpect- edly. See John L. Emery, ‘Welfare of Families of Chil- dren Found Unexpectedly Dead (“Cot Deaths”)’, British Medical Journal, 1972, 1, 612–15. 120Many were convictions for ‘shaken baby syndrome’ and 28 were thought worthy of consideration for appeal. Of these, three were suspected cases of SIDS. J. Rozenberg, ‘Review of 300 Child Deaths Iden- tifies 28 “Unsafe” Cases’, in Daily Telegraph, 22 De- cember 2004. See also: ‘Child Deaths: Unsafe Convictions’, in Guardian, 20 January 2004. 119See for instance: Michael Mansfield, ‘It is Time to Act over the Failings of Forensic Scientists’, in The Times, 13 March 2007; Adrian Keane, ‘Unreliable Evidence is Putting Justice in Jeopardy’, in The Times, 7 Novem- ber 2006; James Le Fanu, ‘Happy, Loving Parents? They Must Be Child Abusers. Two Landmark Court Hearings this Week Will Challenge the Testimony of Medical Experts that has Resulted in Hundreds of In- nocent Mothers and Fathers Being Convicted of Harming their Children’, in Sunday Telegraph, 19 June 2005; R. Verkaik, ‘Appeal Court Clears Mother 118For theimportanceofthis in theAmerican contextsee Bergman, The ‘Discovery’. 121For theUSA,seeBergman, The‘Discovery’; Hufbauer, ‘Federal Funding and Sudden Infant Death Research, 1945–80’. The Second International Conference, 1969 It became a focus for the families of victims, who organised themselves into support groups and raised funds to support research.116 SowhileSIDSmaylackpositivecriteriaorprophylacticandtherapeuticvalue,itwouldbe wrong to regard it as simply a diagnostic dustbin. As Emery noted, the diagnosis has great significance for parents who can be assuaged of guilt that they did something wrong or failed to do something right,and for doctors—who may be concerned that they missed Sudden Infant Death Syndrome in the British Context Page 21 of 22 some sign of disease.117 It also legitimates the phenomena as an area of medical re- search.118 some sign of disease.117 It also legitimates the phenomena as an area of medical re- search.118 However, the medicalisation of sudden infant deaths did not entirely remove legal suspi- cion of infanticide, especially when repeat cases arose within the same family. Without clear aetiology, positive diagnostic criteria or pathological evidence, sudden infant death remained a penumbral area of law, where the opinion of expert medical witnesses was often pivotal in deciding cases. In some instances where expert opinion convicted mothers accused of killing their infant(s), the evidence was subsequently discredited and the convictions overturned—the deaths being attributed to other causes, including SIDS.119 Several such high profile cases led the Attorney General to order a review in 2004.120 at Periodicals Dept on March 6, 2015 http://shm.oxfordjournals.org/ Downloaded from at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from of Killing Children’, in The Independent, 12 April 2005; N. Fleming, ‘Nine Out of Ten Cot Deaths “Occur Naturally”’, in Daily Telegraph, 31 December 2004; ‘Gene Breakthrough that Could Free Murder Mother: Solicitor’s Babies May Have Been Cot Death Victims After All’, in The Daily Mail, 16 July 2001. Conclusion In the case of Britain, SIDS was not an ignored disease. Rather, analysis points to the import- ance of recognising the historicity of SIDS—a diagnosis facilitated by the confluence of changes in medicine and law towards the middle of the twentieth century. The decline in othercategoriesofinfantmortalitymeantthatsuddeninfantdeathsbecameanincreasingly prominent concern. Legislation facilitated greater numbers of autopsies, giving unprece- dented opportunities to test theories of overlaying. And, the growing emphasis on onto- logical concepts of disease meant that it was easier to medicalise such deaths despite the absence of a patient. In sharp contrast to Bergman’s account of the challenges faced in gaining federal funding for SIDS research in the USA, it is clear that, in Britain, there was early support from both the Ministry of Health and the MRC.121 Indeed, by the mid-1960s the MRC had difficulty persuading researchers to undertake further immuno- logical investigations. However,thegrowthofmedicalinterestandtheuseof“SIDS”didsubstantiallyimpacton registration practices. The large-scale diagnostic transfer to SIDS underlines the importance ofadiagnosticlabelinbureaucraticmedicine,andmightberegardedassignificantinreveal- ingtheextentoftheproblem.However,asFroggatnoted,theriskfactorsforSIDSwerechar- acteristic of infant mortality generally, so the category would have been an appealing Page 22 of 22 Angus H. Ferguson catchall for many cases. Over time, the flow was reversed as advances in technology and knowledge facilitated the identification of an alternate specific cause for some cases, and uncertainty over SIDS as a diagnosis led some pathologists to register deaths under other headings. This does not mean that SIDS was nothing more than a diagnostic dustbin. As noted in later sections, although the term had little therapeutic or prophylactic value, it had great sig- nificance in other respects. It could reduce the suspicion cast on parents, and alleviate some of the guilt stemming from fears that they had done something wrong or failed to do some- thing right. In that sense it had great psychological and socio-legal value. As such, it illus- trates the growing authority and status of medicine—producing a medical model of explanation that encroached into the traditional legal frameworks of understanding and re- sponse. At a time when critiques were bringing to light cases of iatrogenic harm, abuses of medical power and the dangers of overly authoritarian and paternalistic approaches to medical research,122 SIDS provides an example of more beneficent medicalisation of life and death. (London: George Allen & Unwin, 1981); Maurice H. Pappworth, Human Guinea Pigs: Experimentation on Man (London: Routledge & K Paul, 1967). 122For examples, see Ivan Illich, Medical Nemesis: The Ex- propriation of Health,(London: Calder and Boyars, 1975); Ian Kennedy, The Unmasking of Medicine Acknowledgements Thanks are due to my colleagues: Lawrence Weaver, Marguerite Dupree, Anne Crowther and Malcolm Nicolson. Their support, positive input and constructive feedback assisted the early development of this paper. I am also grateful to the anonymous referees for their helpful comments. Conclusion The relatives of victims welcomed a medical diagnosis which could provide them with a starting point towards explanation of the sudden death of their infant; could help assuage concerns or guilt that a symptom or sign had been missed; and could counter legal suspicion of infanticide. at Periodicals Dept on March 6, 201 http://shm.oxfordjournals.org/ Downloaded from at Periodicals Dept on March http://shm.oxfordjournals.org/ Downloaded from SIDS wasneither anignoreddiseasenorsimply adiagnosticdustbin.To medicalhistorians it presents a valuable case study of both the historicity of disease and the numerous ways in which it can be identified, defined and understood. Above all, SIDS provides a unique example of an officially recognised disease that, by definition, had no patients, and in which the certainty of the diagnosis was greatest whenever there was least evidence of symptoms, signs or pathology. (London: George Allen & Unwin, 1981); Maurice H. Pappworth, Human Guinea Pigs: Experimentation on Man (London: Routledge & K Paul, 1967). Funding This work was supported by the Wellcome Trust as part of an Enhancement Grant to the Centre for the History of Medicine at the University of Glasgow. Grant number 074301/Z/ 04/Z/AW/HH.
https://openalex.org/W4298348772	https://zenodo.org/records/6965424/files/IJISRT22JUL1407%20(1).pdf	English	null	A Study of the Effects of Corona Virus Disease (COVID-19) in Thailand	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	5,552	A Study of the Effects of Corona Virus Disease (COVID-19) in Thailand Vikrom Ahuja (PhD Candidate) Assumption University, Thailand Sinopharm, and Johnson & Johnson, among others. Various clinical trials at different phases are ongoing across the world. The deadly virus has spread to almost all countries and continents (Marome and Shaw, 2021). Abstract:-The purpose of this paper was to determine the effects of COVID-19 pandemic in Thailand. Specifically, the focus has been given to the economic and social impacts of the virus and associated restrictions. A mixed-methods design was adopted in this study employing both the qualitative and quantitative approaches. A mixture of KIIs and IDIs was utilized to collect the qualitative data from the respondents (n=20). A document review tool was also used to gather quantitative information from periodicals, journals, newspapers, and government reports (n=20). The concept of saturation controlled the number of respondents in this research. Both convenient and purposive sampling techniques were adopted in this study. Ethical considerations including informed consenting, privacy and confidentiality, as well as respect for persons were upheld at all times. The results showed that the COVID-19 pandemic in Thailand led to business closures, job losses, reduced government revenue, and increased inflation, among others. It was also noted that an upsurge in the rates of domestic violence, depression, and boredom was attributed the COVID-19 and related restrictions. It is also possible that the pandemic had some positive effects on other sectors like the ICT. It was recommended that the government should put aside some resources to cushion citizens from unprecedented occurrences and disasters like the COVID-19 pandemic. Sinopharm, and Johnson & Johnson, among others. Various clinical trials at different phases are ongoing across the world. The deadly virus has spread to almost all countries and continents (Marome and Shaw, 2021). The data from the WHO shows that more than 570 million people have been infected with COVID-19 across the world. Approximately 6 million individuals have succumbed to viral illnesses all over the globe (Triukose et al., 2021). However, the good news is that many people recover from this disease within a short period. For instance, data shows that more than 540 million people have recovered from the illness. Globally, the total number of vaccine doses administered is estimated to be 12,219,375,500 (Pakpour and Griffiths, 2020). This paper focuses on the status of COVID-19 in Thailand, particularly the effects on the social and economic lives of the citizens. Volume 7, Issue 7, July – 2022 Volume 7, Issue 7, July – 2022 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 A Study of the Effects of Corona Virus Disease (COVID-19) in Thailand It is a Southeast Asian country that is known for beaches, palaces, and temples. The capital city of Thailand is Bangkok and the population of the country was estimated at 69.8 million people as of the year 2020. The confirmed cases of COVID-19 in Thailand as of 22nd July 2022 are estimated at 4,568,461. The WHO reports that the total number of deaths as a result of COVID-19 in Thailand is 31,073. The Southeast Asian country has administered a total of 139,937,409 vaccine doses (Katewongsa et al., 2021). Keywords:- Coronavirus Disease, COVID-19 Restrictions, Economic Effects, Social Effects, Thailand Keywords:- Coronavirus Disease, COVID-19 Restrictions, Economic Effects, Social Effects, Thailand The COVID-19 pandemic has had severe to moderate effects on many people across the world. The most commonly affected sectors are tourism, hospitality, and transportation, among others. Countries that have relied on tourism as a source of foreign exchange were really affected by the pandemic. The unprecedented effects of COVID-19 have brought many nations down economically. The broad areas of coverage in this paper are the economic and social effects of the COVID-19 pandemic on the growth and development of Thailand (Nicola et al., 2020). Keywords:- Coronavirus Disease, COVID-19 Restrictions, Economic Effects, Social Effects, Thailand The overall purpose of this study is to determine the effects of coronavirus disease in Thailand. A. Purpose of the study The overall purpose of this study is to determine the effects of coronavirus disease in Thailand. A. Purpose of the study A. Economic Effects of COVID-19 Research has shown that the diagnosis of the first case of coronavirus in Thailand and other countries across the world led to different economic effects (Suntronwonget al., 2020). For instance, banks and other lending institutions scaled down their operations due to the unprecedented impacts of the pandemic. Some people who had active loans and facilities were unable to pay due to the tough economic times. Financiers were forced to provide grace periods for the borrowers to take a break (Cakmakl et al., 2020). Research showed that there was an increase in cases of domestic violence during the COVID-19 period. Incidences of human trafficking and child molestation were also recorded in some places across the world. It was also noted that food insecurity and hunger were on the rise in many parts of the globe due to the low agricultural production. Mental health issues comprise another huge problem that has been experienced in some regions. Research has been done to determine the association between depression and COVID-19 pandemic. Some findings showed that watching television and reading news of deaths traumatized many people (John et al., 2020). The coronavirus pandemic has also affected many sectors of the economy in Thailand. For instance, the tourism sector has been hardly hit due to the restrictions. Many countries went on lockdown to curb the spread of the virus. Tourists were unable to move from one country to another freely (Tiapraponget al., 2021). Another sector that was affected by the pandemic is transport. Most airlines were not operational in fear of the spread of the disease (Chen et al., 2021). IV. RESEARCH FRAMEWORK Despite the negative economic impacts, the pandemic was also seen to boost other sectors such as ICT. Many companies resorted to remote working and virtual meetings. ICT organizations such as Microsoft and Zoom made good profits during the period. They got a chance to market and sell their products and services (Goodwin et al., 2020). The conceptual framework adapted from the literature highlights the dependent, independent, and intervening variables of the study. The framework illustrates the cause- and-effect relationship between infectious diseases and the socio-economic development of the country. III. RESEARCH OBJECTIVE The overall objective of this paper is to determine the effects of the COVID-19 pandemic on the economic and social development of Thailand. I. INTRODUCTION The coronavirus disease, abbreviated as COVID-19, was first detected in Wuhan, China, in December 2019. The specific type of virus that causes the disease is called SARS- CoV-2. The World Health Organization declared COVID-19 as a global pandemic in early 2020. It is a highly infectious respiratory disease that spreads when a sick person sneezes, speaks, breathes, sings, and spits to a healthy individual. An infected person experiences mild to moderate symptoms that typically go away after 7 to 10 days (Vijaya et al., 2021). Some people will not require medical attention while others may become critically ill leading to hospitalizations and deaths. The virus spreads faster in closed-door settings and crowded areas. The most common symptoms of COVID-19 are coughs, fever, tiredness, and loss of smell or taste. However, some people experience rare signs such as muscle ache, diarrhea, sore throat, headaches, and skin rashes, among others. There has been a global search for drugs and vaccines that can control the spread of COVID-19. Some of the available vaccines that have been scientifically proven to be effective are AstraZeneca, Moderna, Pfizer, BioNTech, The overall purpose of this study is to determine the effects of coronavirus disease in Thailand. 766 IJISRT22JUL1407 www.ijisrt.com Volume 7, Issue 7, July – 2022 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 II. LITERATURE REVIEW there is the need to restrict such interactions. The new normal of staying indoors and avoiding public spaces led to various social effects on the people (Issac et al., 2021). there is the need to restrict such interactions. The new normal of staying indoors and avoiding public spaces led to various social effects on the people (Issac et al., 2021). E. Reliability Test A reliability test was performed when the number of target participants reached 20. The Cronbach’s Alpha Coefficients was performed and it was found to be 0.78. This figure showed that the data collection instruments had high internal validity and reliability. F. Data Collection Tools The instruments for data collection will be chosen based on the capability to provide relevant information for the quantitative and qualitative arms of the study. People who are deemed to be opinion shapers and decision-makers at the Ministry of Health used Key Informant Interviews (KIIs). All other individuals contacted in the city of Bangkok utilized In-depth Interviews (IDIs). The difference between the two qualitative tools of data collection is the ability to provide key information that no one else can give. V. RESEARCH METHODOLOGY of 20 interviews were conducted since the saturation point was reached. A. Study Design The quantitative arm of the study used a random sampling technique. The researcher searched through various databases such as PubMed and Medline to get the relevant sources of information. All publications related to the effects of the COVID-19 pandemic in Thailand were chosen for analysis. A total of 20 sources of information were obtained and analyzed since the saturation point was reached. The study adopted a mixed-methods research design to achieve the objective. The quantitative arm of the study involved a document review method where previously published data were analyzed. The data was obtained from grey literature such as government reports, WHO facts and figures, journals, periodicals, and other published information. The qualitative arm of the study involved the use of key informants and in-depth interviews to gather information from persons of interest. Specifically, a parallel study design was adopted in this research. This means that the two methods were used concurrently. B. Study Location The research was conducted between January and March 2022 in the city of Bangkok in Thailand. All the participants interviewed were found in the city. The literature reviewed was published between January 2020 and December 2021. They include articles that focus on the effects of the COVID-19 pandemic in Thailand. B. Social Effects of COVID-19 It is always said that humans are social beings who like interacting and mingling with others. However, the COVID- 19 period denied people the freedom to associate and move freely. The virus spreads faster in crowded places, hence, Independent Variables Intervening Variables Dependent Variables The above diagram shows that COVID-19 pandemic in Thailand has led to various effects on the social and economic development of the country. For example, the spread of the virus has the potential of causing economic depression and mental health problems. The restrictions imposed to curb the virus such as lockdown and curfews may cause mental health issues (Yorsaeng et al., 2022).  Spread of the virus  Restrictions imposed  Economic Depression  Mental Health Issues Vaccines Administration Intervening Variables Dependent Variables  Spread of the virus  Restrictions imposed Vaccines Administration The above diagram shows that COVID-19 pandemic in Thailand has led to various effects on the social and economic development of the country. For example, the spread of the virus has the potential of causing economic depression and mental health problems. The restrictions imposed to curb the virus such as lockdown and curfews may cause mental health issues (Yorsaeng et al., 2022). IJISRT22JUL1407 767 www.ijisrt.com Volume 7, Issue 7, July – 2022 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 C. Study Population The qualitative arm of the study targeted participants of different demographics around the city of Bangkok, Thailand. Specifically, they had various socio-economic statuses, age, gender, religion, and education levels, among others. The target participants were all people living in Bangkok city, Thailand. As for the quantitative arm of the study, the researcher adopted a document review tool to collect information from published and non-published information. The guide developed helped in obtaining the data relevant to the effects of the COVID-19 pandemic in Thailand. The data mining procedures were detailed to give useful information for the study. The three tools of data collection (KIIs, IDIs, and document review instruments) were piloted before the commencement of the study. The importance of the piloting phase is to test the reliability and validity of the tool prior to the actual work. It is also necessary to identify any errors and correct them to make sure that accurate data is collected from the field. The piloting phase was conducted in the actual environment. The data obtained from this testing phase was analyzed. a) Inclusion criteria for the qualitative arm of the study a) Inclusion criteria for the qualitative arm of the study  Adults aged between 18 and 65 years of age  Those who consented willingly to take part in the study b) Exclusion criteria for the quantitative arm of the study  Children and elderly people  Non-citizens of Thailand  Critically ill and mentally challenged persons. D. Sampling It is important to ensure that the study picks a representative sample of the target population. The people who will respond to the interview must give an overview of the opinions of the whole group. The results obtained are assumed to be generalizable to the bigger population of Thailand. G. Data Collection Procedures a) Qualitative arm of the study The data collection procedures adopted in this study are from one arm of the research or the other. The field assistants identified potential study participants using the sampling techniques identified above. They were then contacted on the phone, by e-mail, or physically to explain the background of the study. Those who accepted to be part of the study were given informed consent to read and sign. All interviews took place at a location and time that were deemed convenient to the participant. All interviews took between 25 and 35 minutes excluding the time taken during the consenting processes. Interviews were audio-recorded for reference purposes. a) Qualitative arm of the study In this case, a combination of purposive and convenient sampling techniques was used to get the people who were interviewed. The study targeted individuals who would give information related to the effects of the COVID- 19 pandemic in Thailand. For example, individuals from the relevant government ministries dealing with coronavirus directly were interviewed. People running various businesses were also interviewed to understand the effects of the virus in the conduct of their activities. A convenient sampling technique was also used in the sense that those who were available for the interview were contacted. A total 768 IJISRT22JUL1407 www.ijisrt.com Volume 7, Issue 7, July – 2022 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 b) Quantitative arm of the study The process of collecting quantitative data is different from the qualitative aspect. For instance, the researcher identified the keywords to be used in searching through the database. The sources of data were then determined using reputable databases such as PubMed, Medline, and EBSCO. Keywords were then utilized to obtain the relevant sources of information. All articles mined were reviewed to get the required data. The effects of the COVID-19 pandemic in Thailand were documented for analysis purposes. confidentiality of the participants were assured by destroying all forms of identifiers such as names and specific locations of residences. All potential risks and benefits that may accrue from the study were disclosed at the beginning of the research. The informed consent forms contained contact details for IRB that approved the study and the Principal Investigator (PI). As for the quantitative data collection, there were minimal ethical concerns. G. Data Collection Procedures It was only necessary to recognize, acknowledge, and give credit to the authors of the information obtained from the web. I. Data management It was important to ensure that all kinds of data collected and utilized in the course of the study were managed professionally. Firstly, the transfer of data from the source to the storage point was done in a manner that avoids any form of infiltration and leakage. Audio-recorded information was moved using digital recorders. All information kept in hard copies such as informed consent forms and field notes were securely kept in folders. All sets of information are kept in the custody of the principal investigator under a lock and key. At the end of the study period, all kinds of data whether in soft or hard copy will be destroyed permanently. The data kept in computers, hard drives, and digital hard drives will be deleted. The information available in hard copies such as informed consent forms and field notes will be shredded. All types of data will be de-identified for the safety and welfare of the owner. A. Sociodemographic details of participants g p f p p A total of 20 residents of Bangkok city in Thailand were interviewed before the saturation point was reached. The interview was stopped when no new information was obtained by the researcher. It was noted that the themes and subthemes started becoming repetitive when the 20th participant was being interviewed. Table 1 below indicates the socio-demographic characteristics of participants. It was noted that more men (55%) were picked by the sampling technique that was used compared to women who were at 45%. When it comes to the age brackets, results indicated that 40% of the participants were aged 51 years and above. This data shows that many senior people took part in the study. This could be attributed to the sampling technique chosen that was purposive. The researcher wanted to get information from the people who had experience in doing business before and during the COVID-19 pandemic. Many of the participants chosen were key informants who provided the information that no one else could have been privy to. It was also interesting to note that individuals who earn a monthly income of 500 dollars and below were the same as those who get 1,000 dollars above. Both groups were at 35%. Most of the key informants in this study were working for the government either directly or indirectly. The quantitative data were generated and entered in MS Excel. The fields and rows developed were in line with the specific and general objectives of the study. The information was quantified and analyzed using STATA software. VI. RESULTS AND DISCUSSION The findings of the study are reported in a two-fold manner. The data obtained from the qualitative arm of the study was analyzed and reported separately. H. Data analysis All audio-recorded data were transcribed verbatim and typed into the Microsoft Word document. Qualitative software called NVIVO was used to analyze the data obtained from KIIs and IDIs. Themes and sub-themes were generated from the transcripts.Codes were then established from themes. All the relevant themes were put together to ease the interpretation of the results. The socio-demographic characteristics of the respondents were also analyzed and summarized. H. Data analysis All audio-recorded data were transcribed verbatim and typed into the Microsoft Word document. Qualitative software called NVIVO was used to analyze the data obtained from KIIs and IDIs. Themes and sub-themes were generated from the transcripts.Codes were then established from themes. All the relevant themes were put together to ease the interpretation of the results. The socio-demographic characteristics of the respondents were also analyzed and summarized. J. Ethical considerations The study obtained relevant ethical, scientific, and regulatory approvals before its commencement. It is important to ensure that the safety, welfare, and rights of participants are upheld at all times. The detailed informed consent process is an indicator that the people who provided the data were respected. The researcher ensured that the participation is voluntary. No one was coerced to provide the information at any stage. It was also necessary to compensate participants for the time taken to respond to the interview questions. The rate of compensation was commensurate to the wage rate of the participant ranging from 15 to 20 dollars. Those who used transportation means or the Internet were reimbursed accordingly. Privacy and 769 IJISRT22JUL1407 www.ijisrt.com www.ijisrt.com Volume 7, Issue 7, July – 2022 ISSN No:-2456-216 Variables Frequency (f) Percentage (%) Gender Male Female 11 9 55 45 Age brackets (years) 18-30 31-40 41-50 51 and above 4 3 5 8 20 15 25 40 Income per month (dollars) Below 500 501-1000 1000 and above 7 6 7 35 30 35 Table 1: Socio demographic details of participants researcher wanted to get information from verified and trusted sources. It was also necessary to get the data from other places such as the government ministries and departments. It was noted that Thailand’s executive arm was collecting data on the effects of the pandemic on the social and economic livelihoods of the people. It was also interesting to see how newspapers and periodicals had a lot of data on the impacts of COVID-19 pandemic on people’s businesses. The second part of the socio-demographic details reports the findings of the quantitative arm of the study. The researcher utilized various sources of information to get the data relating to the effects of the COVID-19 pandemic in Thailand. All the resources were published between the year 2020 and 2022. It was also necessary to get the data from multiple sources to get the variety needed for the study. Results from the pie chart below show that peer-reviewed articles formed the majority of the sources consulted. The Fig. 1: A Pie Chart Showing Various Sources of Information Cited Number Journals Periodicals Government reports Newspapers Number Fig. 1: A Pie Chart Showing Various Sources of Information Cited “The COVID-19 pandemic led to the closure of my business in Thailand. J. Ethical considerations I am a proud owner of a travel agency, but the business ceased operating when there were no more tourists visiting the country”. {Respondent 15} B. Economic effects of COVID-19 pandemic in Thailand The two variables of interest in this study were the economic and social effects of the pandemic on the people. We begin by reporting the economic impacts of the pandemic on the citizens of Thailand. Twenty people who were interviewed gave their views and opinions on the subject matter. For privacy and confidentiality purposes, codes will be used to identify the respondents. The following excerpt was obtained from the transcripts: The above respondent expressed her concerns over the economic effects of the pandemic. It is noted that the tourism industry was hardly hit by COVID-19. Many people across the world were unable to travel to different destinations due to the fear of contracting the disease. It was also noted that the government had introduced strict IJISRT22JUL1407 770 www.ijisrt.com Volume 7, Issue 7, July – 2022 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 measures to curb the spread of the virus. Participant number 15 owns a travel agency and they had to close the business due to the lack of customers. industries to close their operations. The sustainability of businesses and employees in Bangkok became an issue. When one individual loses a job, the effects will trickle down to many people including parents, children, and other dependents. The following excerpt gives another illustration of the economic effects of COVID-19 pandemic in Thailand. Apart from the qualitative data obtained from interviews, the researcher got some useful information from the literature search. The periodicals, newspapers, government reports, and peer-reviewed articles gave a wide range of facts and figures on the economic effects of COVID-19 pandemic in Thailand. The bar graph below illustrates the effects of the pandemic in different areas of the economy. It was noted that the peer-reviewed journals had a lot of information relating to the topic. This could be attributed to the fact that many journals were found from the database. All the documents that were reviewed indicated that the COVID-19 pandemic led to closure of businesses, job losses, auctioning of products, and reduced sales. One of the interesting variables in this study is auctioning. It was noted that many people living in Thailand decided to sell their products for survival purposes. C. Social effects of COVID-19 pandemic in Thailand “The job loss and lack of finances frustrated my life. My children were unable to go to school because I could not afford the fees. My ailing mother stopped going to the hospital for regular check-up because the facility was full and medical experts were exhausted” {Respondent 19} Another variable of interest in this study was the social effects of the pandemic of the people living in Thailand. We begin this section by reporting the qualitative and quantitative data. The following excerpt illustrates the difficulties that people went through during the time of COVID-19 in Thailand. The above respondent expressed her concerns over the social effects of the pandemic. She lost her job leading to so many other undesirable impacts in her life. Her children were affected since they could not go to good schools. Her mother could not visit the hospital because the facility was overwhelmed with cases of COVID-19. The elderly were not allowed to move freely to minimize interactions and possible exposure to the virus. “I was unable to meet my friends due to the lock-down and restriction of movement that the government introduced. I also missed my foreign friends and family members living in Italy. All flights and other means of transport ceased operating during this time” {Respondent 2} “I was unable to meet my friends due to the lock-down and restriction of movement that the government introduced. I also missed my foreign friends and family members living in Italy. All flights and other means of transport ceased operating during this time” {Respondent 2} “I was unable to meet my friends due to the lock-down and restriction of movement that the government introduced. I also missed my foreign friends and family members living in Italy. All flights and other means of transport ceased operating during this time” {Respondent 2} The above respondent expressed her views regarding the kind of social life that she had to live following the pandemic and related restrictions. It was noted that people were not allowed to move freely due to the cessation of different means of transport. The other sets of data to be reported in this paper are the quantitative ones obtained from the documents reviewed and analyzed. The table below illustrates the social effects of COVID-19 pandemic on the people of Thailand. VII. LIMITATIONS AND CONCLUSION This paper was conducted with some limitations and delimitations. For instance, it is important to acknowledge geographical and time limitations. It was not sufficient to make a general conclusion about the social and economic effects of COVID-19 pandemic in Thailand. Collecting data in one city was not adequate and may not be replicable to the general population worldwide. In conclusion, the COVID-19 pandemic led to undesirable economic and social effects on the livelihoods of the people of Thailand and other parts of the world. [3.] Goodwin, R. et al. (2020). Anxiety and public responses to covid-19: early data from Thailand.Journal of Psychiatric Research. 2020;129:118–121. [4.] Issac A, et al. (2021). An examination of Thailand’s health care system and strategies during the management of the COVID-19 pandemic. Journal of Global Health. 2021;11. [5.] John, A. et al. (2020). The impact of the COVID-19 pandemic on self-harm and suicidal behaviour:Update of living systematic review. F1000Research. 2020;9:1097. REFERENCES The variables obtained from the documents reviewed included the rates of depression, boredom at home, and domestic violence, among others. It was interesting to note that there was an increase in domestic violence during the COVID-19 period. This was partly attributed to the tough economic times. [1.] Cakmakl, C. et al. (2020). COVID-19 and emerging markets: An epidemiological model with International production networks and capital flows [IMF Working Papers WP/20/133, International Monetary Fund]. p y [2.] Chen, R. et al. (2021). The Perspective of Thailand’s Economy After the Effect of Coronavirus-19 Pandemics: Explication by Dynamic I-O Models and Agent-Based Simulations. SAGE Open April-June 2021: 1–17. C. Social effects of COVID-19 pandemic in Thailand Source of data Rates of depression Boredom at home Domestic violence Journals 41% 54% 74% Periodicals 33% 0 51% Newspapers 28% 24% 61% Government reports 39% 69 79% J. Ethical considerations It reached a point where some individuals sold some household items and other precious property to buy essential things like foods. On the other hand, it was difficult to find buyers of different goods and services that were up for sale. The reduced number of buyers and increased products in the market is an indicator of a failing economy. The circulation of money and the purchasing power of the people were very low during the pandemic. All these indicators show that the economy was not doing well. Some of the parameters that the government uses to gauge the progress of a country include the GDP, GNP, income per capita, inflation and interest rates, among others (Vicerra, 2021). “The government used to generate a lot of revenues from hotel levies and taxes. However, for the last two years, we have been running on losses because we have to support citizens in running their businesses by providing subsidies”. {Respondent 4} The above respondent was a key informant working at a government ministry. It is clear from the above excerpt that the government of Thailand has been affected economically following the closure of business. The administration has been forced to provide subsidies to the citizens so that they can continue operating. However, the government has also been struggling due to the reduced taxes and levies from the people. “I lost a job in March 2020 because my company was closed indefinitely. We got a memo indicating that the business could not be sustained due to the lack of tourists and other key stakeholders who have been supporting the firm. The company could only afford to pay salaries for the managers and other essential workers. The downsizing of the firm’s operations led to job losses” {Respondent 11} f 11} The above respondent reported that the company retrenched many people due to the lack of business. It is clear that the COVID-19 pandemic in Thailand forced many Fig. 2: A Bar Graph Illustrating the Effects of the Pandemic as Shown from Different Sources 0 1 2 3 4 5 6 7 8 9 10 Business closure Job losses Auctioning Reduced sales Journals Periodicals Government Reports Newspapers Fig. 2: A Bar Graph Illustrating the Effects of the Pandemic as Shown from Different Sources IJISRT22JUL1407 771 www.ijisrt.com Volume 7, Issue 7, July – 2022 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 VIII. RECOMMENDATION The findings generated in this paper call for the need to adopt a multi-sectoral approach to minimizing the effects of COVID-19 pandemic in Thailand and other parts of the world. It is necessary to incorporate the contribution of various agencies on a local, regional, or global scale. The World Health Organization has been leading in the fight against the disease. However, it is important to have all sectors and agenciesplay a role in curbing the virus. For example, the government should be providing subsidies to cushion citizens from the undesirable effects of the diseases. People need to be creative and innovative to survive during tough times. [6.] Katewongsa, P. et al. (2021). The effects of the COVID-19 pandemic on the physical activity of the Thai population: Evidence from Thailand's Surveillance on Physical Activity 2020. Journal of Sport and Health Science. 10(3), May 2021, Pages 341-348. [7.] Marome, W. and Shaw, R. (2021). COVID-19 Response in Thailand and Its Implications on Future Preparedness. International Journal of Environmental Research and Public Health 2021, 18(3), 1089. [8.] Nicola M, et al. (2020). The socio-economic implications of the Coronavirus pandemic (COVID- IJISRT22JUL1407 772 www.ijisrt.com Volume 7, Issue 7, July – 2022 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 19): A review. InternationalJournal of Surgery. 2020;78: 185–193. 19): A review. InternationalJournal of Surgery. 2020;78: 185–193. [9.] Pakpour, A. H. and Griffiths, M. D. (2020). The fear of COVID-19 and its role in preventive behaviors. J. Concur. Disord, 2 (2020), pp. 58-63. Concur. Disord, 2 (2020), pp. 58-63. [10.] Suntronwong N, et al. (2020). Impact of COVID-19 public health interventions on influenza incidence in Thailand. Pathog. Glob. Health. 2020;00(00):1–3. [11.] Tiaprapong K, et al. (2021). Awareness of COVID-19 influences on the wellness of Thai health professional students: An ambulatory assessment during the early “new normal” informing Policy. PLoS ONE 16(6): e0252681. [12.] Triukose, S. et al. (2021). Effects of public health interventions on the epidemiological spread during the first wave of the COVID-19 outbreak in Thailand. PLoS One. 2021; 16(2): e0246274. [13.] Yorsaeng, R. et al. (2022). The impact of COVID-19 and control measures on public health in Thailand, 2020. PeerJ. 2022; 10: e12960. [14.] Vijaya, B. R. et al. (2021). Influences of covid 19 on the environment and its impact on community health in Thailand. International Journal of Pharmaceutical Research; 13(1):5382-5391, 2021. VIII. RECOMMENDATION [15.] Vicerra P. M. (2021). The Knowledge-Behavior gap on COVID-19 among older people in rural Thailand. Gerontological Geriatric Medicine, 2021;7:233372142199720. Medicine, IJISRT22JUL1407 IJISRT22JUL1407 773 www.ijisrt.com
https://openalex.org/W3123725905	https://europepmc.org/articles/pmc6308975?pdf=render	English	null	Electrochemical Instrumentation of an Embedded Potentiostat System (EPS) for a Programmable-System-On-a-Chip	null	2,018	cc-by	14,860	Received: 2 October 2018; Accepted: 20 November 2018; Published: 18 December 2018 Abstract: Under the main features required on portable devices in electrochemical instrumentation is to have a small size, low power consumption, economically affordable and precision in the measurements. This paper describes the development of a programmable Embedded Potentiostat System (EPS) capable of performing electrochemical sensing over system-on-a-chip platforms. Furthermore, the study explains a circuit design and develops some validation of the entire system. The hardware validation is performed by electrochemical experiments such as Double Step Chronoamperometry (DSC), Linear Sweep Voltammetry (LSV) and Cyclic Voltammetry (CV); moreover, a comparison of the experimental signals between a commercial potentiostat and the EPS was done by analysis of errors on the response signal. Results illustrate that the EPS is capable of handling currents in the range of absolute values of 86.44 to 3000 nA and having control voltages in the range of ±2 V. The device can support from 50 to 2000 samples per second. The EPS capabilities were compared with other compact potentiostats. The programmable EPS is an original approach which hugely reduces the hardware complexity and leads the way to create new applications for Point-of-Care or industrial developments with a reusable full electronics module. Keywords: chronoamperometry; potential sweep methods; reconﬁgurable embedded potentiostat; portable potentiostat; programmable-system-on-a-chip; wireless electronic Sensors 2018, 18, 4490; doi:10.3390/s18124490 sensors sensors Article Electrochemical Instrumentation of an Embedded Potentiostat System (EPS) for a Programmable-System-On-a-Chip Adrián Iván Muñoz-Martínez 1, Omar Israel González Peña 1,* , Jordi Colomer-Farrarons José Manuel Rodríguez-Delgado 1, Alfonso Ávila-Ortega 1 and Graciano Dieck-Assad 1 Adrián Iván Muñoz-Martínez 1, Omar Israel González Peña 1,* , Jordi Colomer-Farrarons 2, José Manuel Rodríguez-Delgado 1, Alfonso Ávila-Ortega 1 and Graciano Dieck-Assad 1 1 Tecnologico de Monterrey, School of Engineering and Sciences, Av. Eugenio Garza Sada Sur No. 2501, Col. Tecnologico, Monterrey 64849, Mexico; adrian.ivan.munoz@gmail.com (A.I.M.-M.); jmrd@itesm.mx (J.M.R.-D.); aavila@itesm.mx (A.Á.-O.); graciano.dieck.assad@itesm.mx (G.D.-A.) 2 Department of Electronics and Biomedical Engineering, Bioelectronics and Nanobioengineering Research Group (SIC-BIO), University of Barcelona, Martí i Franquès, 08028 Barcelona, Spain; jcolomerf@ub.edu * Correspondence: ogonzalez.pena@gmail.com; Tel.: +52-818-358-2000 1 Tecnologico de Monterrey, School of Engineering and Sciences, Av. Eugenio Garza Sada Sur No. 2501, Col. Tecnologico, Monterrey 64849, Mexico; adrian.ivan.munoz@gmail.com (A.I.M.-M.); jmrd@itesm.mx (J.M.R.-D.); aavila@itesm.mx (A.Á.-O.); graciano.dieck.assad@itesm.mx (G.D.-A.) 2 Department of Electronics and Biomedical Engineering, Bioelectronics and Nanobioengineering Research Group (SIC-BIO), University of Barcelona, Martí i Franquès, 08028 Barcelona, Spain; jcolomerf@ub.edu * Correspondence: ogonzalez.pena@gmail.com; Tel.: +52-818-358-2000 jmrd@itesm.mx (J.M.R.-D.); aavila@itesm.mx (A.Á.-O.); graciano.dieck.assad@itesm.mx (G.D.-A.) 2 Department of Electronics and Biomedical Engineering, Bioelectronics and Nanobioengineering Research Group (SIC-BIO), University of Barcelona, Martí i Franquès, 08028 Barcelona, Spain; jcolomerf@ub.edu * C d l @ il T l +52 818 358 2000 Received: 2 October 2018; Accepted: 20 November 2018; Published: 18 December 2018 1. Introduction A potentiostat is a device which can input predetermined voltage/current signals that generate outputs with an electron-related behavior needed to study redox reactions [1]. The potentiostat also relies on a feedback loop usually implemented with advanced electronic components to accurately control and condition electrical potential differences obtained from sensors to ensure reliable information at the output. Before the invention of computers to control voltage and/or current, it was challenging to obtain signal processing in electrochemical instrumentation. Indeed, modern electroanalytical chemistry started with the invention of polarography in the 1920s [2]; since then, the electrochemical instrumentation has been pushing forward according to progress achieved in electronics (Moore’s law) and information technology (Internet of Things). Incorporation of electrochemical sensors continues to gain a presence in research efforts to develop technology in different application ﬁelds such as Sensors 2018, 18, 4490; doi:10.3390/s18124490 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors 2 of 21 Sensors 2018, 18, 4490 Lab-on-a-chip devices or centrifugal microﬂuidic platforms [3]; indeed, these microﬂuidic platforms have proved to be very convenient for clinical diagnosis of glucose and cancer cell detection issues [4–6]. a-chip devices or centrifugal microfluidic platforms [3]; indeed, these microfluidic platforms have proved to be e y o e ie t fo li i al dia o i of lu o e a d a e ell dete tio i ue [4 6] Point of Care Technology (POCT) devices make possible to obtain sample measurements of patients by using wireless communication and under a large distance between patients and professionals of health by using the internet of things technology. This technology offers features such as shortening the sample analysis periods, reducing the size of the ﬁnal device reaching portability. Thus, it is possible to implant POCT devices in humans for continuous monitoring purposes [7,8]. to be very convenient for clinical diagnosis of glucose and cancer cell detection issues [4–6]. Point of Care Technology (POCT) devices make possible to obtain sample measurements of patients by using wireless communication and under a large distance between patients and professionals of health by using the internet of things technology. 1. Introduction This information related to electrochemical detection is necessary to manipulate certain variables like the voltage waveforms. Thus, experimentalists have to adapt the methodologies under development to the available potentiostats in the market; likewise, the lack of this information results in different kinds of problems to develop new measurement approaches for the need for highly customized and ﬂexible electrochemical instruments for hardware and software. determination, PH detection, drug-concentration quantification, among others. The lack of the information about the circuitry is one of the most important disadvantages present in commercially supplied potentiostats [8,10–13]. This information related to electrochemical detection is necessary to manipulate certain variables like the voltage waveforms. Thus, experimentalists have to adapt the methodologies under development to the available potentiostats in the market; likewise, the lack of this information results in different kinds of problems to develop new measurement approaches for the need for highly customized and flexible electrochemical g y This study had proposed a highly customizable and ﬂexible platform consisting of the electronic circuits and the software to drive redox reactions. In addition, it is presented in the study a well-description and characterization on the potentiostat system, which it is necessary for making possible the availability of technological devices [8,10–14]. Figure 1 shows a possible solution to have a highly customized potentiostat system for applications like Lab-on-a-System, wearable monitoring systems and POCT. The idea is to have an embedded system small enough to meet the application requirements. In this case, the Programmable System on a Chip (PSoC) and the Programmable Radio on a Chip (PRoC) have shown their worth [14–17]. The interface system uses LabVIEW in a computer to deal with the user with a versatile graphical environment [18–22]. The communications between computer and PSoC is wireless. The scalability of the system takes place using pattern designs at the software level. new measurement approaches for the need for highly customized and flexible electrochemical instruments for hardware and software. This study had proposed a highly customizable and flexible platform consisting of the electronic circuits and the software to drive redox reactions. In addition, it is presented in the study a well- description and characterization on the potentiostat system, which it is necessary for making possible the availability of technological devices [8,10–14]. Figure 1 shows a possible solution to have a highly customized potentiostat system for applications like Lab-on-a-System, wearable monitoring systems and POCT. 1. Introduction This technology offers features such as shortening the sample analysis periods, reducing the size of the final device reaching portability Thus it is possible to implant POCT devices in humans for continuous monitoring Trends in microelectrode fabrication, microﬂuidics and microelectronic systems have resulted in both challenges: in the design/development of potentiostats and signiﬁcant advances in the capabilities of the potentiostats to collect data at the transient that take place at different time constants associated to a different phenomenon; for instance, in the order of ~(10−15 to 10−11) s, it is possible to observe the ohmic drop of the system, or if a experimentalist will like to observe the time constant associated with molecular diffusion it is required to record the electrochemical signal of the experiment in the order to ~(10−3 to 50) s [9]. Consequently, in the middle on the mass transfer process and the ohmic drop of the system occurs the time constant associated with electrochemical reactions [10]. As a result, the potentiostats have become very sophisticated systems to make possible applications such as DNA identiﬁcation, protein classiﬁcation, neural recording, glucose-level determination, PH detection, drug-concentration quantiﬁcation, among others. portability. Thus, it is possible to implant POCT devices in humans for continuous monitoring purposes [7,8]. Trends in microelectrode fabrication, microfluidics and microelectronic systems have resulted in both challenges: in the design/development of potentiostats and significant advances in the capabilities of the potentiostats to collect data at the transient that take place at different time constants associated to a different phenomenon; for instance, in the order of ~(10−15 to 10−11) s, it is possible to observe the ohmic drop of the system, or if a experimentalist will like to observe the time constant associated with molecular diffusion it is required to record the electrochemical signal of the experiment in the order to ~(10−3 to 50) s [9]. Consequently, in the middle on the mass transfer process and the ohmic drop of the system occurs the time constant associated with electrochemical reactions [10]. As a result, the potentiostats have become very sophisticated systems to make possible applications such as DNA identification, protein classification, neural recording, glucose-level d t i ti PH d t ti d t ti tifi ti th The lack of the information about the circuitry is one of the most important disadvantages present in commercially supplied potentiostats [8,10–13]. 1. Introduction The idea is to have an embedded system small enough to meet the application requirements. In this case, the Programmable System on a Chip (PSoC) and the Programmable Radio on a Chip (PRoC) have shown their worth [14–17]. The interface system uses LabVIEW in a computer to deal with the user with a versatile graphical environment [18–22]. The communications between computer and PSoC is wireless. The scalability of the system takes place using pattern designs at the software level. Figure 1. Block Diagram of the Embedded Potentiostat System (EPS) with its User Interface. Figure 1. Block Diagram of the Embedded Potentiostat System (EPS) with its User Interface. Figure 1. Block Diagram of the Embedded Potentiostat System (EPS) with its User Interface. Figure 1. Block Diagram of the Embedded Potentiostat System (EPS) with its User Interface. Sensors 2018, 18, 4490 3 of 21 Furthermore, it is presented in this work a ﬂexible and integral methodology that includes the characterization and calibration of the potentiostat, thus the electronics of the device were tested by performing three electrochemical techniques and its analysis of errors. This methodology allows for the reconﬁguration of the device to execute different electrochemical techniques allowing a correct functioning of the equipment. • Actuation signals generation. • Actuation signals generation. The PSoC from Cypress Semiconductor is one of the icon devices in an embedded mixed-signal architecture [27]. The selection of this device relies on the incorporation of several features in a single chip. Hence, analog, digital and processing systems are inside of a PSoC with the capacity to address several applications. The main feature of the PSoC is its conﬁgurability. That allows to an experimentalist to have new solutions to the most challenging problems [15–18]. A Full potentiostat system, requires the management of analog and digital signals. A PSoC provides an architecture for the treatment of mixed-signals in one chip [17]. These features bring advantages as fast development times, space reduction and simpliﬁcation of the application. 1.1. Background A potentiostat by itself just controls the potential in an electrochemical cell and more electronic components are necessary to get more information about the electrochemical phenomenon [23,24]. The Digital to Analog Converter (DAC) provides the control signal for the potentiostat. The current measurement circuit reads the electrons ﬂow of the reactions. The Analog to Digital Converter (ADC) turns the analog current values in digital. Thus, the basic potentiostat system deﬁnes the performance of the entire instrument. The function generator can give the waveform values in an analog or digital way. However, a computer generates the digital signals most of the time. Also, the recorder system has to handle digital values because it is the easiest way to save data. The display system can be any device capable of showing information. Though, one of the fastest is a screen. Hence, all these components and the basic potentiostat system let us have a complete device to perform electrochemical experiments. 1.1.1. Embedded Potentiostat System (EPS) The EPS prototype includes some modiﬁcations to incorporate additional electrochemical techniques. The main aspects are the description of the embedded systems and design patterns for programming. An embedded mixed-signal architecture deals with applications where the acquisition, processing and manipulation of the variables are necessary [25]. In a potentiostat system, the electrochemical reaction current is the variable to sense, the voltage at the electrodes is the variable to control and the microcontroller algorithm generates the appropriate waveforms for an electrochemical trial. Thus, a potentiostat application matches with an architecture like this. The main functions to perform by this kind of embedded system are [26]: • Sensing the analog signals. • Transmission and reception of data inside and outside of the embedded system • Firmware execution. 1.1.2. Prototype Implementation and Architecture The potentiostat instrument prototype has two main systems: the embedded and the interface system as shown in Figure 2. The EPS is responsible for the manipulation of the electrochemical cell sending and receiving data wirelessly. The Potentiostat User Interface System (PUIS) deals with the user and controls the EPS behavior. Both systems constitute a Master/Slave design where the EPS is the slave and the PUIS is the master. 4 of 21 4 of 22 Sensors 2018, 18, 4490 S 2018 18 Figure 2. Slave-master scheme: Embedded and User Interface systems (EPS and PUIS) driving the electrochemical cell. Figure 2. Slave-master scheme: Embedded and User Interface systems (EPS and PUIS) driving the electrochemical cell. Figure 2. Slave-master scheme: Embedded and User Interface systems (EPS and PUIS) driving the electrochemical cell Figure 2. Slave-master scheme: Embedded and User Interface systems (EPS and PUIS) driving the electrochemical cell. The modular design allows the completion of the research objectives by using a small embedded system as a slave [27]. Also, the modular design is an excellent feature to customize the system. To implement the potentiostat instrument prototype, the master sends commands to the slave and the slave returns the task response. In this prototype, there is a command for each electrochemical method and the response is a lot of digital values from the trial. The PUIS focuses on managing the recording and display system, on generating the appropriate waveforms and on sending Redox current/voltage values (according to the test) to the PUIS. This multiprocessing capability allows achieving a full potentiostat system The modular design allows the completion of the research objectives by using a small embedded system as a slave [27]. Also, the modular design is an excellent feature to customize the system. To implement the potentiostat instrument prototype, the master sends commands to the slave and the slave returns the task response. In this prototype, there is a command for each electrochemical method and the response is a lot of digital values from the trial. The PUIS focuses on managing the recording and display system, on generating the appropriate waveforms and on sending Redox current/voltage values (according to the test) to the PUIS. This multiprocessing capability allows achieving a full potentiostat system. achieving a full potentiostat system. The EPS is a set of electronic components small enough to be embedded in a potentiostat system application. 1.1.2. Prototype Implementation and Architecture This system has three main aspects: Bluetooth, PSoC and the electrochemical cell as shown in Figure 3A. The PSoC has analog and digital modules to implement as a potentiostat, a microcontroller and it executes the firmware. The PSoC analog hardware allows to the designer the development of a basic potentiostat system with all the electronic components needed by the typical potentiostat system. Moreover, the microcontroller has the code for the generation of the waveforms according to the electrochemical technique selected and the parameters to stop a running experiment. The main algorithm of the PSoC has a State Machine design pattern programmed in C language. This design pattern is highly acceptable by programmers because its implementation is very flexible and easy to follow [28]. Also, the modularity of the pattern makes feasible the additions of states to implement more electrochemical methods in the same PSoC. Thus, the state machine is an excellent choice to have a friendly firmware because it is very explicit p y The EPS is a set of electronic components small enough to be embedded in a potentiostat system application. This system has three main aspects: Bluetooth, PSoC and the electrochemical cell as shown in Figure 3A. The PSoC has analog and digital modules to implement as a potentiostat, a microcontroller and it executes the ﬁrmware. The PSoC analog hardware allows to the designer the development of a basic potentiostat system with all the electronic components needed by the typical potentiostat system. Moreover, the microcontroller has the code for the generation of the waveforms according to the electrochemical technique selected and the parameters to stop a running experiment. The main algorithm of the PSoC has a State Machine design pattern programmed in C language. This design pattern is highly acceptable by programmers because its implementation is very ﬂexible and easy to follow [28]. Also, the modularity of the pattern makes feasible the additions of states to implement more electrochemical methods in the same PSoC. Thus, the state machine is an excellent choice to have a friendly ﬁrmware because it is very explicit. choice to have a friendly firmware because it is very explicit. Besides the PSoC, Figure 3A shows another chip named PRoC that focuses on Bluetooth communication instead of hardware modules. The addition of this device increases the prototype size. However, this element is extremely important for a successful EPS functionality. 1.1.2. Prototype Implementation and Architecture Also, the PRoC is one of the best options considering that it comes from the same manufacturer of the PSoC Besides the PSoC, Figure 3A shows another chip named PRoC that focuses on Bluetooth communication instead of hardware modules. The addition of this device increases the prototype size. However, this element is extremely important for a successful EPS functionality. Also, the PRoC is one of the best options considering that it comes from the same manufacturer of the PSoC. is one of the best options considering that it comes from the same manufacturer of the PSoC. The PUIS schematic of Figure 3B uses Bluetooth communication to send commands to the slave. The commands come from a computer with a program and the PUIS is always waiting for any events at the interface to start the electrochemical experiment. Also, it has a recording system to save the data to a computer. Figure 3B shows that the Bluetooth device is out of the computer and it controls all the communication of the master. Moreover, the Bluetooth version is the BLE 4.2 with a data rate up to 25 Mbps for this prototype [29]. The Bluetooth communication works basically as a bridge allowing the design of a simple communication protocol for sending commands. Thus, it is easy the manipulation of bytes to send tasks to the slave and receive the voltage and the current values The PUIS schematic of Figure 3B uses Bluetooth communication to send commands to the slave. The commands come from a computer with a program and the PUIS is always waiting for any events at the interface to start the electrochemical experiment. Also, it has a recording system to save the data to a computer. Figure 3B shows that the Bluetooth device is out of the computer and it controls all the communication of the master. Moreover, the Bluetooth version is the BLE 4.2 with a data rate up to 25 Mbps for this prototype [29]. The Bluetooth communication works basically as a bridge allowing the design of a simple communication protocol for sending commands. Thus, it is easy the manipulation of bytes to send tasks to the slave and receive the voltage and the current values. manipulation of bytes to send tasks to the slave and receive the voltage and the current values. Recording on a display system is a challenge for any designer. Figure 3. Modular Potentiostat: (A) Slave syst PUIS 1.1.3. Analog and Digital Circuits in the PSoC 1.1.3. Analog and Digital Circuits in the PSoC The Figure 4 shows the digital schematic circuit of the PSoC, which provides a firmware execution in real-time and the communication to the PRoC. The advanced potentiostat circuit with a Trans-Impedance Amplifier (TIA) from Figure 4 has features used to make the electrochemical prototype tests [1,10]. Moreover, the design of the analog circuitry is very important to provide a good performance in the prototype system. The analog hardware from Figure 4 relies on the advanced potentiostat circuit with the TIA and it has some extra features. The Operational Amplifiers marked as Opamp_0 and Opamp_2 control the potential at the WE through the RE, Opamp_1 supplies the energy for the waveform while the DAC throws the waveform values at the proper rate. The Universal Asynchronous Receiver-Transmitter (UART) module communicates with the Bluetooth module to send data and receive commands wirelessly. The Programable Gate Array (PGA) provides a reference voltage of 2.048 V because the RE can be just manipulated in a range of 0 to 4.08 V. Hence, this floating potential gives a chance to work with ±2 V approximately in the electrochemical cell. The TIA and the ADC transform the current into digital values. The DAC brings some restriction to the embedded application. The maximum quantization error is 0.5 mV because every step is of 1 mV. The minimum time for the DAC to change a value at its output is of 4 µs. Hence, the maximum scan rate for the prototype is 250 V/s in a range of 0 to 4.08 V. The DAC needs the The Figure 4 shows the digital schematic circuit of the PSoC, which provides a ﬁrmware execution in real-time and the communication to the PRoC. The advanced potentiostat circuit with a Trans-Impedance Ampliﬁer (TIA) from Figure 4 has features used to make the electrochemical prototype tests [1,10]. Moreover, the design of the analog circuitry is very important to provide a good performance in the prototype system. The analog hardware from Figure 4 relies on the advanced potentiostat circuit with the TIA and it has some extra features. The Operational Ampliﬁers marked as Opamp_0 and Opamp_2 control the potential at the WE through the RE, Opamp_1 supplies the energy for the waveform while the DAC throws the waveform values at the proper rate. 1.1.2. Prototype Implementation and Architecture However, the use of advanced design patterns is very helpful. Thus, LabVIEW allows the creation of user interface systems with advanced programming techniques. The algorithm performed by the computer uses a Producer/Consumer and a State Machine design pattern. A later subsection provides more information about these techniques Recording on a display system is a challenge for any designer. However, the use of advanced design patterns is very helpful. Thus, LabVIEW allows the creation of user interface systems with advanced programming techniques. The algorithm performed by the computer uses a Producer/Consumer and a State Machine design pattern. A later subsection provides more information about these techniques. 5 of 21 f 5 of 21 Sensors 2018, 18, 4490 Figure 3 Modular Potentiostat: (A) Slave system of the EPS (B) Master system with the structure Figure 3. Modular Potentiostat: (A) Slave system of the EPS. (B) Master system with the structure PUIS. Figure 3. Modular Potentiostat: (A) Slave system of the EPS. (B) Master system with the structure PUIS. Figure 3. Modular Potentiostat: (A) Slave syst PUIS 1.1.3. Analog and Digital Circuits in the PSoC The Universal Asynchronous Receiver-Transmitter (UART) module communicates with the Bluetooth module to send data and receive commands wirelessly. The Programable Gate Array (PGA) provides a reference voltage of 2.048 V because the RE can be just manipulated in a range of 0 to 4.08 V. Hence, this ﬂoating potential gives a chance to work with ±2 V approximately in the electrochemical cell. The TIA and the ADC transform the current into digital values. The DAC brings some restriction to the embedded application. The maximum quantization error is 0.5 mV because every step is of 1 mV. The minimum time for the DAC to change a value at its output is of 4 µs. Hence, the maximum scan rate for the prototype is 250 V/s in a range of 0 to 4.08 V. The DAC needs the digital waveform value in 12 bits to make the conversion. Also, the DAC requires a buffer at the output to keep the right potential and supply the energy to the potentiostat control signal. 6 of 21 6 of 22 Sensors 2018, 18, 4490 Sensors 2018, 18, x Figure 4. Digital peripherals and analog front end devices of the PSoC. Digital to Analog Converter (DAC), Analog to Digital converter (ADC), Input-Output Port (UART), Trans-Impedance Amplifier (TIA). Figure 4. Digital peripherals and analog front end devices of the PSoC. Digital to Analog Converter (DAC), Analog to Digital converter (ADC), Input-Output Port (UART), Trans-Impedance Ampliﬁer (TIA). tal peripherals and analog front end devices of the PSoC. Digital to Analog Converter (DAC), Analog to Digital converter (ADC), Input-Output Port al peripherals and analog front end devices of the PSoC. Digital to Analog Converter (DAC), Analog to Digital converter (ADC), Input-Output Port Impedance Ampliﬁer (TIA) vices of the PSoC. Digital to Analog Converter (DAC), Analog to Digital converter (ADC), Input-Output Po ices of the PSoC. Digital to Analog Converter (DAC), Analog to Digital converter (ADC), Input-Output Po Figure 4. Digital peripherals and analog front end devices of the PSoC. Digital to Analog Converter (DAC), Analog to Digital converter (ADC), Input-Output Port (UART), Trans-Impedance Amplifier (TIA). Figure 4. Digital peripherals and analog front end devices of the PSoC. Digital to Analog Converter (DAC), Analog to Digital converter (ADC), Input-Output Port (UART), Trans-Impedance Ampliﬁer (TIA). 2.1. Electrochemical Equipment 2. Materials and Methods The EPS uses two kits as it is shown on Figure 5A from Cypress Semiconductors: CY8CKIT-059 and CY8CKIT-042-BLE, the CY8CKIT-059 kit has the chip CY8C5888LTI-LP097; the CY8CKIT-042-BLE kit has four devices but the prototype just needs the PRoC and the USB dongle. The measurements of the EPS are compared to a commercial potentiostat system (CH Instruments, model 700E). The EPS was operated to recording 2000 data per second which is the maximum samples that the equipment can measure. The three cables on Figure 5A at the bottom right part of the protoboard were connected to the three electrodes of the electrochemical cell on Figure 5B. 2.1. Electrochemical Equipment The EPS uses two kits as it is shown on Figure 5A from Cypress Semiconductors: CY8CKIT-059 and CY8CKIT-042-BLE, the CY8CKIT-059 kit has the chip CY8C5888LTI-LP097; the CY8CKIT-042- BLE kit has four devices but the prototype just needs the PRoC and the USB dongle. The measurements of the EPS are compared to a commercial potentiostat system (CH Instruments, model 700E). The EPS was operated to recording 2000 data per second which is the maximum samples that the equipment can measure. The three cables on Figure 5A at the bottom right part of the protoboard were connected to the three electrodes of the electrochemical cell on Figure 5B. Figure 5. (A) Prototype of the EPS. (B) Electrochemical cell (height = 35 mm, diameter = 60 mm), solution volume of 50 mL of K₃[Fe(CN)₆] and KCl. Figure 5. (A) Prototype of the EPS. (B) Electrochemical cell (height = 35 mm, diameter = 60 mm), solution volume of 50 mL of K3[Fe(CN)6] and KCl. Figure 5. (A) Prototype of the EPS. (B) Electrochemical cell (height = 35 mm, diameter = 60 mm), solution volume of 50 mL of K₃[Fe(CN)₆] and KCl. Figure 5. (A) Prototype of the EPS. (B) Electrochemical cell (height = 35 mm, diameter = 60 mm), solution volume of 50 mL of K3[Fe(CN)6] and KCl. Figure 3. Modular Potentiostat: (A) Slave syst PUIS 1.1.3. Analog and Digital Circuits in the PSoC Sensors 2018, 18, 4490 7 of 21 The TIA module, which it is shown at the top of Figure 4 and the Delta-Sigma Analog to Digital Converter (∆∑ADC) deﬁne the sensitivity of the current measurement. The TIA has eight resistors to have eight different quantization levels. The maximum current value is obtained by using the values for the operation, thus it comes from the minimum resistor of the TIA module (20 kΩ) and the ADC voltage range at its input (±1.024 V) as the equation one describes. However, the missing data needs to be calculated through a characterization. Sensors 2018, 18, x 7 of 22 The TIA module, which it is shown at the top of Figure 4 and the Delta-Sigma Analog to Digital Converter (∆∑ ADC) define the sensitivity of the current measurement. The TIA has eight resistors to have eight different quantization levels. The maximum current value is obtained by using the l f h i h i f h i i i f h TIA d l (20 kΩ) d h I =Vref R = ±1.024 V 20000 Ω= ±51.2 µA (1) t comes from the minimum resistor of the TIA module (20 kΩ) and the t (±1.024 V) as the equation one describes. However, the missing data h a characterization. (1) The ∆∑ADC has several features that deﬁne the behavior of this module in the prototype. The conversion mode is a single sample. The ADC has 18 bits and it takes 414 µS approximately to perform one conversion. The clock frequency is around 3071 kHz but the output rate is slower because it uses oversampling to get a better signal quality. The input range is ±1.024 V and the ADC has a buffer to avoid any measurement error by impedance mismatching. IൌVref R ൌേ1.024 V 20000 Ω ൌേ51.2 μA (1) The ∆∑ ADC has several features that define the behavior of this module in the prototype. The conversion mode is a single sample. The ADC has 18 bits and it takes 414 µS approximately to perform one conversion. The clock frequency is around 3071 kHz but the output rate is slower because it uses oversampling to get a better signal quality. The input range is ±1.024 V and the ADC 2.3. Experimental Design The electrochemical techniques used in the EPS are LSV, CV and DSC. Before comparing the commercial potentiostat with the EPS, the WE were cleaned by immerse it in 0.1 M of HNO3 (Sigma Aldrich) for approximately 10 min, later the WE was rinsing with distilled water; after that, the WE received an electrochemical pretreatment to activate its surface by running a sequence of different scan rates of CV and by using 0.1 M of HCl (Sigma Aldrich); The CV sequences of the activation surface is shown on Table 1. Table 1. Sequences for the surface activation on the WE. Sequence 1 Sequence 2 Sequence 3 Sequence 4 Scan Rate 500 mV/s 250 mV/s 100 mV/s 50 mV/s Cycles 50 25 10 5 Method Cyclic Voltammetry Initial Voltage 0.25 V Maximum Voltage 0.65 V Minimum Voltage −0.15 V Initial Scan Direction anodic direction Table 1. Sequences for the surface activation on the WE. The process of surface activation is initiated with a high scan rate (500 mV/s) and through lower scan rates until a scan rate of (50 mV/s) is reached. In the process of surface activation, all CVs were done on the windows of scan potentials of (−0.15 to 0.65) V versus Ag/AgCl where the initial voltage was set at 0.25 V versus Ag/AgCl. Likewise, the number per cycles of each sequence of CV is higher (50 cycles) at the highest scan rate and it decreases at lower scan rates until reaching (5 cycles). All experiments were carried out at room temperature ~25 ◦C and the potential recorded was against the Ag/AgCl saturated. The Randles-Sevcik equation presented below relates to the scan rate, the molecular diffusion and the bulk concentration of the analyte with the current peak from a CV or LSV experiments [34,35]. Ip = 2.69 × 105 n3/2(Dv)1/2ACbulk (4) (4) Here, Ip is the maximum current (A), n is the number of electrons per mole oxidized or reduced, D is de diffusion coefﬁcient (cm2/s), v is the scan rate of the CV or LSV (V/s), A is the working electrode area (cm2) and Cbulk is the bulk concentration of the oxidized or reduced specie (mol cm−3). Table 2 shows the parameters of the Randles-Sevcik equation and the current peaks of the two concentrations tested in an ideal Nernstian reversible system and under the assumption of semi-inﬁnite linear diffusion. 2.2. Analyte, Electrolyte and Electrodes 2.2. Analyte, Electrolyte and Electrodes All experiments were performed on a volume of 50 mL on a electrochemical cell (height = 35 mm, diameter = 60 mm) of potassium ferricyanide K3[Fe(CN)6ሿ; this analyte is common to use to test potentiostats [12,30–32], since its kinetics is well known and it describes an electrochemical reversible behavior [23,33,34]. Ferricyanide can be reduced to ferrocyanide as Equation (2) shows; the backward direction of the reaction corresponds to the ferrocyanide oxidation to ferricyanide as Equation (3) describes. F ሺCNሻ3- F ሺCNሻ4- (2) All experiments were performed on a volume of 50 mL on a electrochemical cell (height = 35 mm, diameter = 60 mm) of potassium ferricyanide K3[Fe(CN) 6]; this analyte is common to use to test potentiostats [12,30–32], since its kinetics is well known and it describes an electrochemical reversible behavior [23,33,34]. Ferricyanide can be reduced to ferrocyanide as Equation (2) shows; the backward direction of the reaction corresponds to the ferrocyanide oxidation to ferricyanide as Equation (3) describes. FeሺCNሻ6 3 +e-→FeሺCNሻ6 4 Fe(CN)3− 6 +e−→Fe(CN)4− 6 FeሺCNሻ6 3 +e →FeሺCNሻ6 4 Fe(CN)3− 6 +e−→Fe(CN)4− 6 (2) Sensors 2018, 18, 4490 8 of 21 Fe(CN)4− 6 →Fe(CN)3− 6 +e− (3) (3) Fe(CN)4− 6 →Fe(CN)3− 6 +e− In the experiments two analyte concentrations of 1 mM and 10 mM of K3[Fe(CN)6] (Sigma-Aldrich, Saint Louis, MO, USA, CAS: 13746-66-2) were used to evaluate the EPS. The electrolyte support used was 0.5 M KCl (Fermont, presentation no. 24842). The reference electrode (RE) used is Ag/AgCl (BASi model MF-2052). A platinum wire (BASi model MW-4130) and a disk glassy carbon electrode (BASi model MF-2012, diameter φ = 3 mm) was used as the Counter Electrode (CE) and the Working Electrode (WE), respectively, as it is shown in Figure 5B. 2.3. Experimental Design The setup condition for each experiment was related to the number of experimental conditions. The conditions rely on the previous investigation where similar values were used [10,14]. The only changes between conditions were the scan rate value. Hence, conditions allow us to evaluate the EPS at different currents magnitudes and scan rates. In addition, a comparison was done of the EPS signal with a commercial potentiostat. Table 3 describes the setup parameters for the conditions tested on CVs. 9 of 21 Sensors 2018, 18, 4490 Table 2. Randles-Sevcik parameters and expected maximum current from CV and LSV experiments. Table 2. Randles-Sevcik parameters and expected maximum current from CV and LSV experiments. Solution 1 Solution 2 K3[Fe(CN)6], analyte concentration 1 mM 10 mM KCl, Electrolyte support concentration 0.5 M # of e−per mole oxidized or reduced 1 Analyte Diffusion Coefﬁcient 7.23 µcm2/s [36] Scan Rate 10 mV/s Room Temperature 25 ◦C Surface Area of WE 0.071 cm2 Randles-Sevcik Current Peak 5.11 µA 51.1 µA Table 3. CV conditions for each experiment. Table 3. CV conditions for each experiment. Table 3. CV conditions for each experiment. Table 3. CV conditions for each experiment. Condition 1 Condition 2 Condition 3 Condition 4 Scan Rate 10 mV/s 100 mV/s 250 mV/s 500 mV/s Initial Voltage 0.25 V Minimum Voltage −0.15 V Maximum Voltage 0.65 V Recorded cycle for comparison ﬁfth Initial Scan Direction anodic direction Analyte concentration 1 mM K3[Fe(CN)6] Electrolyte support concentration 0.5 M KCl Scan Rate Initial Voltage Minimum Voltage Maximum Voltage Recorded cycle for comparison Initial Scan Direction Analyte concentration Electrolyte support concentration Table 4 describes the conditions for the DSC experiments. The small changes between the ﬁrst and the last step allow us to explore the changes in the current measurements on the prototype and it can be related to the lowest limit of detection on the device. The pulse width was set to 62 s since at that time the current measurement reaches the steady-state response. The last step is practically the open circuit potential of 0.308 V for 1 mM K3[Fe(CN)6] in Table 4. Table 4. DSC conditions for each experiment. Cond. 5 Cond. 6 Cond. 7 Cond. 8 Cond. 2.3. Experimental Design 9 First Step 0.325 V 0.315 V 0.305 V 0.295 V 0.195 V Last Step 0.310 V Pulse Width 62 s Quite time 62 s Analyte 1 mM K3[Fe(CN)6] Electrolyte 0.5 M KCl T bl 5 d b h d f h LSV Th d l h Table 4. DSC conditions for each experiment. Table 4. DSC conditions for each experiment. Table 5 describes the conditions for the LSV experiments. The conditions rely on the previous investigation where similar values were used [10,14], where the only changes were the scan rates. Table 5 describes the conditions for the LSV experiments. The conditions rely on the previous investigation where similar values were used [10,14], where the only changes were the scan rates. Table 5. LSV conditions for the experiments. Cond. 10 Cond. 11 Cond. 12 Cond. 13 Cond. 14 Cond. 15 Initial Voltage 0.65 V −0.15 V 0.65 V −0.15 V 0.65 V −0.15 V Final Voltage −0.15 V 0.65 V −0.15 V 0.65 V −0.15 V 0.65 V Scan Rate 10 mV/s 10 mV/s 100 mV/s 100 mV/s 500 mV/s 500 mV/s Analyte 1 mM K3[Fe(CN)6] Electrolyte 0.5 M KCl Table 5. LSV conditions for the experiments. Table 5. LSV conditions for the experiments. 3. Results and Discussion Results of the experimental conditions in Tables 3–5 are shown in the graphs from Figures 6–8. In the LSV and CV, the voltage values are versus the RE and it is indicated as EREF on the abscissa 10 of 21 gures 6–8 e absciss Sensors 2018, 18, 4490 Results of t I h LSV d C axis. All electrochemical experiments follow the sign convention used on the commercial potentiostat (chemistry convention); therefore, the peak currents observed on CVs in Figure 5 with negative magnitude, correspond to the oxidation in the Equation (3); contrary, the positive magnitude of the current corresponds to a reduction in the Equation (2). A minor discrepancy on the signal of the prototype occurred at high currents; however, the results from the prototype are close to the commercial potentiostat in most of the graphs when it is considered a proper range to work for the prototype. A minor drawback of the potentiostat prototype is the ﬁlter; this capacitor introduces a shift phase and it is possible to been observed when the scan rate is fast as Figure 6D shows. This ﬁlter is necessary because noise appears in the measurements specially when very low currents are monitored. Thus, this component allows us to reduce the detection limits sacriﬁcing a little of the potentiostat prototype bandwidth. axis. All electrochemical experiments follow the sign convention used on the commercial potentiosta (chemistry convention); therefore, the peak currents observed on CVs in Figure 5 with negativ magnitude, correspond to the oxidation in the Equation (3); contrary, the positive magnitude of th current corresponds to a reduction in the Equation (2). A minor discrepancy on the signal of th prototype occurred at high currents; however, the results from the prototype are close to th commercial potentiostat in most of the graphs when it is considered a proper range to work for th prototype. A minor drawback of the potentiostat prototype is the filter; this capacitor introduces shift phase and it is possible to been observed when the scan rate is fast as Figure 6D shows. Thi filter is necessary because noise appears in the measurements specially when very low currents ar monitored. Thus, this component allows us to reduce the detection limits sacrificing a little of th potentiostat prototype bandwidth. Figure 6. 3. Results and Discussion Cyclic voltammetry at different scan rates of 1 mM K₃[Fe(CN)₆] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter ϕ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively: (A) 10 mV/s (experiment under the conditions 1). (B) 100 mV/s (experiment under the conditions 2). (C) 250 mV/s (experiment under the conditions 3). (D) 500 mV/s (experiment under the conditions 4). -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 -3.0x10-6 -2.0x10-6 -1.0x10-6 0.0 1.0x10-6 2.0x10-6 3.0x10-6 4.0x10-6 A Current (A) Voltage vs ERef (V) PSoC Current CHI Current 10 mV/s -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 -1.2x10-5 -8.0x10-6 -4.0x10-6 0.0 4.0x10-6 8.0x10-6 1.2x10-5 1.6x10-5 C Current (A) Voltage vs ERef (V) PSoC Current CHI Current 250 mV/s -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 -1.5x10-5 -1.0x10-5 -5.0x10-6 0.0 5.0x10-6 1.0x10-5 1.5x10-5 2.0x10-5 D Current (A) Voltage vs ERef (V) PSoC Current CHI Current 500mV/s -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 -8.0x10-6 -6.0x10-6 -4.0x10-6 -2.0x10-6 0.0 2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6 1.0x10-5 B Current (A) Voltage vs ERef (V) PSoC Current CHI Current 100mV/s Figure 6. Cyclic voltammetry at different scan rates of 1 mM K3[Fe(CN)6] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter φ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively: (A) 10 mV/s (experiment under the conditions 1). (B) 100 mV/s (experiment under the conditions 2). (C) 250 mV/s (experiment under the conditions 3). (D) 500 mV/s (experiment under the conditions 4). 3. Results and Discussion -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 -3.0x10-6 -2.0x10-6 -1.0x10-6 0.0 1.0x10-6 2.0x10-6 3.0x10-6 4.0x10-6 A Current (A) PSoC Current CHI Current 10 mV/s -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 -8.0x10-6 -6.0x10-6 -4.0x10-6 -2.0x10-6 0.0 2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6 1.0x10-5 B Current (A) PSoC Current CHI Current 100mV/s B Current (A) -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 -6 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0-6 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Voltage vs ERef (V) -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 -1.2x10-5 -8.0x10-6 -4.0x10-6 0.0 4.0x10-6 8.0x10-6 1.2x10-5 1.6x10-5 C Current (A) Voltage vs ERef (V) PSoC Current CHI Current 250 mV/s C t (A) -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 -1.5x10-5 -1.0x10-5 -5.0x10-6 0.0 5.0x10-6 1.0x10-5 1.5x10-5 2.0x10-5 D Current (A) Voltage vs ERef (V) PSoC Current CHI Current 500mV/s 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Voltage vs ERef (V) Voltage vs ERef (V) Voltage vs ERef (V) Voltage vs ERef (V) Voltage vs ERef (V) Current (A) C Current (A) -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 x10 5 Voltage vs ERef (V) -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Voltage vs ERef (V) Voltage vs ERef (V) Figure 6. Cyclic voltammetry at different scan rates of 1 mM K₃[Fe(CN)₆] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter ϕ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively: (A) 10 mV/s (experiment under the conditions 1). (B) 100 mV/s (experiment under the conditions 2). (C) 250 mV/s (experiment under the conditions 3). (D) 500 mV/s (experiment under the diti 4) Figure 6. Cyclic voltammetry at different scan rates of 1 mM K3[Fe(CN)6] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter φ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively: (A) 10 mV/s (experiment under the conditions 1). 3. Results and Discussion (B) 100 mV/s (experiment under the conditions 2). (C) 250 mV/s (experiment under the conditions 3). (D) 500 mV/s (experiment under the conditions 4). Figure 6. Cyclic voltammetry at different scan rates of 1 mM K₃[Fe(CN)₆] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter ϕ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively: (A) 10 mV/s (experiment under the conditions 1). (B) 100 mV/s (experiment under the conditions 2). (C) 250 mV/s (experiment under the conditions 3). (D) 500 mV/s (experiment under the diti 4) Figure 6. Cyclic voltammetry at different scan rates of 1 mM K3[Fe(CN)6] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter φ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively: (A) 10 mV/s (experiment under the conditions 1). (B) 100 mV/s (experiment under the conditions 2). (C) 250 mV/s (experiment under the conditions 3). (D) 500 mV/s (experiment under the conditions 4). conditions 4). In Figure 7, it is shown different DSCs at different first step potentials described in Table 4. I Figure 7A,B, the initial step corresponds to the oxidation and on the second step it is shown In Figure 7, it is shown different DSCs at different ﬁrst step potentials described in Table 4. In Figure 7A,B, the initial step corresponds to the oxidation and on the second step it is shown a reduction; contrary, in Figure 7C–E) the process has been inverted. reduction; contrary, in Figure 7C–E) the process has been inverted. In Figure 7B,C, the current recorded at longer times describes more evidently an oscillation when the system is close to reach a relaxed response; This oscillation can be related to the perturbation step signal, which was very close to the open circuit potential; as a result, in Figure 7B,C, the ratio 11 of 21 Sensors 2018, 18, 4490 In Figu of the peak currents divided by the current measured at the steady-state provides a less abrupt ratio compared to when the system is under a large perturbation signal of a given step of potential. 3. Results and Discussion sig a , ic as e y c ose o e ope ci cui po e ia ; as a esu , i igu e ,C, e a io o e peak currents divided by the current measured at the steady-state provides a less abrupt ratio compared to when the system is under a large perturbation signal of a given step of potential. 0 20 40 60 80 100 120 140 -4.0x10-6 -3.0x10-6 -2.0x10-6 -1.0x10-6 0.0 1.0x10-6 2.0x10-6 3.0x10-6 4.0x10-6 5.0x10-6 E 1st Step Potential = 0.195 V Current (A) time (s) PSoC Current CHI Current 0 20 40 60 80 100 120 140 -6.0x10-8 -4.0x10-8 -2.0x10-8 0.0 2.0x10-8 4.0x10-8 6.0x10-8 8.0x10-8 B Current (A) time (s) PSoC Current CHI Current 1st Step Potential = 0.315 V 0 20 40 60 80 100 120 140 -2.0x10-7 -1.5x10-7 -1.0x10-7 -5.0x10-8 0.0 5.0x10-8 1.0x10-7 1.5x10-7 2.0x10-7 A Current (A) time (s) PSoC Current CHI Current 1st step potential = 0.325 V 0 20 40 60 80 100 120 140 -1.0x10-6 -5.0x10-7 0.0 5.0x10-7 1.0x10-6 D Current (A) time (s) PSoC Current CHI Current 1st Step Potential = 0.295 V 0 20 40 60 80 100 120 140 -4.0x10-8 -2.0x10-8 0.0 2.0x10-8 4.0x10-8 6.0x10-8 8.0x10-8 1.0x10-7 C Current (A) time (s) PSoC Current CHI Current 1st Step Potential = 0.305 V Figure 7. Double Step Chronoamperometry at different ﬁrst step potentials of 1 mM K3[Fe(CN)6] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter φ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively. All DSC were done with a pulse width of 62 s and the last step was setup at 0.310 V vs. Ag/AgCl: (A) ﬁrst step potential 0.325 V vs. Ag/AgCl (experiment under the conditions 5). (B) ﬁrst step potential 0.315 V vs. Ag/AgCl (experiment under the conditions 6). (C) ﬁrst step potential 0.305 V vs. Ag/AgCl (experiment under the conditions 7). (D) ﬁrst step potential 0.295 V vs. Ag/AgCl (experiment under the conditions 8). (E) ﬁrst step potential 0.195 V vs. Ag/AgCl (experiment under the conditions 9). Figure 8 shows the LSV experiments described in Table 5. 3. Results and Discussion In Figure 8A,C,E, it is shown that L 0 20 40 60 80 100 120 140 -6.0x10-8 -4.0x10-8 -2.0x10-8 0.0 2.0x10-8 4.0x10-8 6.0x10-8 8.0x10-8 B Current (A) time (s) PSoC Current CHI Current 1st Step Potential = 0.315 V 0 20 40 60 80 100 120 140 -2.0x10-7 -1.5x10-7 -1.0x10-7 -5.0x10-8 0.0 5.0x10-8 1.0x10-7 1.5x10-7 2.0x10-7 A Current (A) time (s) PSoC Current CHI Current 1st step potential = 0.325 V 0 20 40 60 80 100 120 140 -1.0x10-6 -5.0x10-7 0.0 5.0x10-7 1.0x10-6 D Current (A) time (s) PSoC Current CHI Current 1st Step Potential = 0.295 V 0 20 40 60 80 100 120 140 -4.0x10-8 -2.0x10-8 0.0 2.0x10-8 4.0x10-8 6.0x10-8 8.0x10-8 1.0x10-7 C Current (A) time (s) PSoC Current CHI Current 1st Step Potential = 0.305 V 0 20 40 60 80 100 120 140 -6.0x10-8 -4.0x10-8 -2.0x10-8 0.0 2.0x10-8 4.0x10-8 6.0x10-8 8.0x10-8 B Current (A) time (s) PSoC Current CHI Current 1st Step Potential = 0.315 V 0 20 40 60 80 100 120 140 -2.0x10-7 -1.5x10-7 -1.0x10-7 -5.0x10-8 0.0 5.0x10-8 1.0x10-7 1.5x10-7 2.0x10-7 A Current (A) time (s) PSoC Current CHI Current 1st step potential = 0.325 V Current (A) B 0 20 40 60 80 100 120 140 -1.0x10-6 -5.0x10-7 0.0 5.0x10-7 1.0x10-6 D time (s) PSoC Current CHI Current 1st Step Potential = 0.295 V 0 20 40 60 80 100 120 140 -1.0x10-6 -5.0x10-7 0.0 5.0x10-7 1.0x10-6 D Current (A) time (s) PSoC Current CHI Current 1st Step Potential = 0.295 V 0 20 40 60 80 100 120 140 -4.0x10-8 -2.0x10-8 0.0 2.0x10-8 4.0x10-8 6.0x10-8 8.0x10-8 1.0x10-7 C Current (A) time (s) PSoC Current CHI Current 1st Step Potential = 0.305 V Current (A) 0 20 40 60 80 100 120 140 -4.0x10-6 -3.0x10-6 -2.0x10-6 -1.0x10-6 0.0 1.0x10-6 2.0x10-6 3.0x10-6 4.0x10-6 5.0x10-6 E 1st Step Potential = 0.195 V Current (A) time (s) PSoC Current CHI Current Figure 7. Double Step Chronoamperometry at different ﬁrst step potentials of 1 mM K3[Fe(CN)6] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter φ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively. All DSC were done with a pulse width of 62 s and the last step was setup at 0.310 V vs. Ag/AgCl: (A) ﬁrst step potential 0.325 V vs. 3. Results and Discussion Ag/AgCl (experiment under the conditions 5). (B) ﬁrst step potential 0.315 V vs. Ag/AgCl (experiment under the conditions 6). (C) ﬁrst step potential 0.305 V vs. Ag/AgCl (experiment under the conditions 7). (D) ﬁrst step potential 0.295 V vs. Ag/AgCl (experiment under the conditions 8). (E) ﬁrst step potential 0.195 V vs. Ag/AgCl (experiment under the conditions 9). Figure 7. Double Step Chronoamperometry at different ﬁrst step potentials of 1 mM K3[Fe(CN)6] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter φ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively. All DSC were done with a pulse width of 62 s and the last step was setup at 0.310 V vs. Ag/AgCl: (A) ﬁrst step potential 0.325 V vs. Ag/AgCl (experiment under the conditions 5). (B) ﬁrst step potential 0.315 V vs. Ag/AgCl (experiment under the conditions 6). (C) ﬁrst step potential 0.305 V vs. Ag/AgCl (experiment under the conditions 7). (D) ﬁrst step potential 0.295 V vs. Ag/AgCl (experiment under the conditions 8). (E) ﬁrst step potential 0.195 V vs. Ag/AgCl (experiment under the conditions 9). Figure 8 shows the LSV experiments described in Table 5. In Figure 8A,C,E, it is shown that LSV under a cathodic scan corresponds to a reduction; on the other hand, Figure 8B,D,F shows the anodic direction on the LSV associated with an oxidation. A little discrepancy of the phase response was observed at the maximum scan rate of 500 mV/s for the cathodic and anodic directions with respect 12 of 21 Sensors 2018, 18, 4490 to the commercial potentiostat response; the small difference can be associated with the same signal observed at the highest scan rate of the CV experiment. Sensors 2018, 18, x 13 of 22 g p Figure 8. Linear sweep voltammetry at different scan rates at different first step potentials of 1 mM K₃[Fe(CN)₆] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter ϕ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively. All voltages are reported vs. Ag/AgCl. (A) Scan rate 10 mV/s, initial and final voltage are 0.65 V and −0.15 V, respectively (experiment under the conditions 10). 3. Results and Discussion (B) Scan rate 10 mV/s, initial and final voltage are −0.15 V and 0.65 V, respectively (experiment under the conditions 11). (C) Scan rate 100 mV/s, initial and final voltage are 0.65 V and −0.15 V, respectively -0.2 0.0 0.2 0.4 0.6 -2.0x10-5 -1.5x10-5 -1.0x10-5 -5.0x10-6 0.0 5.0x10-6 1.0x10-5 F Current (A) Voltage vs ERef (V) PSoC Current CHI Current 500 mV/s Positive Direction -0.2 0.0 0.2 0.4 0.6 -8.0x10-6 -6.0x10-6 -4.0x10-6 -2.0x10-6 0.0 2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6 D Current (A) Voltage vs ERef (V) PSoC Current CHI Current 100 mV/s Positive Direction -0.2 0.0 0.2 0.4 0.6 -5.0x10-6 0.0 5.0x10-6 1.0x10-5 1.5x10-5 2.0x10-5 2.5x10-5 E Current (A) Voltage vs ERef (V) PSoC Current CHI Current 500 mV/s Negative Direction -0.2 0.0 0.2 0.4 0.6 -2.0x10-6 0.0 2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6 B Current (A) Voltage vs ERef (V) PSoC Current CHI Current 10 mV/s Positive Direction -0.2 0.0 0.2 0.4 0.6 -2.0x10-6 -1.0x10-6 0.0 1.0x10-6 2.0x10-6 3.0x10-6 4.0x10-6 A Current (A) Voltage vs ERef (V) PSoC Current CHI Current 10 mV/s Negative Direction -0.2 0.0 0.2 0.4 0.6 -2.0x10-6 0.0 2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6 1.0x10-5 C Current (A) Voltage vs ERef (V) PSoC Current CHI Current 100 mV/s Negative Direction Figure 8. Linear sweep voltammetry at different scan rates at different ﬁrst step potentials of 1 mM K3[Fe(CN)6] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter φ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively. All voltages are reported vs. Ag/AgCl. (A) Scan rate 10 mV/s, initial and ﬁnal voltage are 0.65 V and −0.15 V, respectively (experiment under the conditions 10). (B) Scan rate 10 mV/s, initial and ﬁnal voltage are −0.15 V and 0.65 V, respectively (experiment under the conditions 11). (C) Scan rate 100 mV/s, initial and ﬁnal voltage are 0.65 V and −0.15 V, respectively (experiment under the condition 12). (D) Scan rate 100 mV/s, initial and ﬁnal voltage are −0.15 V and 0.65 V, respectively (experiment under the conditions 13). (E) Scan rate 500 mV/s, initial and ﬁnal voltage are 0.65 V and −0.15 V, respectively (experiment under the conditions 14). (F) Scan rate 500 mV/s, initial and ﬁnal voltage are −0.15 V and 0.65 V, respectively (experiment under the conditions 15). 3. Results and Discussion -0.2 0.0 0.2 0.4 0.6 -2.0x10-6 -1.0x10-6 0.0 1.0x10-6 2.0x10-6 3.0x10-6 4.0x10-6 A Current (A) Voltage vs ERef (V) PSoC Current CHI Current 10 mV/s Negative Direction -0.2 0.0 0.2 0.4 0.6 -2.0x10-6 0.0 2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6 B Current (A) PSoC Current CHI Current 10 mV/s Positive Direction Current (A) Voltage vs ERef (V) -0.2 0.0 0.2 0.4 0.6 -8.0x10-6 -6.0x10-6 -4.0x10-6 -2.0x10-6 0.0 2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6 D Current (A) Voltage vs E (V) PSoC Current CHI Current 100 mV/s Positive Direction Voltage vs ERef (V) Ref -0.2 0.0 0.2 0.4 0.6 -2.0x10-6 0.0 2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6 1.0x10-5 C Current (A) Voltage vs ER f (V) PSoC Current CHI Current 100 mV/s Negative Direction Current (A) D -0.2 0.0 0.2 0.4 0.6 -2.0x10-5 -1.5x10-5 -1.0x10-5 -5.0x10-6 0.0 5.0x10-6 1.0x10-5 F Voltage vs ERef (V) PSoC Current CHI Current 500 mV/s Positive Direction Voltage vs ERef (V) Current (A) -0.2 0.0 0.2 0.4 0.6 -5.0x10-6 0.0 5.0x10-6 1.0x10-5 1.5x10-5 2.0x10-5 2.5x10-5 E Current (A) Voltage vs ERef (V) PSoC Current CHI Current 500 mV/s Negative Direction Voltage vs ERef (V) E Current (A) Voltage vs ERef (V) Figure 8. Linear sweep voltammetry at different scan rates at different first step potentials of 1 mM K₃[Fe(CN)₆] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter ϕ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively. All voltages are reported vs. Ag/AgCl. (A) Scan rate 10 mV/s, initial and final voltage are 0.65 V and −0.15 V, respectively (experiment under the conditions 10). (B) Scan rate 10 mV/s, initial and final voltage are −0.15 V and 0.65 V, respectively (experiment under the conditions 11). (C) Scan rate 100 mV/s, initial and final voltage are 0.65 V and −0.15 V, respectively Figure 8. Linear sweep voltammetry at different scan rates at different ﬁrst step potentials of 1 mM K3[Fe(CN)6] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter φ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively. All voltages are reported vs. Ag/AgCl. (A) Scan rate 10 mV/s, initial and ﬁnal voltage are 0.65 V and −0.15 V, respectively (experiment under the conditions 10). Analysis of Results An error analysis will show the differences between both devices for each experiment quantitatively. The absolute error express how far is the measured value of the real as the Equation (5) describes. In these experiments, the real values are from the commercial potentiostat while the measured values are from the prototype. The mean error refers to the average of the absolute errors in an experiment as Equation (6) agrees. The highest error is a value very close to the maximum error because it comes from the standard deviation (σ) of the absolute errors as Equation (7) shows. Absolute Error = \|\|ValueMeasured\| −\|ValueReal\|\| (5) Mean Error = ∑ Sample Number i=1 (Absolute Error)i Sample Number (6) Highest Error = 3σ + Mean Error (7) Absolute Error = \|\|ValueMeasured\| −\|ValueReal\|\| (5) Mean Error = ∑ Sample Number i=1 (Absolute Error)i Sample Number (6) Highest Error = 3σ + Mean Error (7) (5) (6) (7) The previous equations do not have any reference to describe the error and with this peculiarity, it cannot be clear how bad is that error. Thus, the full scale will be the reference with a value according to the peak to peak amplitude of the Redox current signal from the commercial potentiostat. The Mean Error Percent (MEP) describes how big the mean error is against the peak to peak amplitude as Equation (8) illustrates. The Highest Error Percent (HEP) describes how big this highest error is against the peak to peak amplitude as Equation (8) shows. Hence, these indicators will describe the error of the prototype measurements with a solid reference. Mean Error Percent = Mean Error Peak to Peak Amplitude ×100 (8) Highest Error Percent = Highest Error Peak to Peak Amplitude×100 (9) (8) (9) It is shown in Table 6 that the most relevant indicators for the error analysis. The MEP describes the error percent to expect in given measure. The HEP describes the maximum error percent to expect in an electrochemical trial. The CV errors are higher than those from the DSC and that can come from two factors: the full scale and the scan rate. However, it is difﬁcult to know which of each has more weight because they are related. Table 6. Error analysis summary of the CV, DSC and LSV experiments in Figures 6–8 and by using Equations (5–9). Experimental conditions are reported in Tables 3–5. 3. Results and Discussion (B) Scan rate 10 mV/s, initial and ﬁnal voltage are −0.15 V and 0.65 V, respectively (experiment under the conditions 11). (C) Scan rate 100 mV/s, initial and ﬁnal voltage are 0.65 V and −0.15 V, respectively (experiment under the condition 12). (D) Scan rate 100 mV/s, initial and ﬁnal voltage are −0.15 V and 0.65 V, respectively (experiment under the conditions 13). (E) Scan rate 500 mV/s, initial and ﬁnal voltage are 0.65 V and −0.15 V, respectively (experiment under the conditions 14). (F) Scan rate 500 mV/s, initial and ﬁnal voltage are −0.15 V and 0.65 V, respectively (experiment under the conditions 15). Sensors 2018, 18, 4490 13 of 21 13 of 21 Analysis of Results Experiment under the: MEP, % HEP, % Mean Error, A Highest Error, A Full Scale, A Method Conditions 1 2.887 5.226 1.98 × 10 −7 3.58 × 10 −7 6.852 × 10 −6 CV Conditions 2 1.691 5.721 2.88 × 10 −7 9.75 × 10 −7 1.704 × 10 −5 CV Conditions 3 2.226 7.653 5.62 × 10 −7 1.93 × 10 −6 2.527 × 10 −5 CV Conditions 4 3.397 11.178 1.18 × 10 −6 3.89 × 10 −6 3.481 × 10 −5 CV Conditions 5 0.319 5.185 5.35 × 10 −9 8.69 × 10 −8 1.676 × 10 −6 DSC Conditions 6 0.587 5.386 3.6 × 10 −9 3.30 × 10 −8 6.134 × 10 −7 DSC Conditions 7 0.780 5.480 5.04 × 10 −9 3.54 × 10 −8 6.461 × 10 −7 DSC Conditions 8 0.562 6.169 1.03 × 10 −8 1.13 × 10 −7 1.827 × 10 −6 DSC Conditions 9 0.414 4.758 7.73 × 10 −8 8.87 × 10 −7 1.865 × 10 −5 DSC Conditions 10 2.019 5.994 9.55 × 10 −8 2.83 × 10 −7 4.728 × 10 −6 LSV Conditions 11 0.585 5.464 4.88 × 10 −8 4.56 × 10 −7 8.341 × 10 −6 LSV Table 6. Error analysis summary of the CV, DSC and LSV experiments in Figures 6–8 and by using Equations (5–9). Experimental conditions are reported in Tables 3–5. 14 of 21 Sensors 2018, 18, 4490 Table 6. Cont. Experiment under the: MEP, % HEP, % Mean Error, A Highest Error, A Full Scale, A Method Conditions 12 2.837 8.429 3.12 × 10 −7 9.26 × 10 −7 1.098 × 10 −5 LSV Conditions 13 5.081 17.825 6.2 × 10 −7 2.18 × 10 −6 1.220 × 10 −5 LSV Conditions 14 4.243 14.483 9.94 × 10 −7 3.39 × 10 −6 2.342 × 10 −5 LSV Conditions 15 7.655 19.173 1.72 × 10 −6 4.32 × 10 −6 2.252 × 10 −5 LSV The experimental conditions 6 and 7 allow knowing the resolution of the equipment that can be related to the Lower Limit of Detection (LLD), thus in these trials the prototype measured the smallest signal value. From the conditions 6 and 7, it is possible to calculate the 5% of the mean error. Therefore, its values reﬂex the LLD to have an expected accuracy of 95% in the measurements compared with the commercial potentiostat. Analysis of Results Cyclic voltammetry at 10 mV/s, 10 mM K₃[Fe(CN)₆] and 0.5 M KCl as the electrolyte suppo the working electrode, the counter electrode and the reference electrode were a disk glassy carb electrode (diameter ϕ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively; experim under the conditions 16: (Blue line) CV response by the EPS. (Orange line) CV response by i l t ti t t Figure 9. Cyclic voltammetry at 10 mV/s, 10 mM K3[Fe(CN)6] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter φ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively; experiment under the conditions 16: (Blue line) CV response by the EPS. (Orange line) CV response by the commercial potentiostat. The HLD comes from the current values of the CV; specifically, condition 16 shows how prototype cannot handle much current. The main difference between experimental conditions 1 16 is the concentration difference in the analyte (10 times in the order of magnitude). The Figu describes how the EPS response is under a different phase than the commercial potentiostat. condition one provides the HLD of ±3 µA according to the possible resolution of the syst H t i l ith l t t ti b t (1 d 10) M K [F (CN) ] ld The HLD comes from the current values of the CV; speciﬁcally, condition 16 shows how the prototype cannot handle much current. The main difference between experimental conditions 1 and 16 is the concentration difference in the analyte (10 times in the order of magnitude). The Figure 9 describes how the EPS response is under a different phase than the commercial potentiostat. The condition one provides the HLD of ±3 µA according to the possible resolution of the system. However, trials with analyte concentration between (1 and 10) mM K3[Fe(CN)6] could prove a greater current range. Analysis of Results With that criterion, “the prototype can handle currents above 86.44 nA and below of −86.44 nA,” as Table 7 shows to have an accuracy above of the 95%. Table 7. Lower Limit of Detection analysis. * Data did not evaluate in the study. Mean Error, A LLD from 5% of the Mean Error, A Conditions 6 3.6 × 10 −9 * Conditions 7 5.04 × 10−9 * Conditions Average 4.32 × 10−9 86.44 × 10−9 Table 7. Lower Limit of Detection analysis. * Data did not evaluate in the study. To obtain the Higher Limit of Detection (HLD) an additional experiment was carried out at the experimental conditions described in Table 8 (condition 16) and it is presented in Figure 9. Table 8. Condition to explore the highest limit of detection. Table 8. Condition to explore the highest limit of detection. Conditions 16 Scan Rate 10 mV/s Initial Voltage 0.40 V Minimum Voltage −0.10 V Maximum Voltage 0.53 V Cycle Fifth Initial Scan Direction Positive Analyte 10 mM K3[Fe(CN)6] Electrolyte Support 0.5 M KCl 15 of 21 16 of Sensors 2018, 18, 4490 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 -3x10-5 -2x10-5 -1x10-5 0 1x10-5 2x10-5 3x10-5 4x10-5 PSoC Current CHI Current Current (A) Voltage vs ERef (V) 10 mV/s Figure 9. Cyclic voltammetry at 10 mV/s, 10 mM K₃[Fe(CN)₆] and 0.5 M KCl as the electrolyte suppor the working electrode, the counter electrode and the reference electrode were a disk glassy carbo electrode (diameter ϕ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively; experimen under the conditions 16: (Blue line) CV response by the EPS. (Orange line) CV response by th commercial potentiostat. Figure 9. Cyclic voltammetry at 10 mV/s, 10 mM K3[Fe(CN)6] and 0.5 M KCl as the electrolyte support; the working electrode, the counter electrode and the reference electrode were a disk glassy carbon electrode (diameter φ = 3 mm), a platinum wire and Ag/AgCl electrode, respectively; experiment under the conditions 16: (Blue line) CV response by the EPS. (Orange line) CV response by the commercial potentiostat. -0.1 0.0 0.1 0.2 0.3 0.4 0.5 -3x10-5 -2x10-5 -1x10-5 0 1x10-5 2x10-5 3x10-5 4x10-5 PSoC Current CHI Current Current (A) 10 mV/s Current (A) Voltage vs ERef (V) Figure 9. , current ran 4. Conclusions 4. Conclusions Table 9 shows the electrochemical conditions in which the response signal of the prototyp congruent with the commercial potentiostat. This table is a summary of the experiments to appreci the capacity of the EPS. The concentration used provides information about the voltage and Table 9 shows the electrochemical conditions in which the response signal of the prototype is congruent with the commercial potentiostat. This table is a summary of the experiments to appreciate the capacity of the EPS. The concentration used provides information about the voltage and the current range of the electrochemical techniques studied. In addition, the study provides a guide to test the scan rate and the range of the sample per second on the EPS. current range of the electrochemical techniques studied. In addition, the study provides a guide test the scan rate and the range of the sample per second on the EPS. Out of the ranges of Table 9, the behavior of the EPS is erratic or unknown. The potentiostat lo the voltage control with analyte concentrations above 10 mM K₃[Fe(CN)₆]. As a result, the E Out of the ranges of Table 9, the behavior of the EPS is erratic or unknown. The potentiostat loses the voltage control with analyte concentrations above 10 mM K3[Fe(CN)6]. As a result, the EPS capacity established in this study allows us to have several applications for the medical, biotechnology, environmental areas. 16 of 21 16 of 21 Sensors 2018, 18, 4490 Table 9. Electrochemical conditions evaluated. * Data did not evaluate in the study. CV LSV DSC Analyte Concentration 1 mM K3[Fe(CN)6] 1 mM K3[Fe(CN)6] 1 mM K3[Fe(CN)6] Voltage Range −0.15 to 0.65 V −0.15 to 0.65 V 0.195 to 0.325 V Peak Current Range −3.0 to 4.0 µA −2.5 to 3.5 µA * Maximum Step * * 0.310 to 0.195 V Minimum Step * * 0.310 to 0.315 V Maximum Current at 62 s * * 1.2 µA Minimum Current at 62 s * * 43.22 nA Scan Rate Range 10 to 500 mV/s 10 to 500 mV/s * SPS Range 50 to 2000 SPS 50 to 2000 SPS 50 to 2000 SPS Table 9. Electrochemical conditions evaluated. * Data did not evaluate in the study. Table 10 describes the principal features of the EPS. The prototype has a capacity of ±2 V to control the voltage. , current ran 4. Conclusions The range of currents was established from the experiments discussed previously. The number of samples per seconds comes from the ADC selected in the PSoC. The scan rate range relies on the architecture of the PSoC and the algorithm that controls the waveform generator. The parameters described in Table 10 point out that the PSoC is a suitable device to work as a prototype of a portable EPS. Table 10. Principal electric features of the EPS. Voltage Range: ±2 V Samples per Second: 50 to 2000 Current Range: ±3 µA Scan Rate: 10 to 500 mV/s Table 10. Principal electric features of the EPS. Voltage Range: ±2 V Samples per Second: 50 to 2000 Current Range: ±3 µA Scan Rate: 10 to 500 mV/s Table 10. Principal electric features of the EPS. Tables 11 and 12 give information about the power consumption and the values to compensate for having more accurate results. The offset voltages are summarized in Table 9 and the bias current at the inverting input establishes the minimum current to read in the device. The power consumption provides a clear idea of the battery requirements. Table 11. Additional features of the EPS with a source voltage of 5 volts. Table 11. Additional features of the EPS with a source voltage of 5 volts. ADC Offset Voltage: −61.056 µV EPS consumption at Stand By: 137.5 mW TIA Offset Voltage: −3.36034 mV EPS consumption at 2000 SPS: 207.5 mW ADC Offset Voltage: −61.056 µV EPS consumption at Stand By: 137.5 mW TIA Offset Voltage: −3.36034 mV EPS consumption at 2000 SPS: 207.5 mW Table 12. TIA resistor calculated and bias current at the inverting input. TIA Resistors Value Calculated Bias Current at the Inverting Input R1 18,971.46 Ω 3.40 × 10−8 A R2 28,636.77 Ω 2.06 × 10−8 A R3 38,272.14 Ω 1.67 × 10−8 A R4 77,433.47 Ω 6.77 × 10−9 A R5 116,828.09 Ω 5.35 × 10−9 A R6 244,583.12 Ω 2.59 × 10−9 A R7 490,370.41 Ω 1.27 × 10−9 A R8 981,623.90 Ω 6.89 × 10−10 A P t ti l A li ti f th EPS D l d Table 12. TIA resistor calculated and bias current at the inverting input. Potential Applications of the EPS Developed Potential Applications of the EPS Developed al Applications of the EPS Developed According to Periasamy et al., it is possible to use their glucose biosensor in a linear concentration range of 6.3 to 20.09 mM [37] and the concentration can go from 2 to 22 mM in humans [38]. The biosensor has a sensitivity of 2.47 µA/(mM cm2). In addition, the output current range of the biosensor with a ﬁxed area of 2 mm × 2 mm is of 0.62 to 1.98 µA. Hence, this prototype can handle that sensor because the EPS input current is wider than the biosensor output. Sensors 2018, 18, 4490 17 of 21 17 of 21 Apetrei et al. developed a sensor with a sensitivity of 37.1 nA/µM with an area of 0.867 cm2 in a linear range of 1–300 µM to detect melatonin [39]. With the EPS it is possible to detect melatonin in a concentration of 43 µM, since the detection range is around 2.5–80.0 µM. Other works have developed sensors with a sensitivity of 35 mA/(M cm2) for H2O2 [40–42]. In an amount of 10–200 µM, it can provoke a senescence-like state if a human cell gets in contact with it [42]. Thus, in a ﬁxed area of 7.2 mm × 7.2 mm it is possible to detect it in a range of 5–165 µM with the EPS. Jaiswal et al. developed a biosensor for the determination of nitrite (NO2−) [43]. In that study, they found two linear ranges of 0.1 to 1 µM and 1 to 1000 µM having two different sensitivities of 1.25 µA/(µM cm2) and 0.005 µA/(µM cm2), respectively. As a result, the EPS can be useful in the detection of nitrite in a range of 0.1 to 833 µM with an electrode area of 0.72 cm2 and by considering the two slopes in this range. Furthermore, the EPS can accomplish suitable features such as being a compact device, have a low power consumption, economically affordable, ﬂexible for being programmed according to with the required necessity, suitable for being integrated over system-on-a-chip platforms, it provides accuracy in the range of measured currents. In addition, since there is a setup of slave-master on the EPS, then it becomes attractive to use this technology to install a network of different EPS to transmit via wireless communication the sensing data to the Potentiostat User Interface System (PUIS). Potential Applications of the EPS Developed Finally, Table 13 presents a comparison of different compact potentiostats that have been studied to visualize their parameters in comparison to the parameters that can offer the EPS studied. Table 13 describes important features that should have the new generation in the electrochemical instrumentation, such as being small in size in order to be portable, economically affordable, precision in the measurements, low power consumption and wireless. The potentiostat designed, constructed and characterized in this work is demonstrated to be competitive with the previous work in potentiostats that has been shown recently (Table 13). Nonetheless, this work presented one of the ﬁrst potentiostat constructed by using embedded electronics and it is the ﬁrst of being a CYPRESS. For designing this potentiostat, the kits have a value of approximately cost (~100$ USD), therefore this approach to constructing a potentiostat can be very convenient versus other routes; also, the programmable circuit can vastly reduce the hardware complexity. Thus, it can lead the way to creating new applications for Point-of-Care with a reusable full electronics module. In addition, this work contributes to providing information about the architecture (digital peripherals and analog front end devices) required to construct a potentiostat since this information is scarce, due to the main providers being companies who protect their circuit design. Finally, an integral methodology that includes the characterization and calibration of the potentiostat has been presented, thus an analysis of errors on the measurements in this device were tested and three electrochemical techniques were performed. 18 of 21 Sensors 2018, 18, 4490 Table 13. Electrochemical Instruments Comparison. * Data not available. Table 13. Electrochemical Instruments Comparison. * Data not available. Electrochemical Instrument Jafari et al. [44] Dorta-Quinones et al. [45] Bozorgzadeh et al. [46] Dryden et al. [10] EmStat 3+ Embedded/OEM [47] WaveNow AFTP1 [48] Sun et al. [11] Giordano et al. [49] Muñoz et al. Potential Applications of the EPS Developed (This Article) Highest Current Detection Limit 350 nA 430 nA 950 nA 22 mA 100 mA 100 mA 200 µA 50 µA 3 µA Lowest Current Detection Limit 8.6 pA * * 600 fA 1 pA 80 nA 1 nA 100 nA 86 nA Electronic Chip Area 3 mm × 3 mm 1.5 mm × 1.0 mm 3.16 mm × 3.16 mm * * * * * * PCB Area * 4.7 cm × 1.9 cm * 8 cm × 8 cm 5.5 cm × 4.1 cm 16.5 cm × 10 cm 3.9 cm × 1.62 cm 9.7 cm × 5.7 cm ~(8 cm × 6 cm) Maximum Samples Per Second 2 ksps 10 ksps 10 ksps 30 ksps Less than 1 Ksps 1 Ksps 200 ksps * 2 Ksps ADC Effective Number of Bits 9 bits 10.95 bits * 21.3 bits at 1.45 ksps * * * * 18 Wireless Connectivity Ultra-Wideband Transmitter Ultra-Wideband Transmitter FSK Transmission with Manchester Encoding No Bluetooth or Wiﬁ No No Bluetooth Bluetootth Number of Techniques Implemented 1 1 2 More than 3 9 33 3 4 At least 3 Channels 54 1 1 1 up to 16 1 2 1 1 SoC Present Yes Yes Yes No * * No No Yes Capabilities additional to a potentiostat No No No Yes Yes Yes Yes No Yes, ﬂexibility to reconﬁgure the analog front end Maximum Power Consumption 1543.3 µW for each channel 30 µW Approximately 0.4 mW Less than 1 W 2.5 W 10 W 111 mW *** 207 mW at 5 V 19 of 21 Sensors 2018, 18, 4490 19 of 21 Author Contributions: O.I.G.P. wrote the code for the EPS and performed experiments to evaluate the electronics on the EPS and for the electrochemical experiments, A.I.M.-M.; established the requirements and characteristics that the potentiostat must have, performed experiments for the electrochemical experiments, proposed the experimental methodology and setup conditions, analyzed the results, evaluated all functionality and performance of the potentiostat, wrote the article, corresponding author O.I.G.P.; proposed the methodology for evaluating the errors and precision on the EPS, J.C.-F.; developed the architecture circuit design on the potentiostat, J.M.R.-D.; reviewed the code written for the EPS, A.Á.-O.; reviewed the overall functionality of the EPS from the point of view of the embedded electronics, funding acquisition, G.D.-A. 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Potential Applications of the EPS Developed Funding: This research was funded by Tecnologíco de Monterrey and was funded by The National Council of Science and Technology of Mexico (CONACYT). Acknowledgments: The National Council of Science and Technology of Mexico (CONACYT) and the Tecnologico de Monterrey provided ﬁnancial support to conduct this study. Acknowledgments: The National Council of Science and Technology of Mexico (CONACYT) and the Tecnologico de Monterrey provided ﬁnancial support to conduct this study. Conﬂicts of Interest: The authors declare no conﬂict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W2147630672	https://researchonline.ljmu.ac.uk/id/eprint/5671/1/27434-95703-1-PB.pdf	English	null	Perceptions of Auditing and the Provision of Non-Audit Services: Case Study in Libya	International journal of business and management	2,013	cc-by	10,042	Sawan, N, Alzeban, A and Hamuda, K Perceptions of Auditing and the Provision of Non-Audit Services: Case Study in Libya http://researchonline.ljmu.ac.uk/id/eprint/5671/ Article LJMU has developed LJMU Research Online for users to access the research output of the University more effectively. Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Users may download and/or print one copy of any article(s) in LJMU Research Online to facilitate their private study or for non-commercial research. You may not engage in further distribution of the material or use it for any profit-making activities or any commercial gain. The version presented here may differ from the published version or from the version of the record. Citation (please note it is advisable to refer to the publisher’s version if you intend to cite from this work) Sawan, N, Alzeban, A and Hamuda, K (2013) Perceptions of Auditing and the Provision of Non-Audit Services: Case Study in Libya. International Journal of Business and Management, 8 (14). ISSN 1833-3850 LJMU Research Online Sawan, N, Alzeban, A and Hamuda, K Perceptions of Auditing and the Provision of Non-Audit Services: Case Study in Libya http://researchonline.ljmu.ac.uk/id/eprint/5671/ Article LJMU has developed LJMU Research Online for users to access the research output of the University more effectively. Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Users may download and/or print one copy of any article(s) in LJMU Research Online to facilitate their private study or for non-commercial research. You may not engage in further distribution of the material or use it for any profit-making activities or any commercial gain. The version presented here may differ from the published version or from the version of the record. Citation (please note it is advisable to refer to the publisher’s version if you intend to cite from this work) Sawan, N, Alzeban, A and Hamuda, K (2013) Perceptions of Auditing and the Provision of Non-Audit Services: Case Study in Libya. International Journal of Business and Management, 8 (14). ISSN 1833-3850 LJMU Research Online LJMU Research Online Article Sawan, N, Alzeban, A and Hamuda, K (2013) Perceptions of Auditing and the Provision of Non-Audit Services: Case Study in Libya. International Journal of Business and Management, 8 (14). ISSN 1833-3850 Sawan, N, Alzeban, A and Hamuda, K (2013) Perceptions of Auditing and the Provision of Non-Audit Services: Case Study in Libya. International Journal of Business and Management, 8 (14). ISSN 1833-3850 LJMU has developed LJMU Research Online for users to access the research output of the University more effectively. Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Users may download and/or print one copy of any article(s) in LJMU Research Online to facilitate their private study or for non-commercial research. You may not engage in further distribution of the material or use it for any profit-making activities or any commercial gain. The version presented here may differ from the published version or from the version of the record. Please see the repository URL above for details on accessing the published version and note that access may require a subscription. For more information please contact researchonline@ljmu.ac.uk http://researchonline.ljmu.ac.uk/ International Journal of Business and Management; Vol. 8, No. 14; 2013 ISSN 1833-3850 E-ISSN 1833-8119 Published by Canadian Center of Science and Education 1. Introduction The non-audit service by incumbent auditors has been intensively debated in the literature. Since the early 1980s, the work undertaken by audit firms has gradually changed such that the revenue from audit services has fallen, while that from the provision of other services has increased (Abu Bakar et al, 2005; Abdel-Khalik, 1990; Craswell, 1999; DeFond et al, 2002; Firth, 1997a). This trend has caused some major worries, concerned with the pricing of individual services where the auditor performs several different services, and there is potential for compromising auditor independence. The AICPA defines Non-Audit Service (NAS) as: “…the function of providing professional advisory (consulting) services, the primary purpose of which is to improve the client’s use of its capabilities and resources to achieve the objective of the organisation” (cited in Patten and Nuckols, 1978). 2. Literature Review Although there have no studies recently dealing with the relationship between non-audit service and audit quality in Libya, however, the topic still raises interest and calls for further and more in-depth research. The following provides an overview of the key literature in order to gain a deeper understanding of the topic under consideration. There has been extensive debate (e.g. Barkess and Simnett, 1994; Canning and Gwilliam, 1999; DeFond et al, 2002; Firth, 1997a) on the merits of audit firms providing non-audit services, the main concern being that NAS is normally expressed in terms of economic dependency and mutuality of interest, and this is why audit fees must be disclosed in annual reports. If NAS become sufficiently important, either in total or in relation to an individual client, the economic dependence of the audit firm on these services and clients may result in the loss of impartiality and objectivity (Simunic, 1984). Abstract This paper examines whether non audit service are associated with audit quality. In relation to the impact of non-audit service on audit quality, the provision of NAS to audit clients was found to provide auditors with greater experience of the client’s industry and greater access to the client’s accounting system. Additionally, such an arrangement was considered to enhance audit quality, but simultaneously it was also believed that a separation of NAS from audit services was desirable since auditors are perceived to have greater credibility when the demarcation is clear. A questionnaire was used to collect data. To confirm and support the questionnaire findings semi-structured interviews were conducted the data used for this study was collected from two sources: the demand side (Libyan oil companies) and the supply side (audit firms working in Libya). Keywords: auditor independence, audit quality, non-audit service, oil company, audit firms Perceptions of Auditing and the Provision of Non-Audit Services: Case Study in Libya Nedal Sawan1, Abdulaziz Alzeban2 & Khaled Hamuda3 1 Liverpool Business School, United Kingdome 2 King Abdulaziz University, Saudi Arabia 3 Al-Aziza Institute, Libya Correspondence: Nedal Sawan, Liverpool Business School, United Kingdome. E-mail: ne2000dal@hotmail.com Received: May 13, 2013 Accepted: June 7, 2013 Online Published: June 26, 2013 doi:10.5539/ijbm.v8n14p168 URL: http://dx.doi.org/10.5539/ijbm.v8n14p168 2.1 Arguments against the Provision of Non-Audit Services (NAS) Much public interest has arisen in the provision of NAS (Simunic, 1984), and as noted by Craswell (1999), regulators internationally have considered the potential problems it can cause. Actually, Beattie and Fearnley (2002) found no evidence to support the contention that the provision of audit and NAS resulted in decreased audit quality, but they did confirm that it might adversely affect the perception of audit independence because suspicions are, that where an audit firm acts as both consultant and auditor, there may be an incentive not to report any 168 International Journal of Business and Management Vol. 8, No. 14; 2013 www.ccsenet.org/ijbm shortcomings in consultancy advice which have been discovered during the course of the audit in order to avoid damage to its reputation (Simunic 1984). shortcomings in consultancy advice which have been discovered during the course of the audit in order to avoid damage to its reputation (Simunic 1984). Additionally, the economic relationship between the audit client and the auditor may be such that the auditor will ignore dubious management accounting practices since the act of calling these practices into question could result in the loss of the current audit fee as well as any future NAS undertakings. Another disadvantage of providing NAS to audit clients occurs when NAS provide auditors with client-specific rents, in which case companies may be able to obtain more favourable financial reports by threatening to switch auditor. The potential for public perception of impaired auditor independence when a firm provides both audit and NAS is confirmed by several researchers (Hay et al, 2006; Simunic, 1984, Beck et al, 1988a, Canning and Gwilliam, 1999; Craswell, 1999; Abu Bakar et al, 2005; Alleyne and Devonish, 2006), who note that the high fees involved suggest too great a mutual reliance between auditor and auditee. However, as noted by Mautz and Sharaf (1961) half a century ago, the interests of auditor and auditee become the same when both audit and NAS are provided, and this inevitably leads to an end to the accountant’s independence. Consequently, they advocate the use of auditors for auditing purposes only, and the complete separation of audit and NAS. Clikeman (1998) echoes these arguments, confirming that the provision of NAS requires the provider to be an ally of management, whereas the provision of audit services demands total professional detachment from management. 2.1 Arguments against the Provision of Non-Audit Services (NAS) Clearly, this is a very difficult position to achieve because, as argued by DeBerg et al (1991), the economic bond between auditor and client is strengthened by the provision of both audit and NAS, and as Largay (2002) observes, with a significant growth in this respect, auditors may become more willing to protect clients in order to avoid dismissal and lose fees. Such assumptions have been tested by Firth (2002) who took a sample of 1,112 non-financial companies listed on the international stock exchange, and found there was indeed a link between clean audit reports and high NAS fees. Nonetheless, it was acknowledged by Firth (2002), that the findings were inconclusive since other variables may have caused the result, such as for instance, extra NAS may have been employed by a struggling company in order to help clear up problems it was facing, and the consequent resolution of those problems by the auditor could result in a genuinely clean audit report. In such cases, NAS are helpful to the audit process (Firth 2002). Lennox (1999b), however, argues that NAS have both benefits and disadvantages because whilst they increase the likelihood of problems being found, they also enhance the probability of reducing auditor independence. It is obvious that the provision of both audit and NAS may well result in a conflict of interests because of the economic relationship, which could easily lead to the client’s expectation that the auditor will be amenable to compromise. Indeed, Flint (1988: 81) argues that ”the auditors may become unduly sympathetic to a directorial or managerial attitude or interpretation of events, or the work may involve the creation of systems and information rather than assessing the adequacy of the systems and information which have been created by the directors or managers”. Because of these strong potential problems, Lee (1993) stresses the need for total impartiality and the need to make decisions without considering potential adverse financial results, when both audit and NAS are commissioned. Many researchers argue that it is not possible for this level of objectivity to be sustained (Brandon et al, 2004; Frankel et al, 2002; Glezen and Millar, 1985; Jenkins and Krawczyk, 2001; Lowe and Pany, 1995; 1996; Raghunandan, 2003; Wines, 1994) because the auditors would in effect be auditing their own work, and perhaps acting in management role. 2.2 Arguments in Favour of the Provision of Non-Audit Services As already seen, contrasting findings do show that there is no detrimental effect on audit quality and audit independence (Antle et al, 1997; Glezen and Millar, 1985; Gul, 1989; Raghunandan, 2003; Scheiner, 1984), but nevertheless, many countries prefer the relevant professional bodies to acknowledge the potential conflict and formulate guidelines (Lee, 1993). Researchers who support the provision of joint services argue that this arrangement ameliorates audit independence (Antle et al, 1997), because it provides the auditor with better knowledge of a client (through ‘knowledge spillover’), and may well enhance the likelihood of problem discovery, and hence audit quality (Goldman and Barlev, 1974; Wallman, 1996). Another argument is that the switching of auditors creates disharmony since the close relationship between auditor and client is lost in the process. Indeed, Hartley and Ross (1972) found from their survey that only 6% of respondents considered the provision of NAS as negatively affecting auditor independence. Likewise, Firth (1980) argues that the provision of NAS causes an insignificant risk to auditor independence, and in research conducted by Glezen and Millar (1985), it was found that stockholders were not concerned about the joint provision of audit and NAS. Palmrose’s (1988) study of US corporations showed that they preferred to use their commissioned audit firms for NAS, irrespective of the amount of NAS involved, and there was no perceived risk to auditor independence. Svanström and Sundgrenwe (2012) confirm a positive association between perceived quality of audit services and the likelihood of a client purchasing non-audit services from the auditor. Gul (1989) actually found that New Zealand bankers perceived the effects of NAS as being a positive influence on auditor independence, and Moizer (1997) considered that a direct, positive relationship existed between an audit firm’s economic interests in a client and that client’s dependence on it. Further, Hussey (1999) indicated that financial directors in the UK were in favour of the provision of NAS to audit clients. Many benefits such as a reduction of total costs, increased technical auditing quality, and enhanced competition are cited (Arrunada, 1999; Goldman and Barlev, 1974; Wallman, 1996), all of which could improve audit quality, and lead to cost savings in both markets (audit and NAS) which could be passed on to the customer. Furthermore, the provision of NAS results in an increase in client- and firm-specific assets, which always have a positive effect on independence. concentrate on different types of NAS. Finally, Gaynor et al (2006), studying audit committee members, demonstrate that the provision of NAS has definitely been held to damage independence, because since the 2002 SEC ruling that audit committees must pre-approve and disclose all NAS provided by their regular auditor, the audit committee members are less likely to allow joint provision of both audit and NAS, even if these services actually improved audit quality. Given that it is the responsibility of audit committees to maintain investor trust, they are reluctant to allow both audit and NAS to be conducted by the same firm as they recognise completely the inherent contradiction in the role and expectations of the auditor. 2.1 Arguments against the Provision of Non-Audit Services (NAS) The strong economic bond is believed to affect “their mental attitude, impartiality and objectivity, and independence of thought and act” (Flint, 1988:82), heightening the risk that auditors may surrender to management pressure when they provide ongoing NAS (Antle, 1984; Canning and Gwilliam, 1999; DeAngelo, 1981a; Magee and Tseng, 1990; Simunic, 1984). Indeed, Brandon et al (2004) found a negative relationship between the size of NAS fees paid to external auditors and a firm’s bond rating. Recent research (Felix et al, 2005) confirms that the amount of client pressure upon auditors providing them with NAS increases, and that auditors become less concerned with the quality of internal audits, and are subsequently influenced in decisions made when collecting audit evidence. Indeed, Quick and Ben-Rasmussen (2005) found from their questionnaire survey with state authorised auditors, managing directors, bank loan officers, private shareholders and business journalists, that produced a response rate of 73.1%, that all of the groups of respondents with the exception of auditors themselves and managing directors, perceived auditor independence to be impaired when both audit and NAS are performed for a particular client. That said, the researchers do point out that because auditors do not consider their provision of NAS to impair their independence, “independence in mind is not affected” (Quick and Ben-Rasmussen 2005:148). However, in this study, NAS were treated broadly and as there is a possibility that specific NAS may have different effects upon auditor independence, future research should 169 International Journal of Business and Management Vol. 8, No. 14; 2013 www.ccsenet.org/ijbm concentrate on different types of NAS. 3. Methods and the Sampling Unit A mixed methods approach was utilised in this study to gather a range of views from all the professional groups involved in Libyan Auditing. Questionnaires were used to collect data concerning the perceptions of two sources: the demand side (Libyan oil companies) and the supply side (audit firms working in Libya). The data for the Libyan oil companies was gathered from three different types of respondents: internal auditors, financial managers and accounts managers. The reason for choosing these three groups of respondents, rather than other employees in the company, was the fact that the literature of auditing indicates that the external auditor usually has more contact with these groups than any others. For the audit firms, data was gathered from employees at all levels in the audit firm: managing partners, audit supervisors and auditors. The rationale for choosing oil companies was because of their high level of organisation and the fact that most of these companies employ staffs who hold degrees from the United States or Britain – these two facts enabled the researcher to access the right people and obtain the appropriate data. The magnitude of the activities of the oil companies, and hence the scale of the accounting systems, represent an attraction for large numbers of qualified accountants, who hold qualifications and different accounting backgrounds, a fact that allowed the researcher to have access to a large community of accountants, with diverse careers and work experience. In order to refute and support the questionnaire findings, semi-structured interviews were conducted. The sample for the interviews was broadly similar to the sample for the questionnaire, which involved a sample from Libyan oil companies, namely, internal auditors, financial managers, accounts managers, and a sample from audit firms working in Libya, namely, managing partners, auditors and audit supervisors. Regulators working in the LAAA were also included in the sample of the interviews, partly due to the fact that, after analysing the questionnaire survey, the researcher found it necessary to interview regulators to clarify some grey areas found in the analysis, and more importantly, to triangulate the sample of the study and to obtain different opinions from different dimensions. 2.2 Arguments in Favour of the Provision of Non-Audit Services Another perceived advantage (Grout et al, 1994), is the potential for auditors to diversity their portfolio of activity, which ultimately results in a reduction on their reliance on a single customer. It is also noted that the provision of NAS can allow audit firms to develop their capabilities and reputation. Antle (1999) argues that the incentive to sell on profitable non-audit services prevents auditors being dishonest, and De Fond et al (2002) confirm that it is against the best interests of the auditor to sacrifice his/her audit quality in order to enhance a consulting relationship. Moreover, there is a greater risk that government will bring in producing legislation relating to self-regulation if audit firms damage their reputation and thus reduce the credibility of the profession as a whole (Hillison and Kennelly, 1988). Frankel et al (2002) suggest that an auditor’s reputational capital can be enhanced by the provision of NAS, and consequently the auditor would not be prepared to jeopardise this in surrendering to any particular client’s demands to overlook financial problems. The economic theory of auditor independence that postulates the potential for compromise of independence where incentives to do so are present, was tested by Chung and Kallapur (2003), who used a sample of proxy statements from 1,871 Big Four clients, to determine ratios of client fees to total audit firm revenue (to establish economic dependence) and the ratio of the clients’ NAS services fees to total audit firm revenues. Regression techniques revealed no association between abnormal accruals and the client importance ratios, providing evidence that is inconsistent with the economic theory of auditor independence, but that is in line with the arguments of Goldman and Barlev (1974), who assert that NAS increase an auditor’s value to the client, thereby placing the auditor in a stronger position to resist client pressure. Chung and Kallapur (2003) obtained similar findings to those of Mitra (2007), who conducted a cross-sectional regression analysis of abnormal accrual adjustment of oil and gas companies, finding that abnormal accrual adjustments were not related to fees paid for NAS. Mitra (2007) argues 170 International Journal of Business and Management Vol. 8, No. 14; 2013 www.ccsenet.org/ijbm that industry specialisation and reputation protection are sufficient incentive to ensure auditors remain independent and that they actually strengthen independence. 2.2 Arguments in Favour of the Provision of Non-Audit Services All of these research studies, concur with the review of the NAS literature made by Francis (2006), that suggests there is no direct evidence that audit quality is compromised by the provision of NAS to audit clients. Santan (2007), in a recent study, also finds no support for the SEC’s contention that the provision of NAS places audit quality and auditor independence at risk. 3. Methods and the Sampling Unit The total number of interviews conducted with the oil companies was ten (three with internal auditors, four with financial managers, and three with accounts managers), and the total number of interviews conducted with the audit firms working in Libya, was thirteen (four with managing partners, six with audit supervisors, and three with auditors). Lastly, two interviews were conducted with regulators working for the LAAA. This process aims to enhance and supplement the questionnaire findings providing an in-depth clarification and understanding of the effects that the selected factors have on evidence obtained by Libyan auditors. Content analysis was used to analyse the collected data from the interviews. The first part of the questionnaire was designed to obtain the views of external, internal, state and taxation auditors relating to the effects of the professional and academic qualifications of the auditor on quality of evidence. The second section aimed to gather the participant’s opinions regarding the effects of the consistency of evidence on audit evidence. The final section asked the participants about the effects of amount of evidence on quality of evidence. A 5-point Likert-scale ranging from strongly undermines evidence to strongly enhances evidence was utilised to measure perceptions regarding quality of audit evidence (Saunders et al., 2007). For the purpose of this study, 147 questionnaires were distributed to the oil companies. Of these, 52 went to Internal Auditors, 50 to Financial Managers, and 45 to Accountants Managers. Additionally, 300 questionnaires were sent to the audit firms working in Libya, 100 to Managing Partners, 100 to Audit Supervisors and 100 to Auditors. The samples from the demand side (internal auditors, financial managers and accounts managers), represent the agent of the principal, and conduct business on behalf of the principal. Hence, a monitoring mechanism is needed to assess their performance (Jensen and Meckling, 1976). The samples from the supply side (managing partners, audit supervisors and auditors), represent the main subjects of the issue of interest who provide certification and/or information credibility assessment to the stakeholders (Humphrey, 1997). Hasan (2000) point out that audit firms and their clients evaluate audit quality in different ways, and it was, therefore important to receive responses from both sections of the research population. Considerable effort was made in order to avoid problems of non-response and to ensure the completeness of the 171 International Journal of Business and Management Vol. 8, No. 4. Results and Discussion 4. Results and Discussion 4. Results and Discussion 3. Methods and the Sampling Unit 14; 2013 www.ccsenet.org/ijbm questionnaire which, was designed using mainly closed questions which are easy for respondents to answer. The questionnaire sample consisted of all listed oil companies in the NOC and 100 audit firms working in Libya. According to the pilot study results, the wording of the questionnaire was clear and straightforward, the instrument was of a reasonable length, and there were no complaints about layout. Most of the questionnaires were personally administered (Managing Partner - 52 questionnaires, Audit Supervisor - 52 questionnaires, Auditor - 56 questionnaires, Internal Auditor - 45 questionnaires, Financial Manager - 47 questionnaires, and Accounts Manager - 35 questionnaires), and some questionnaires were delivered personally by the researcher and returned by mail. Therefore, anonymity and other ethical considerations relating to mailed questionnaires were avoided as far as possible. The overall response rate to the questionnaire was extremely encouraging at 64% (see Table 1). Remenyi et al. (2002) suggesting that a response rate above 60% is considered to be exemplary. The response rates for Internal Auditors and Financial Managers were 86.5% and 94% respectively, higher than those of Accounts Managers, Managing Partners, and Audit Supervisors, which were 77.7%, 52% and 52% respectively. Some were unable to complete the questionnaires, and the researcher was unable to contact respondents outside Tripoli because of poor communication facilities, included in which is the official postal system. Table 1. Questionnaire survey response rate Group Distributed questionnaires Useable questionnaires Response rate Managing Partner Audit Supervisor Auditor Internal Auditor Financial Manager Accounts Manager Total 100 100 100 52 50 45 447 52 52 56 45 47 35 287 52 52 56 86.5 94 77.7 64 Table 1. Questionnaire survey response rate 4.1 Quantitative Findings 14; 2013 www.ccsenet.org/ijbm client gives the auditor more experience of the client’s industry and more access to the client’s accounting system, achieved the highest mean score of 3.98, with 72.5% of respondents either agreeing or strongly agreeing. client gives the auditor more experience of the client’s industry and more access to the client’s accounting system, achieved the highest mean score of 3.98, with 72.5% of respondents either agreeing or strongly agreeing. The statement with the next highest mean score (3.58) was the prohibition of the provision of NAS to an audit client is only to maintain the perception of independence, which gained either agreement or strong agreement from 58.8% of respondents. The statement the provision of NAS to an audit client reduces the probability of a threat to switch auditor achieved a mean value of (3.53), with just over half (51.9%) of respondents either agreeing or strongly agreeing with it. The statement with the fourth highest mean score (3.34) was Providing NAS to an audit client by a separate department gives the auditor more credibility. Just under half (47.5%) of respondents either agreed or strongly agreed with this statement, possibly reflecting the confidence of the respondents from large audit firms and local firms in their ability to separate their staff according to the type of services they were commissioned to perform. Indeed, the provision of NAS by staff from separate departments has been practised by large audit firms for decades. In contrast, the statement with the lowest mean score (2.43) was the provision of NAS impairs audit quality, which attracted agreement or strong agreement from only 16.9% of respondents. The statement with the second lowest mean score (2.57) was the provision of NAS to an audit client leads to economic dependency on that client and causes a conflict of interests for the auditor. Just less than a third (30%) of respondents either agreed or strongly agreed with this statement. The statement with the third lowest mean score (3.26) was only certain types of NAS impair audit quality and in this respect, 48.8% of audit firm respondents either agreed or strongly agreed with it. Since the overall responses of oil companies and audit firms were not completely identical, an attempt was made to isolate the determinants of the disparities. 4.1 Quantitative Findings 4.1 Quantitative Findings The analysis of respondents’ perceptions about the relationship between the provision of NAS and audit quality. It shows that 84.2% of oil company respondents either agreed or strongly agreed with the statement the provision of NAS to an audit client gives the auditor more experience of the client’s industry and more access to the client’s accounting system. This statement achieved the highest mean score (4.24). Providing NAS to an audit client by a separate department gives the auditor more credibility had the highest second mean score (3.50), with (60.7%) of oil company respondents either agreeing or strongly agreeing with it. This result might reflect the respondents’ confidence in the safeguards of auditor independence from the segregation of duties by splitting the provision of audit and NAS into separate departments, which is consistent with the arguments proposed by Canning and Gwilliam (1999) and Pany and Reckers (1984). Sequentially, the third highest mean score (3.39) was achieved by the statement the prohibition of the provision of NAS to an audit client is only to maintain the perception of independence. Again, 48% either agreed or strongly agreed with it. The statement with the fourth highest mean score (3.20) was the provision of NAS to an audit client reduces the probability of switch threat change auditor, with half (50.4%) of respondents either agreeing or strongly agreeing with it. In contrast, a third of the oil company respondents (33.1%) either agreed or strongly agreed with the statement the provision of NAS to an audit client leads to economic dependency on that client and causes a conflict of interests for the auditor. It achieved the lowest mean score of (2.50). The statement with the second lowest mean score 2.83) was the provision of NAS impairs audit quality, with which 23.7% of respondents either agreed or strongly agreed. The statement with the third lowest mean score (3.04) was only a certain type of NAS impairs audit quality. Over a third (45.7%) of respondents either agreed or strongly agreed with this statement. With regard to responses from the audit firms, Table 2 shows that the statement the provision of NAS to an audit 172 International Journal of Business and Management Vol. 8, No. 4.1 Quantitative Findings Therefore, the extent to which position and type of audit firm might explain differences in oil company and audit firm respondents’ opinions, was once again tested using the Kruskal-Wallis and Mann Whitney U tests. Table 2, which documents the results, shows two significant differences at the 1% level, as measured by the Mann Whitney U test. On examination of the level of agreement, oil company respondents were significantly higher than audit firm respondents in respect of the statement that the provision of NAS impairs audit quality. This difference in the responses is interesting, and perhaps suggests that audit firm respondents viewed the provision of NAS from the profit perspective, whereas oil companies viewed the provision of NAS as not impairing audit quality, but needing some restriction. The level of agreement from oil company respondents was significantly higher than that from audit firm respondents in respect of the statement highlighting the advantages of the provision of NAS to an audit client as being that it gives the auditor more experience of the client’s industry and more access to the client’s accounting system. This result might reflect a preference of oil company respondents to receive the provision of NAS from their external auditor. However, in general, the above results are consistent with prior literature (Abdel-Khalik, 1990; Craswell, 1999; De Fond et al, 2002) that identifies the provision of NAS to audit clients as having the potential to increase the auditor’s client knowledge, and therefore, enhance the probability that problems would be discovered. Therefore, for a given level of independence, NAS may increase audit quality. Further, NAS may increase a client’s dependence on its auditor, thereby reducing the credibility of the switch threat. Another commonly-mentioned advantage of providing NAS to audit clients is the potential cost advantage to the client arising from knowledge spillovers, which are transfers of knowledge that may occur when NAS are provided by the incumbent auditor. 173 International Journal of Business and Management Vol. 8, No. 14; 2013 www.ccsenet.org/ijbm Table 2. Distribution of the evaluations given by the different statements regarding the Non-Audit Services (NAS) and audit quality Statement Audit Firms Oil Companies SD% D% NV% A% SA% MeanMedian SD% D% NV% A% SA% Mean Median Sig The provision of NAS to an audit client gives the auditor more experience of the client’s industry and more access to the client’s accounting system. 4.1 Quantitative Findings 0 5.0 22.5 41.9 30.6 3.98 4.00 0 10.5 5.5 34.6 49.6 4.24 4.00 * The provision of NAS to an audit client leads to economic dependency on that client and causes a conflict of interests for the auditor. 29.4 14.4 26.2 30 0 2.57 3.00 33.9 15.7 17.3 33.1 0 2.50 3.00 Only certain types of NAS impair audit quality. 14.4 17.5 19.4 25 23.8 3.26 3.00 20.5 20.5 13.4 26 19.7 3.04 3.00 The prohibition of the provision of NAS to an audit client is only to maintain the perception of independence. 5.6 16.3 19.4 31.9 26.9 3.58 4.00 7.1 16.5 28.3 26 22 3.39 3.00 Providing NAS to an audit client by a separate department gives the auditor more credibility. 6.9 19.4 26.3 27.5 20 3.34 3.00 8.7 15 15.7 39.4 21.3 3.50 4.00 The provision of NAS impairs audit quality. 17.5 45 20.6 11.3 5.6 2.43 2.00 11 33.9 31.5 8.7 15 2.83 3.00 * The provision of NAS to an audit client reduces the probability of a threat to switch auditor. 5 11.9 31.3 28.8 23.1 3.53 4.00 15.7 22 11 27.6 22.8 3.20 4.00 ,* indicates distribution of responses is significantly different at the 1%, 5% levels, respectively using the Mann Whitney U test. The provision of NAS to an audit client gives the auditor more experience of the client’s industry and more access to the client’s accounting system. The provision of NAS to an audit client leads to economic dependency on that client and causes a conflict of interests for the auditor. Only certain types of NAS impair audit quality. The prohibition of the provision of NAS to an audit client is only to maintain the perception of independence. Providing NAS to an audit client by a separate department gives the auditor more credibility. 4.1.1 The Perceptions of NAS Based on Position Table 3, shows that when comparing the responses of oil company staff and audit firm staff using the Kruskal- Wallis test, one significant difference (at the 5% level) is observed. Financial managers, accounts managers and internal auditors had significantly higher mean scores (4.32, 4.31 and 4.09) than audit supervisors and auditors (3.96 and 3.98) for the statement The provision of NAS to an audit client gives the auditor more experience of the client’s industry and more access to the client’s accounting system. This finding may be due to the belief of financial managers and internal auditors, who may feel that the non-provision of audit services improves audit quality, and believes that if NAS are not provided, more audit hours are needed for new auditor to become familiar with the company system. This result is consistent with that obtained by other researchers who support the provision of joint services, arguing that this arrangement ameliorates audit independence (Antle et al, 1997), because it provides the auditor with better knowledge of a client (through ‘knowledge spillover’), and may well enhance the likelihood of problem discovery, and hence audit quality (Goldman and Barlev, 1974; Wallman, 1996). 174 International Journal of Business and Management Vol. 8, No. 14; 2013 www.ccsenet.org/ijbm Table 3. Distribution of the evaluations given by the different statements regarding the Non-Audit Services (NAS) based on position Statement Audit Firm Staff Oil Company Staff Sig Managing Partner Audit Supervisor Auditor Internal Auditor Financial Manager Accounts Manager Mean Median Mean Median Mean Median Mean Median Mean Median Mean Median The provision of NAS to an audit client gives the auditor more experience of the client’s industry and more access to the client’s accounting system. 4.00 4.00 3.96 4.00 3.98 4.00 4.09 4.00 4.32 5.00 4.31 5.00 ** The provision of NAS to an audit client leads to economic dependency on that client and causes a conflict of interests for the auditor. 2.48 3.00 2.67 3.00 2.55 3.00 2.82 3.00 2.38 2.00 2.23 2.00 Only certain types of NAS impair audit quality 3.50 4.00 3.23 3.00 3.07 3.00 3.24 3.00 3.09 3.00 2.71 2.00 The prohibition of the provision of NAS to an audit client is only to maintain the perception of independence. 3.88 4.00 3.29 3.50 3.57 4.00 3.29 3.00 3.23 3.00 3.74 4.00 Providing NAS to an audit client by a separate department gives the auditor more credibility. 4.1.1 The Perceptions of NAS Based on Position 3.33 3.00 3.27 3.00 3.43 4.00 3.76 4.00 3.13 3.00 3.66 4.00 The provision of NAS impairs audit quality. 2.27 2.00 2.48 2.00 2.52 2.00 2.82 3.00 2.72 2.00 2.97 3.00 The provision of NAS to an audit client reduces the probability of a threat to switch auditor. 3.50 3.00 3.40 3.50 3.68 4.00 3.16 3.00 3.21 4.00 3.23 3.00 ,* indicates distribution of responses is significantly different at the 1%, 5% levels, respectively using the Kruskal-Wallis test. The provision of NAS to an audit client gives the auditor more experience of the client’s industry and more access to the client’s accounting system. The provision of NAS to an audit client leads to economic dependency on that client and causes a conflict of interests for the auditor. 4.1.2 The Perceptions of NAS Based on Type of Audit Firms The analysis by type of audit firm, as reported in Table 4, shows that no significant differences were found at the 5% level, and that just one significant difference was found at the 1% level between local audit firms, local firms affiliated to an international firm, and local firms affiliated to one of the Big Four audit firms (non-Big Four and Big Four) using the Kruskal Wallis test. The level of agreement with the statement: Only certain types of NAS impair audit quality was significantly higher from local audit firms and international audit firms (non-Big Four) (3.58 and 2.86) than from the Big Four firm respondents (2.62). This result might reflect the fact that the Big Four firm respondents believe that all types of NAS can potentially impair the level of audit quality, or pose a threat to auditor independence. This finding is inconsistent with the reports of Abu Bakar et al (2005) and Alleyne and Devonish (2006) who found that individual non-audit services may affect auditor independence influence audit quality perceptions differently and that the provision of NAS is a significant threat to perceptions of auditor independence. 175 International Journal of Business and Management Vol. 8, No. 14; 2013 www.ccsenet.org/ijbm Table 4. Distribution of the evaluations given by the different statements regarding Non-Audit Services (NAS) based on type of audit firm Statement Local Audit Firm International Audit Firm Big Four Audit Firm Sig Mean Median Mean Median Mean Median The provision of NAS to an audit client gives the auditor more experience of the client’s industry and more access to the client’s accounting system. 4.00 4.00 4.07 4.00 3.71 4.00 The provision of NAS to an audit client leads to economic dependency on that client and causes a conflict of interests for the auditor. 2.63 3.00 2.53 3.00 2.38 2.00 Only certain types of NAS impair audit quality. 3.58 4.00 2.86 3.00 2.62 2.00 * The prohibition of the provision of NAS to an audit client is only to maintain the perception of independence. 3.63 4.00 3.56 4.00 3.43 3.00 Providing NAS to an audit client by a separate department gives the auditor more credibility. 3.38 3.00 3.42 4.00 3.05 3.00 The provision of NAS impairs audit quality. 2.39 2.00 2.58 2.00 2.29 2.00 The provision of NAS to an audit client reduces the probability of a threat to switch auditor. 4.2 Qualitative Findings The majority of interviewees did not perceive any reduction in the audit quality where NAS was provided. Indeed, they confirmed that they would have recommended their clients to use their firms for such services, arguing that the audit firm’s experience and knowledge of their businesses would result in better advice being obtained. Other interviewees argued that they would prefer to see audit firms provide NAS to their clients because it would ensure that the standard applied to the audit would be higher and that a better audit opinion would result. On this issue, a Big Four managing partner remarked: “Sometimes, yes, we suggest to clients that they come to us for NAS because accountants are the best people to provide these services because they already know that company’s history and business … and by providing NAS as well, the auditor learns more about the client and can give more informed advice.” The questionnaire survey findings, revealed that only a minority of oil company (23.7%) and audit firm (16.9%) respondents either agreed or strongly agreed with the statement: The provision of NAS impairs audit quality. Similarly, most oil companies and audit firms (72%) disagreed that audit quality would be impaired if the auditor were to provide NAS to audit clients, and pointed out that both parties would benefit from the arrangement. It was indicated that the provision of NAS would complement audit duties, as the auditor would be exposed to the audit client’s business transactions and subsequently gain a better understanding of the client’s overall enterprise. An audit supervisor in a local audit firm affiliated to an Arab audit firm summarised the view by saying: “If you can gain more information about your client, you can give a better audit opinion. Your appreciation of the client’s business is greater so you can consider all the factors, and then provide a better service.” Moreover, a Big Four auditor commented: “An audit could be of a higher standard overall because the auditor is also providing NAS. 4.1.2 The Perceptions of NAS Based on Type of Audit Firms 3.50 4.00 3.63 4.00 3.48 5.00 ,* indicates distribution of responses is significantly different at the 1%, 5% levels, respectively using the Kruskal-Wallis test. 4.2 Qualitative Findings In this case the auditor would not want to lose the client by not giving good quality all-round service so the audit might be of an even higher standard … but actually, the auditor knows the company better than other consultants, and it is true that the company would want someone who did know their business and their systems … they wouldn’t have to spend a long time learning everything about the company because they already have all the background.” Another local audit supervisor added: “The auditor is in a unique position of understanding the client’s financial operations and its business better than 176 International Journal of Business and Management Vol. 8, No. 14; 2013 www.ccsenet.org/ijbm anybody else. I don't have any evidence that would lead me to be concerned that the audit is compromised.” se. I don't have any evidence that would lead me to be concerned that the audit is compromised.” y y y p A small minority of oil companies and audit firms (28%) interviewed did, however, believe that audit quality would be impaired by the joint provision of audit and NAS to audit clients. Their argument was that auditors would build close relationships with their clients with the aim of securing other business opportunities and may use audit services as a ‘loss leader’ to leverage the audit services into other consulting engagements. This belief was well summarised by an internal auditor of one of the oil companies, who argued that: A small minority of oil companies and audit firms (28%) interviewed did, however, believe that audit quality would be impaired by the joint provision of audit and NAS to audit clients. Their argument was that auditors would build close relationships with their clients with the aim of securing other business opportunities and may use audit services as a ‘loss leader’ to leverage the audit services into other consulting engagements. This belief was well summarised by an internal auditor of one of the oil companies, who argued that: “Not all the NAS can impair auditors’ independence, as there are some certain types that can impair it and others would not. 4.2 Qualitative Findings You would have to have an independent person performing the audit, one separate from the person who recommended the system.” However, an audit supervisor from one of Big Four firms stressed the reality of the situation, saying: “ In the smaller firms, separation can not happen and the auditor is less reliable as a consequence … In the larger firms, they are more specialised but in the smaller firms, you have the auditors providing a lot of tax expertise and then that is audited by them. I think that is where you have the least independence.” Also, some of the interviewees indicated that this kind of arrangement would ease information exchange, which may not be achieved if NAS were provided by other firms. Perhaps, audit firms could effectively utilise their personnel who have a good understanding of the client’s business to speed up the process and subsequently produce a high quality financial statement. In summary, it can be seen that the general consensus of opinion among interviewees was that the provision of NAS alongside audit services was only a serious threat to audit quality and independence where the two types of service were performed by the same department. However, the minority who disagreed with this perspective, believed that some types of NAS, especially those including decision-making, impaired audit quality and independence. Nonetheless, this result is not consistent with that of Shockley (1981) as his findings did not support the need for a separation of the consulting and audit function. 4.2 Qualitative Findings For instance, if the service provided is not related to decision making, independence would not be an issue.” Because the majority of interviewees had a positive attitude towards the provision of NAS by audit firms, it led to a discussion on the manner in which they perceived that such services were provided by audit firms. In all cases, they had assumed that both roles were performed by separate departments. A number of them stressed the importance of such separation as a means of ensuring that the audit quality was not compromised. The vast majority of oil companies and audit firms (96%) interviewed agreed that audit quality and auditor independence would not be threatened if the provision of audit and NAS to an audit client was performed by staff from different departments, a perception that is consistent with the results from the questionnaire survey. The interviews disclosed that this mode of NAS provision would allow segregation of duties in monitoring activities, widely known as the ‘Chinese Wall’ (Mikol and Standish, 1998; Sori, 2005). It was believed that different partners would handle the different departments, which would result in greater monitoring activities and mean that the audit division might not lose sight of the material issues On this issue, one of the auditors in one a Big Four firm remarked: “I would be worried if NAS and audit services were being provided by the same department because this is too close, and not good for the auditor’s independence, but I think this does not happen. Different departments offer these services.” The importance of the demarcation of functions was also confirmed by a managing partner of local audit firm, who remarked: “I would see the separation of functions as very important and not prejudicing their independence … You would assume that the audit firm is very rigorous in the separation of the two roles. You would have to have an independent person performing the audit, one separate from the person who recommended the system.” “I would see the separation of functions as very important and not prejudicing their independence … You would assume that the audit firm is very rigorous in the separation of the two roles. 5. Summary and Review of Findings The provision of non-audit services (NAS) that has been debated in the literature as a cause of impaired audit quality, was considered by the majority of this study’s respondents not to be a serious concern, and the statement that the provision of NAS would impair the level of audit quality did not secure very much agreement. In fact, the majority of respondents believed that the provision of NAS to audit clients gives the auditor more experience of the client’s industry as well as more access to the client’s accounting system, thereby functioning in a complimentary capacity. Hence, such an arrangement was considered to increase the auditor’s overall client knowledge, and therefore enhance the probability that problems would be discovered. In such circumstances, the provision of NAS was argued to have the potential for actually increasing audit quality. Furthermore, from the auditor’s perspective, 177 International Journal of Business and Management www.ccsenet.org/ijbm Vol. 8, No. 14; 2013 the provision of NAS was considered favourable, since the arrangement has the potential to increase a client’s dependence on its auditor, thereby reducing the credibility of the switch threat, although such dependence, when viewed from a different perspective, could be perceived as a negative feature. Another commonly-mentioned advantage of the joint provision of audit and NAS, is the benefit arising from knowledge spillovers, which are transfers of knowledge that may occur when NAS are provided by the incumbent auditor It was, however, agreed by the majority of the respondents that the maintenance of audit quality would require the provision of NAS to come from a separate department from the audit department. This degree of faith was interesting, and testified to the respondents’ belief in the ‘Chinese Wall’ approach, which works to minimise communication between the audit and NAS providers despite them being under the ultimate control of the same organisation. Given that the government licenses individual auditors, and these people have to ensure that their personal image is protected, it is argued that auditors would not behave in a way as to compromise this, and in fact, welcome greater monitoring of their own activities. The general feeling among interviewees was that they would recommend their own firms to their clients for the provision of NAS, since they believed this would not result in any reduction in the audit quality, and indeed, enhance it. 5. Summary and Review of Findings In respect of the provision of NAS, it was felt by the majority of respondents that whilst this was essentially considered by them to be a good thing, audit firms should, nonetheless, disclose the type and amount of NAS provided to their audit clients, and likewise, oil companies should disclose in their annual reports, the amount of audit and NAS fees paid. Among the standards to be set by the LAAA, should be the requirement for the separation of duties in respect of the provision of audit services and NAS, and for audit firms’ percentage of NAS income from a single client not to exceed 25% of the fees obtained from that client. Additionally, the LAAA should require audit firms to disclose the type and amount of audit and NAS fees obtained from all clients. References Abdel-Khalik, A. (1990). The Jointness of Audit Fess and Demand for MAS: A Self- Selection Analysis. 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This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). 181
https://openalex.org/W2971910704	https://prism.ucalgary.ca/bitstream/1880/110879/1/12889_2019_Article_7521.pdf	English	null	Methods for detecting seasonal influenza epidemics using a school absenteeism surveillance system	BMC public health	2,019	cc-by	12,946	Ward et al. BMC Public Health (2019) 19:1232 https://doi.org/10.1186/s12889-019-7521-7 Ward et al. BMC Public Health (2019) 19:1232 https://doi.org/10.1186/s12889-019-7521-7 Abstract Background: School absenteeism data have been collected daily by the public health unit in Wellington-Dufferin- Guelph, Ontario since 2008. To date, a threshold-based approach has been implemented to raise alerts for community-wide and within-school illness outbreaks. We investigate several statistical modelling approaches to using school absenteeism for influenza surveillance at the regional level, and compare their performances using two metrics. Methods: Daily absenteeism percentages from elementary and secondary schools, and report dates for influenza cases, were obtained from Wellington-Dufferin-Guelph Public Health. Several absenteeism data aggregations were explored, including using the average across all schools or only using schools of one type. A 10% absence threshold, exponentially weighted moving average model, logistic regression with and without seasonality terms, day of week indicators, and random intercepts for school year, and generalized estimating equations were used as epidemic detection methods for seasonal influenza. In the regression models, absenteeism data with various lags were used as predictor variables, and missing values in the datasets used for parameter estimation were handled either by deletion or linear interpolation. The epidemic detection methods were compared using a false alarm rate (FAR) as well as a metric for alarm timeliness. Results: All model-based epidemic detection methods were found to decrease the FAR when compared to the 10% absence threshold. Regression models outperformed the exponentially weighted moving average model and including seasonality terms and a random intercept for school year generally resulted in fewer false alarms. The best-performing model, a seasonal logistic regression model with random intercept for school year and a day of week indicator where parameters were estimated using absenteeism data that had missing values linearly interpolated, produced a FAR of 0.299, compared to the pre-existing threshold method which at best gave a FAR of 0.827. Conclusions: School absenteeism can be a useful tool for alerting public health to upcoming influenza epidemics in Wellington-Dufferin-Guelph. Logistic regression with seasonality terms and a random intercept for school year was effective at maximizing true alarms while minimizing false alarms on historical data from this region. Keywords: Absenteeism surveillance system, Influenza, Seasonal logistic regression, Disease modelling, Epidemic detection Correspondence: mward06@uoguelph.ca 1Department of Mathematics and Statistics, University of Guelph, Stone Road, N1G 2W1 Guelph, Canada Full list of author information is available at the end of the article © The Author(s). Background seasonal influenza outbreak or epidemic, or measuring correlation between absenteeism and influenza. The stud- ies that attempt to detect the start of an outbreak generally use techniques from one of three categories: thresholds (either fixed or individualized), models adapted from techniques traditionally used in statistical process control, and regression models. Influenza is one of the leading causes of death in Canada, with seasonal influenza resulting in 6000 - 20,000 hos- pitalizations and an average of 11.3 deaths per 100,000 population each year [1, 2]. Early detection of the onset of a seasonal influenza epidemic at the community level is important so that appropriate public health intervention measures can be taken. For example, the World Health Organization suggests several behavioural interventions for preventing the spread of influenza A (pH1N1) such as staying at home when ill and hand-washing [3]. Pub- lic health units can increase communications of these messages if they receive warning sufficiently early in an influenza epidemic [3], which may mitigate the sever- ity of the epidemic due to public awareness. Ideally, the timing of this messaging should occur close enough to influenza season so that the public feels there is cause to follow suggestions, but as early as possible to maximise the effectiveness of mitigation measures. In the first category, a study conducted in Quebec dur- ing the 2009 pH1N1 pandemic found the 10% threshold across all schools failed to detect outbreaks early enough for an intervention to be executed, either at the school- level or for the surrounding community [15]. The study also found that during an early wave of the pandemic, only around one third of schools met the absenteeism thresh- old, despite it being unlikely that none of the remaining schools had experienced an outbreak, indicating the 10% threshold may be too high for many schools and is not effective for early outbreak detection [15]. Mann et al. (2011) took a more individualized approach where an alarm could be raised when a school either surpassed 8% absenteeism or if absenteeism exceeded one standard deviation of the previous 30 day mean [16]. This study attempted to catch school-level outbreaks, however of the 89 schools that produced an alarm only nine were truly in the midst of an outbreak [16]. Background Syndromic surveillance uses non-traditional indicators, such as over-the-counter medication sales [4, 5], ambu- lance dispatch data [6–8], and emergency department data [9, 10] for early detection of outbreaks or epidemics. Indirect health-related indicators such as these have been found to improve timeliness (or sensitivity) of surveillance systems, often resulting in an epidemic being detected sooner than it would have been if only clinical data were monitored [11]. However, the non-specific nature of this type of data can also lead to an increase in alarms that are not related to the disease of interest, reflecting decreased specificity for the surveillance system [11]. Therefore, the challenge of syndromic surveillance lies in finding an epi- demic detection method that can produce a manageable number of false positive alarms, while still remaining sen- sitive enough to provide public health units with warning far enough in advance of the reporting of laboratory- confirmed cases to be useful. Examples of studies that use statistical process con- trol techniques include Besculides et al. (2005), who used a cumulative sum (CUSUM) method to monitor absen- teeism in New York City [17]. They examined three school years’ worth of absenteeism data and were able to detect changes in absenteeism for several community-wide epi- demics of influenza-like illness [17]. However, they con- cluded that the absenteeism data still resulted in too much noise and did not ultimately recommend its implemen- tation [17]. Similarly, Xu et al. (2017) applied CUSUM models to absenteeism from four schools in Tianjin, China [18]. They were able to detect 10 within-school out- breaks over the course of two school years, although they did not report the number of false alarms that were raised [18]. Abstract 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Correspondence: mward06@uoguelph.ca Correspondence: mward06@uoguelph.ca 1Department of Mathematics and Statistics, University of Guelph, Stone Road, N1G 2W1 Guelph, Canada Full list of author information is available at the end of the article Correspondence: mward06@uoguelph.ca 1Department of Mathematics and Statistics, University of Guelph, Stone Road, N1G 2W1 Guelph, Canada Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ward et al. BMC Public Health (2019) 19:1232 Ward et al. BMC Public Health (2019) 19:1232 Page 2 of 16 Ward et al. BMC Public Health (20 Methods The goal of the methods discussed in this section was to detect a seasonal influenza epidemic within WDG ear- lier than it would be detected by waiting for reports of laboratory-confirmed influenza cases. For the purpose of this study, a seasonal influenza epidemic is defined as beginning when more than one case is observed within a seven-day period for the first time in any given influenza season. It is meant to reflect the increase in the occurrence of cases that occurs as the peak of an influenza season approaches, rather than the beginning of an influenza season. The school absenteeism surveillance program at Wellington-Dufferin-Guelph Public Health (WDGPH) has utilized an on-line form to collect daily counts of students absent from schools in the WDG region since 2008. An absenteeism-based influenza surveillance program was piloted by WDGPH independently of the federal government in 2008 and became more established during the during the 2009–2010 pH1N1 epidemic. When absenteeism within a reporting school reaches 10%, WDGPH follows up with the school in question to investigate whether the increased absenteeism is related to illness and to advise on mitigating measures if it is. Cause of absenteeism is unavailable for most schools so WDGPH must use total all-cause absenteeism rather than symptomatic absenteeism with the threshold. Data from WDGPH were available from January 2008; however, at that time point, the 2007–2008 influenza sea- son had already begun. Thus, the study period covered September 2008 to June 2018, not including the 2009– 2010 school year. This year corresponded to the 2009 pH1N1 pandemic and was not used because, unlike in other years, the start of the epidemic preceded the start of the school year. Nine school years/ influenza sea- sons remained available to which the epidemic detection methods could be applied, although there were a lim- ited number of elementary schools and no secondary schools reporting during the 2008–2009 school year as the program was still being piloted. The 10% threshold used by several public health depart- ments for absenteeism-based syndromic surveillance is an arbitrary threshold that is generally used for detection of any significant or widespread epidemic or outbreak within communities and schools. Influenza surveillance with school absenteeism School absenteeism surveillance is of particular impor- tance to public health because children aged five to fif- teen years have been found to have the highest rates of influenza infection [12], and children under eighteen years old are the most likely family members to transmit influenza to the home [13]. Since most children spend a significant part of their time at school, schools likely play a significant role in spreading influenza to the wider com- munity [13]. Furthermore, school absenteeism has been found to be significantly higher during influenza season than during the rest of the winter [14]. The final main modelling technique used for the early detection of seasonal influenza epidemics based on school absenteeism data is regression. A negative binomial regression model for predicting influenza epi- demics using non-cause specific absenteeism in New York City was previously found not to be useful at giving advance notice [19]. However, Zhou et al. (2015) com- pared five statistical process control methods with linear and Poisson regression to see which one would provide the optimal alarm system for influenza where they use U.S. national syndromic (but not absenteeism) data. They reported that the regression models somewhat improved timeliness and sensitivity, especially for high influenza counts [20]. Several studies have used different epidemic detection techniques within influenza surveillance systems using school absenteeism data. These studies are often inter- ested in either raising an alarm for the beginning of a Page 3 of 16 Ward et al. BMC Public Health (2019) 19:1232 Ward et al. BMC Public Health and proposed methods for detecting seasonal influenza epidemics using school absenteeism. The models used in the study were evaluated against influenza data from the community, as historically, there appeared to be no notice- able correlation between the number of cases of any other reportable disease in WDG and levels of school absen- teeism. However, in most years there appeared to be a noticeable peak in absenteeism shortly before the peak in the incidence of reported cases of laboratory-confirmed seasonal influenza within the community. Influenza surveillance in Wellington-Dufferin-Guelph The Wellington-Dufferin-Guelph (WDG) region in Ontario, Canada covers two counties (Wellington, includ- ing the City of Guelph, and Dufferin). It encompasses approximately 4147 km2 and had a recorded population of 284,461 people in 2016 [21, 22]. At the time, the respective population densities of Dufferin and Wellington counties were 41.5 people/km2 and 83.7 people/km2 [21, 22]. Influenza surveillance with school absenteeism Nearly half of the total population resided within the City of Guelph (in Wellington County), which covers 87 km2 and contains 53 elementary and secondary schools. Outside of Guelph, WDG is largely rural with six towns and nine townships. Methods While some correlation has been noticed between the trends in absenteeism during an influenza season and trends in local school absenteeism both in WDG and elsewhere, there is no evidence that the 10% threshold is the best measure of unusual dis- ease activity in a community or school. Further, the 10% threshold does not take into account the varying baseline absenteeism levels between different schools. For these reasons, there is a need to develop statistically sound approaches to using absenteeism data as a predictor of school or community disease activity. In the WDG region school years typically begin during the first week of September, and never before Septem- ber 1st, so for consistency in analysis the school year was assumed to begin on September 1st each year. All data cleaning, analysis, and visualization was performed in R version 3.5.0 [24]. Data sources Absenteeism data No data were available for days when students were not required to attend school: weekends, statutory and school board holidays, and school breaks (for example, winter holidays, March break, and the sum- mer holidays). In addition, schools that reported on fewer than five days throughout the study period were omit- ted from analysis. Extreme points where absenteeism was greater than 50%, and observations where elemen- tary school population sizes were less than 45 or greater than 820 and secondary school population sizes were less than 443 or greater than 1902 (the smallest and largest consistently reported population sizes), were assumed to represent data entry errors and were therefore deleted from the dataset. did not provide this additional information. From the all-cause absenteeism, percentage absenteeism was calcu- lated using total student population at the school as the denominator. Over the study period, 90 unique elemen- tary schools and 14 unique secondary schools reported absenteeism data on at least one day. The number of schools reporting for a given day ranged from only one school to more than 40 schools in October-December 2010. The median number of schools that reported on school days was 13. No data were available for days when students were not required to attend school: weekends, statutory and school board holidays, and school breaks (for example, winter holidays, March break, and the sum- mer holidays). In addition, schools that reported on fewer than five days throughout the study period were omit- ted from analysis. Extreme points where absenteeism was greater than 50%, and observations where elemen- tary school population sizes were less than 45 or greater than 820 and secondary school population sizes were less than 443 or greater than 1902 (the smallest and largest consistently reported population sizes), were assumed to represent data entry errors and were therefore deleted from the dataset. The reference date was the report date for the second of two laboratory-confirmed influenza cases reported within seven days of each other for the first time in an influenza season The reference date was the report date for the second of two laboratory-confirmed influenza cases reported within seven days of each other for the first time in an influenza season coefficient was used to examine strength of relation- ship between averaged elementary and secondary school absenteeism and influenza case counts. Epidemic detection methods Epidemic detection methods were applied to data prospectively. The first available year of data was used to train the models, and therefore was not used in model evaluation. Each school year was evaluated using models that had been trained on all data that temporally preceded that year. The school absenteeism featured missing val- ues as described in the “Data sources” subsection. Both deletion and linear interpolation with the zoo package [28] were considered for treating these missing values. In addition, there were cases where a school reported more than once on a single date. We explored using either the maximum or the median of the reported values for that school to replace the absenteeism observations for those dates and schools. Every combination of the types of miss- ing value and multiple entry handling was considered for each of the epidemic detection methods described in this section. Data sources Absenteeism data Cross-correlation up to 15 lags was also calculated using the ccf function in R, with absenteeism and influenza counts ranked to approximate Spearman correlation. Differences in distribution between elementary and secondary school absenteeism were examined using the Mann-Whitney-Wilcoxon and Kolmogrov-Smirnov tests and, since both test results indicated a significant differ- ence in the two distributions with a p-value < 2.2 × 10−16, data from elementary and secondary schools were analyzed separately when fitting models. Different aggre- gation methods of absenteeism were explored, including using the average absenteeism of all schools of one type that reported on a day, using the average of the three most frequently reporting schools, and using data only from the most frequently reporting school. Sample means were calculated for each aggregation, along with 95% boot- strapped percentile intervals for the population (regional) means. Autocorrelation was accounted for in interval cal- culation; observations were sampled from blocks where block size was chosen from a geometric distribution with mean 20 and a sample size of 10000 [25], using the boot package in R [26, 27]. Data sources Absenteeism data This study evaluates several model-based alternatives to the 10% threshold for raising an epidemic alarm using school absenteeism data, with the goal of reducing these false alarms. The models include a statistical process con- trol method, the exponentially weighted moving average (which approximates a Shewart chart or a CUSUM chart when different parameters are used [23]), as well as varia- tions of logistic regression. In addition, two new metrics, false alarm rate (FAR) and accumulated days delay (ADD), are introduced to allow for epidemic detection method performance evaluation and comparison of the existing Elementary and secondary schools within the WDG area are asked to report their absences to WDGPH each school day by 3:00 p.m. using an on-line form. The data obtained from WDGPH contained anonymized school identifica- tion numbers, the school population size, and the number of students absent for each day. Data from a limited num- ber of schools also included the number of students absent due to illness, and specifically due to respiratory, gas- trointestinal, and other symptoms; however, most schools Ward et al. BMC Public Health (2019) 19:1232 Ward et al. BMC Public Health (2019) 19:1232 Ward et al. BMC Public Health (2019) 19:1232 Page 4 of 16 Page 4 of 16 Table 1 Reference dates representing the beginning of each seasonal influenza epidemic investigated in the study School Year Influenza Epidemic Reference Date 2008–2009 January 20th, 2009 2010–2011 December 14th, 2010 2011–2012 January 9th, 2012 2012–2013 October 26th, 2012 2013–2014 November 27th, 2013 2014–2015 December 8th, 2014 2015–2016 November 17th, 2015 2016–2017 December 15th, 2016 2017–2018 December 6th, 2017 The reference date was the report date for the second of two laboratory-confirmed influenza cases reported within seven days of each other for the first time in an influenza season Table 1 Reference dates representing the beginning of each seasonal influenza epidemic investigated in the study did not provide this additional information. From the all-cause absenteeism, percentage absenteeism was calcu- lated using total student population at the school as the denominator. Over the study period, 90 unique elemen- tary schools and 14 unique secondary schools reported absenteeism data on at least one day. The number of schools reporting for a given day ranged from only one school to more than 40 schools in October-December 2010. The median number of schools that reported on school days was 13. 10% threshold method The influenza dataset contained information about influenza cases in WDG that were laboratory-confirmed, and the dates on which WDGPH was notified of a case (Report Date) were used in analyses, with report date being chosen because this represented the date on which WDGPH first becomes aware of a case of influenza in the normal course of events. The dataset comprised usable data from nine influenza seasons, with seasonal epidemic start dates (reference dates) that ranged from late October to late January (Table 1, Fig. 1). The Spearman correlation The method currently in use by WDGPH is a 10% absen- teeism cut-off for raising an alarm for an outbreak (which may be related to influenza or a different disease). As it is currently used, whenever 10% or greater of the popu- lation at an individual school is absent, WDGPH follows up with that school to investigate possible illness. We explored raising an alarm for a region-wide epidemic with the 10% threshold method by using aggregated rather than individual school absenteeism data. Therefore an alarm Ward et al. BMC Public Health (2019) 19:1232 Page 5 of 16 Ward et al. BMC Public Health Fig. 1 Influenza and absenteeism in WDGPH. Average absenteeism and laboratory-confirmed influenza cases (“Flu Cases”) reported to WDGPH for the WDG region from January 2008 to June 2018 “Data sources” subsection, and either untransformed, log- transformed, or square root-transformed. Additionally, t indexes the days on which absenteeism data is available. For the first observed day, zt−1, or z0, was set to be the expected mean μ0 absenteeism when the process is “in- control” [32]. Here, μ0 was set as the mean absenteeism of the training years and thus was reset for each school year. was raised if absenteeism averaged across certain schools reached 10%. The threshold approach cannot be used with interpolated missing values as there are no parameters to be estimated, and it cannot incorporate any additional data or factors to aid in predicting the start of an epidemic. Exponentially weighted moving average Originally developed for use in econometrics, the expo- nentially weighted moving average (EWMA) and other statistical process control methods have been used in sev- eral influenza surveillance studies [29, 30]. The average of an epidemic-related variable is calculated, where obser- vations in the past are given successively lower weights for determining the current test statistic [31]. Weights are represented by a parameter, λ, which can take values between 0 and 1 [31]. A value of λ close to 0 approximates a CUSUM chart, where all past observations are given equal weighting, while a λ value close to 1 approximates a Shewart chart, in which only the most recent observation is considered [23]. This gives the equation: g y y The variance of zt can be found by expanding Eq. 1 to obtain: zt = λ t−1 j=0 (1 −λ)jxt−j + (1 −λ)tz0, (2) (2) and taking the variance of Eq. 2. This gives: σ 2 zt = σ 2 λ 2 −λ [ 1 −(1 −λ)2t] where σ 2 is the in-control variance of xt. It was fixed at 1 for simplicity. The EWMA statistic is compared to a con- trol limit and when zt falls outside the bound of the control limit, an alarm is raised. Typically, both lower and upper control limits would be used. However, in the context of biosurveillance only the upper limit (UCL) is meaningful. The UCL is given by: zt = λxt + (1 −λ)zt−1 (1) (1) where xt is the value of the observed variable for day t, zt is the EWMA statistic for day t, and zt−1 is the EWMA statistic for the day before day t. In this study, x was absen- teeism aggregated in one of the ways described in the UCL = μ0 + kσ 2 zt and an alarm was raised when zt > UCL. The parameters λ and k can be chosen theoretically to fix the error rate Ward et al. BMC Public Health (2019) 19:1232 Ward et al. BMC Public Health (2019 Page 6 of 16 account the seasonal pattern that influenza tends to fol- low. Thus, a second regression model that captures the seasonality pattern through the inclusion of trigonometric functions as covariates was considered [35]. The seasonal logistic regression model is given by: and make the UCL essentially equivalent to a one-sided 95% confidence limit. However, Buckeridge et al. Exponentially weighted moving average (2005) found that in practice this results in unacceptable false alarm rates for most biosurveillance systems [33]. There- fore, in our study a range of values for the two parameters were used to fit the model, and the optimal values of λ and k were selected based on their performance on prediction with the evaluation metrics described in the “Evaluation metrics” subsection. Twenty values of λ between 0.05 and 1, and 20 values of k between 0.5 and 10 for untransformed data or 0.05 and 1 for transformed data were considered. logit(ρt) = β0 + β1xt + β2xt−1 + ... + βl+1xt−l + βl+2sin 2πt∗ T∗ + βl+3cos 2πt∗ T∗ , (4) (4) where t∗represents the calendar day of the year on which xt was observed, and T∗equals 365.25. When added together, the sine and cosine terms represent the har- monic motion of the response across the time axis, with a 1 Logistic regression models Distributed-lag regression models are used to analyse time series where the predictor variable is expected to correlate with a change of the response variable over a distributed period of time [34]. In the context of this study, it is likely that the first true influenza cases in the community preceded the first laboratory confirmed case each year, since not everyone who contracts an influenza infection will seek treatment from healthcare professionals [12]. In addition, there is a delay between the time when the first symptomatic case seeks health care and when results of the laboratory test for influenza are available. Because of this, it would be expected that an increase in absenteeism would be observed several days in advance of the report of any corresponding laboratory-confirmed cases to public health, and a distributed lag model would be appropriate to capture this phenomenon. Although EWMA models also take past observations of absenteeism into account, using a regression model allows for the inclusion of addi- tional predictors. period of T∗and amplitude of β2 l+2 + β2 l+3 1 2 [35]. period of T∗and amplitude of β2 l+2 + β2 l+3 2 [35]. period of T∗and amplitude of β2 l+2 + β2 l+3 2 [35]. In addition to the lagged absenteeism predictors, ver- sions of these models including an indicator variable for day of the week (DOW) were also considered, to account for possible weekly effects such as increased absenteeism each Monday or Friday. The parameters for the above models were estimated using the glm function in R. For each of these regres- sion models, as well as the mixed logistic regression and GEE models described below, lag lengths of 0 (only the current day’s absenteeism used) to 15 were considered. Alarms were raised when the predicted probability of at least one case being reported to WDGPH surpassed a defined threshold, . Eleven possible thresholds between 0.1 and 0.6 were considered. Autoregressive GEE models Alarms were raised by EWMA models if the EWMA statistic, zt, surpassed the UCL, and by the regres- sion models if the predicted probability of at least one laboratory-confirmed case occurring on day t surpassed the probability threshold . Ideally, an alarm would be raised one to two weeks ahead of the start of a seasonal epidemic, so alarms were considered to be true if they occurred in the 15 calendar day period between the refer- ence day of the epidemic and 14 days prior to the reference day, inclusive. An alarm raised prior to the reference day was considered to be false. Alarms raised between the day after the reference day and the final day of the school year were ignored. An alternative to generalized linear mixed models is to use a generalized estimating equation (GEE), which models data at the population level as opposed to the individ- ual level [38]. School year was still included as a random effect, but a first order autoregressive correlation struc- ture was also specified [38]. Due to the infectious nature of influenza, it is more likely there will be a new case if there has already been a case in the preceding days. Absen- teeism follows a similar pattern, as illness spreads from child to child, so there are reasons to believe that obser- vations which are closer together will be more highly cor- related than those further apart and this can be modelled by a correlation structure. Two first order autoregressive GEEs were investigated in this study. The first is given by: Two metrics were used to optimize model parameters and evaluate the performance of the epidemic detection methods. The first, FAR, was calculated as: logit(μtj) = β0 +β1xtj +β2x(t−1)j +...+βl+1x(t−l)j. (7) FAR = nf nf +1, if a true alarm was raised 1, if no true alarms were raised, (9) FAR = nf nf +1, if a true alarm was raised 1, if no true alarms were raised, (9) Instead of estimating an individual probability of at least one case occurring, the model predicts the mean probabil- ity averaged across all observations with the same absen- teeism values [39]. Therefore, μj represents the mean response for the population that has the same absenteeism pattern. The second model adds sine and cosine terms to create a seasonal variation on Eq. Evaluation metrics For the purposes of this study, seasonal influenza epi- demics were defined to begin when WDGPH was notified of two cases within a seven day period for the first time within an influenza season. Therefore these two cases could have been reported to WDGPH on the same day, or up to six days apart from each other. The reference day of the epidemic was the date of the second of these cases. Note that for the purposes of our analyses, the start of a seasonal influenza epidemic (i.e., the “reference day”) was defined differently from the usual definition of the start of an influenza season (the reporting of the first laboratory- confirmed case). Instead, the start of a seasonal influenza epidemic was defined as the report date of the second of two cases which had been reported to public health within seven days of each other. This was done in order to reflect the approaching peak of the season, as opposed to the rel- atively sporadic cases that often occur early in an influenza season. logit(πtj) = β0 + β1xtj + β2x(t−1)j + ... + βl+1x(t−l)j + βl+2sinj 2πt∗ T∗ + βl+3cosj 2πt∗ T∗ + γj, (6) (6) with again γj ∼ N(0, τ 2). This model attempted to account for both the possible dependence of observations on school year as well as the seasonality of influenza. To fit the mixed models, we used the glmer command from the lme4 package in R [37]. Because the school year being modeled needed to be represented in the training dataset in order to estimate the intercept, data from September of the year of interest were included in the training data for each random intercept model. Mixed logistic regression models Distributed-lag models use the value of a predictor vari- able for day t as well as for each day until l (the desired number of lags) days before day t. In this study, the out- come of interest was whether or not at least one case of (laboratory-confirmed) influenza would be reported to WDGPH on a given day, and thus logistic regression was used. Under this model the log-odds that at least one case occurs on day t is given by: To account for the effect of school years, models that included a random intercept for school year were also considered. Mixed regression models allow for intracor- relation among observations at a given measurement unit, such as multiple observations within an individ- ual, a geographical location, or a time period [36]. For this study, including a random intercept for the observed absenteeism with a given school year acknowledges that absenteeisms, or their relationship to influenza may be correlated or similar within one year but vary over differ- ent years. Residual variance is divided into one component for the yearly level, and one component for the daily level [36]. logit(ρt) = log ρ 1 −ρ =β0+β1xt+β2xt−1+...+βl+1xt−l, (3) (3) Two mixed logistic regression models were examined in this study. The first added a random intercept for year to Eq. (3), giving: where ρ is the probability of at least one case occur- ring on day t, t = 1, . . . , T; T is the total number of days with absenteeism data available; xt is the percent- age of students absent on the given day, and xt−i gives the percentage of students absent on the ith day with absen- teeism data available before day t. Although this model accounts for the potential delay between influenza cir- culation and reporting, it does not specifically take into logit(πtj) = β0+β1xtj+β2x(t−1)j+...+βl+1x(t−l)j+γj. (5) The random component of the intercept is represented by γj and follows a normal distribution with mean 0 and variance τ 2, and j indexes school year. (2019) 19:1232 Page 7 of 16 Page 7 of 16 Ward et al. BMC Public Health (2019) 19:12 The second mixed model was an adaptation of Eq. (4): The second mixed model was an adaptation of Eq. (4): Autoregressive GEE models (7): (9) where nf is the number of false alarms produced during that school year. The FAR produces a value between 0 and 1, where 0 would indicate that no false alarms and at least one true alarm were raised in a year. An FAR value of 1 or close to 1 would indicate that no true alarms were raised in a year, or that there was a large number of false alarms. logit(μtj) = β0 + β1xtj + β2x(t−1)j + ... + βl+1x(t−l)j + βl+2sinj 2πt∗ T∗ + βl+3cosj 2πt∗ T∗ . (8) The second metric was ADD. The ADD was used to give a sense of timeliness for true alarms. It was calculated as: (8) ADD = τoptimal −τobs, if a true alarm was raised τmax, if no true alarms were raised (10) ADD = τoptimal −τobs, if a true alarm was raised τmax, if no true alarms were raised ADD = τoptimal −τobs, if a true alarm was raised τmax, if no true alarms were raised max (10) In both equations, j indexes school year, and t, t∗, and T∗are as previously defined. The geeglm function from the geepack package in R was used to fit the GEE models [38]. (10) where τoptimal is 14 (or the ideal number of calendar days of advance notice before an epidemic reference day) Page 8 of 16 Ward et al. BMC Public Health (2019) 19:1232 Ward et al. BMC Public Health summary statistics for the different school aggregations considered in the epidemic detection methods are pre- sented in Table 2. and τobs is the number of calendar days before the epi- demic reference day that the first true alarm raised for that season was declared. For example, if two true alarms were raised prior to the seasonal epidemic one year, one 12 days before the reference day and one 10 days prior, τobs = 12. In the event that no true alarms were raised, a large value was assigned to represent the system rely- ing only on laboratory-confirmed cases. This value, τmax, was set to the number of days between the first day of the school year for which absenteeism data were available and the epidemic reference day, and so differed by year. See Fig. 2 for an illustration of the definitions of terms used in computing ADD. Epidemic detection methods Using the maximum versus the median reported num- ber of absences to replace entries when a school reported more than once within one day had little to no effect in most models, therefore only the results based on the maximum reported absence are presented in this section. Autoregressive GEE models Spearman correlation between influenza counts and average absenteeism was fairly weak, particularly for sec- ondary schools. For elementary school absenteeism the correlation was 0.371, and for secondary school absen- teeism it was 0.161. Cross-correlation was highest when elementary school absenteeism lagged behind influenza counts by six days (0.405) and when secondary school absenteeism was lagged by 11 days (0.181). After removing missing and misreported values, the data aggregation type that had the most days of usable data was absenteeism averaged across all schools (ES.SS- allavg) with 1709 school days available out of the 3223 total calendar days in the study period. The average for all elementary schools (ES-allavg) had a similar number of school days available (1697), while the aggregation type with the fewest usable days was the top reporting sec- ondary school (SS-top) with 1133 days available. This school did not begin reporting until the 2010–2011 school year and stopped reporting before the 2017–2018 school year. The top reporting elementary school (ES-top) had 1384 usable days. Ideally, an epidemic detection method would have an ADD of 0, meaning a true alarm was observed 14 days before the epidemic reference day. An ADD of 14 would mean that the first true alarm was observed on the epi- demic reference day. An ADD greater than 14 indicates no true alarm was observed that school year. Therefore any ADD value less than 14 indicates that the method was able to provide an alarm prior to when the epidemic would have been declared based on laboratory-confirmed influenza reports alone. Metrics were calculated for each school year of every epidemic detection method, and then were averaged over all school years. For model-based methods, estimated parameters were chosen based on minimizing the average FAR. The models fitted with those optimized estimated parameters were then compared primarily by looking at which method could produce the lowest FAR, and among models that produced similar FARs, which method had the timeliest alarms as indicated by a low average ADD value. 10% threshold method The 10% threshold method applied to the aggregated absenteeism data had limited ability to accurately identify epidemics ahead of laboratory confirmation. At best, this method produced an FAR of 0.661 with a corresponding ADD of 28.75 days when the average absenteeism across all secondary schools was used (Table 3). Allowing an Preliminary data analysis Mean daily all-cause absenteeism was 5.94%[95% CI = (5.58%, 6.31%)] for elementary schools and 7.76%[95% CI = (6.96%, 8.55%)] for secondary schools. Additional Fig. 2 Evaluation metrics time-line. Illustration of terms used in the definitions of the evaluation metrics, ADD and FAR ig. 2 Evaluation metrics time-line. Illustration of terms used in the definitions of the evaluation metrics, ADD and FAR Page 9 of 16 Ward et al. BMC Public Health (2019) 19:1232 Ward et al. BMC Public Health Table 2 Summary of the different data aggregation types under consideration for use in epidemic detection methods Data Type Description Mean 95% CI Elementary (ES) ES-top Daily absenteeism for the elementary school that reported the most days throughout the study period. 8.34% (7.89%, 8.79%) ES-3avg Daily absenteeism averaged over the three elemen- tary schools that reported the most days throughout the study period. 6.39% (5.96%, 6.82%) ES-allavg Daily absenteeism averaged over all the elementary schools that reported. 5.94% (5.58%, 6.31%) Secondary (SS) SS-top Daily absenteeism from the secondary school that reported the most days throughout the study period. 3.35% (3.17%, 3.53%) SS-3avg Daily absenteeism averaged over the three secondary schools that reported the most days throughout the study period. 8.15% (7.06%, 9.40%) SS-allavg Daily absenteeism averaged over all the secondary schools that reported. 7.76% (6.96%, 8.55%) ES.SS-allavg Daily absenteeism averaged across all elementary and secondary schools that reported. 6.19% (5.83%, 6.56%) The sample means and bootstrapped (R = 10000) percentile 95% confidence intervals are given one of these two absenteeism aggregations (Table 4). Even amongst the 50 lowest FAR-producing models, approx- imately half used absenteeism averaged over either all the schools or all elementary schools (Fig. 3). In par- ticular, ES-allavg consistently produced results with low FARs. Table 5 shows the models that produced the lowest FAR for each absenteeism aggregation type and method of handling missing values; models that used ES- allavg absenteeism data had the lowest FARs regardless of whether deletion or interpolation in the training datasets was used. Secondary school absenteeism data was found to have lower predictive ability than elementary school data. None of the ten lowest FAR-producing model- based methods used secondary school absenteeism alarm to be raised when any individual school reached 10% absenteeism generally resulted in lower ADD values, but very high FAR (0.827 at best). Preliminary data analysis The most effective use of the threshold method gave true alarms for six out of eight evaluable school years, but up to 18 false alarms in five of the years. Although there was a high number of false alarms, the true alarms produced by this method were well-timed. Out of the six years where the start of a sea- sonal influenza epidemic was detected, only one of them had less then 10 days notice prior to the reference day. See Table 2 for aggregation abbreviations See Table 2 for aggregation abbreviations Statistical models See Table 2 for aggregation abbreviations these terms included tended to have considerably lower FARs than the non-seasonal models. Additionally, the ES- allavg data gave the most consistently low FARs with the regression-based models, with all other aggregation types performing variably well depending on what other factors were included in the model. Table 4 shows that, based on FAR, the ten best performing epidemic detection meth- ods were all regression-based models with seasonality terms, all of which incorporated some form of elemen- tary school absenteeism data. Between the mixed and GEE models, eight out of the ten best performing epi- demic detection methods included a random intercept for school year. However, there was no clear pattern for day of week indicator or interpolated/ deleted missing values in the absenteeism training data. Linear interpolation of the missing absenteeism data in the training data for the model-based methods did not consistently improve the number of true alarms or reduce false alarms compared to when days missing values were simply deleted. Similarly, the inclusion of a categorical variable for day of the week improved FAR in some cases and worsened it in others. unless it was averaged together with elementary school data (Table 4). Of the various modelling techniques that were consid- ered for use as influenza epidemic detection methods, the EWMA models were the least represented amongst the best performing methods. None of the ten low- est FAR-producing epidemic detection methods used EWMA modelling, and even in the 50 best methods there were very few EWMA models. At best, the EWMA models were able to give an FAR of 0.438 and ADD of 32 days, these being obtained when the square-root transformed average of all elementary school absenteeism data with missing values deleted was used. Transform- ing the data by either taking the square root or log of absenteeism did not generally improve results com- pared to the untransformed data. Overall, EWMA models outperformed the 10% threshold method but were less successful than the regression-based models at achiev- ing an acceptable balance between false and timely true alarms. The best performing models were based on variations of the logistic regression model. Figure 4 summarizes the effect that the inclusion of various factors and dif- ferent data aggregation types had on FAR across all the regression-based models. Statistical models The absenteeism data aggregations that resulted in the lowest FAR values for the model-based methods were those that incorporated the largest numbers of schools into their averages (ES-allavg and ES.SS-allavg). Of the ten models that produced the lowest FARs, all but two used Table 4 Epidemic detection methods with the ten lowest FARs Model Data Type Parameters FAR ADD Seasonal Mixed, D.O.W. ES-allavg (Int.) l = 7, = 0.20 0.299 15.13 Seasonal Mixed ES-allavg (Del.) l = 11, = 0.25 0.313 23.63 Seasonal Mixed, D.O.W. ES-allavg (Del.) l = 15, = 0.25 0.333 21.50 Seasonal LR ES-allavg (Del.) l = 5, = 0.25 0.344 23.00 Seasonal GEE ES-top (Int.) l = 1, = 0.15 0.350 14.29 Seasonal GEE ES-allavg (Del.) l = 7, = 0.25 0.350 23.13 Seasonal GEE ES-3avg (Del.) l = 15, = 0.25 0.375 29.38 Seasonal LR ES.SS-allavg (Del.) l = 11, = 0.30 0.375 31.75 Seasonal GEE, DOW ES-allavg (Del.) l = 11, = 0.30 0.375 33.13 Seasonal GEE, DOW ES.SS-allavg (Del.) l = 8, = 0.30 0.375 33.13 Del. = Missing values deleted, Int. = Missing values linearly interpolated See Table 2 for aggregation abbreviations Table 3 Evaluation metrics for the threshold-based epidemic detection methods where alarms are raised when absenteeism reaches 10%. Missing observations were not treated Data Type FAR ADD ES-top 0.799 37.75 ES-3avg 0.685 60.89 ES-allavg 0.722 70.33 SS-top 1.00 93.71 SS-3avg 0.664 28.88 SS-allavg 0.661 28.75 ES.SS-allavg 0.889 78.33 See Table 2 for aggregation abbreviations Table 3 Evaluation metrics for the threshold-based epidemic detection methods where alarms are raised when absenteeism reaches 10%. Missing observations were not treated Ward et al. BMC Public Health (2019) 19:1232 (2019) 19:1232 Page 10 of 16 Ward et al. BMC Public Health Fig. 3 Characteristics of best-performing models. Representation of the proportion of a) model types and b) absenteeism data aggregation types within the 50 lowest FAR-producing epidemic detection methods. See Table 2 for aggregation abbreviations Fig. 3 Characteristics of best-performing models. Representation of the proportion of a) model types and b) ab within the 50 lowest FAR-producing epidemic detection methods. See Table 2 for aggregation abbreviations Fig. 3 Characteristics of best-performing models. Representation of the proportion of a) model types and b) absenteeism data aggregation types within the 50 lowest FAR-producing epidemic detection methods. Statistical models Seasonality terms seemed to have the greatest effect on the FAR, as models with The detection method with the lowest FAR was the sea- sonal mixed model with a day of week indicator, using ES-allavg absenteeism data with values linearly interpo- lated in the training sets. The optimized parameters were l = 7 days and = 0.20. Under this model, the start Ward et al. BMC Public Health (2019) 19:1 Ward et al. BMC Public Health Page 11 of 16 Table 5 Best performing statistical models by data type, when missing values are either deleted or linearly interpolated Data Type Model Parameters FAR ADD ES-top Deleted Seasonal Mixed, DOW l = 7-8, = 0.30 0.411 26.71 Interpolated Seasonal GEE l = 1, = 0.15 0.350 14.29 ES-3avg Deleted Seasonal GEE l = 15, = 0.25 0.375 29.38 Interpolated Seasonal GEE l = 6, = 0.20 0.433 22.75 ES-allavg Deleted Seasonal Mixed l = 11, = 0.25 0.313 23.63 Interpolated Seasonal Mixed, DOW l = 7, = 0.20 0.299 15.13 SS-top Deleted Seasonal Mixed l = 4, = 0.10 0.461 14.67 Interpolated LR, DOW l = 4, = 0.25 0.454 9.17 SS-3avg Deleted Seasonal GEE, DOW l = 0, = 0.25 0.420 21.00 Interpolated Seasonal Mixed l = 1, = 0.15 0.422 21.57 SS-allavg Deleted Seasonal GEE, DOW l = 0, = 0.25 0.420 21.43 Interpolated Seasonal GEE, DOW l = 0, = 0.25 0.420 21.43 ES.SS-allavg Deleted Seasonal LR l = 11, = 0.30 0.375 31.75 Interpolated Seasonal LR l = 4, = 0.25 0.411 21.86 The metrics for the model with the lowest FAR are shown in bold. See Table 2 for aggregation abbreviations was similar to the best performing model for the years where it did capture epidemics. of each seasonal epidemic was captured with the excep- tion of the 2012–2013 epidemic, and false alarms were only raised in two years. Figure 5 illustrates the timing of these alarms relative to the start of the influenza epi- demic, where each panel represents a different school year where an alarm had the potential to be raised. True alarms tended to occur close to the start date of the seasonal epi- demic. Statistical models False alarms appeared to coincide with early cases of influenza that occurred far enough apart so as not to be classified as the start of the seasonal epidemic. Table 6 shows the number of false and true alarms, along with ADD, that were produced every school year in the study period when this model was used. Discussion Effects of different model factors and absenteeism data aggregations on FAR averaged over all regression model types with optimized parameters. Each row represents a different aggregation for absenteeism data and each column represents either a data handling method or whether an additional predictor aside from absenteeism was included in the model. The pairs of columns separated by spaces can be compared to view the effect on FAR across the different data aggregations, where a lighter shade indicates preferable (lower) FAR. The value within each cell is the mean FAR Fig. 4 Characteristics of regression models. Effects of different model factors and absenteeism data aggregations on FAR averaged over all regression model types with optimized parameters. Each row represents a different aggregation for absenteeism data and each column represents either a data handling method or whether an additional predictor aside from absenteeism was included in the model. The pairs of columns separated by spaces can be compared to view the effect on FAR across the different data aggregations, where a lighter shade indicates preferable (lower) FAR. The value within each cell is the mean FAR Fig. 4 Characteristics of regression models. Effects of different model factors and absenteeism data aggregations on FAR averaged over all regression model types with optimized parameters. Each row represents a different aggregation for absenteeism data and each column represents either a data handling method or whether an additional predictor aside from absenteeism was included in the model. The pairs of columns separated by spaces can be compared to view the effect on FAR across the different data aggregations, where a lighter shade indicates preferable (lower) FAR. The value within each cell is the mean FAR have been proposed for use with non-normally distributed data (see [40] for a review of several), however a chal- lenge remains in choosing an appropriate subset of data to represent the “in-control” process; the distribution of absenteeism when influenza is not present in the com- munity. In this study, the “in-control” mean was set to be the mean of all absenteeism data from the training years, however that average would include observations during an influenza season. Without knowing more infor- mation about additional circulating infectious diseases or other factors affecting absenteeism (which could be unique to different schools) it is difficult to choose a specific time period to use as the “in-control” process. Discussion This paper proposed and tested several possible model- based epidemic detection methods as alternatives to the 10% absenteeism threshold method currently being used in WDG. For this study, an ideal model would be able to detect a seasonal epidemic earlier than it would be caught using reported laboratory-confirmed case counts alone, while not raising many false alarms. Overall, we found that all of the tested model-based approaches achieved these characteristics to a higher degree than the school absenteeism threshold method did. Parameter estimation was slower for models where missing absenteeism values in the training data had been linearly interpolated rather than deleted. The yearly results for the best model that used deletion are reported in Table 7. This model was also a seasonal mixed model using the ES-allavg absenteeism data, but did not include a day of week indicator. In comparison to the best epidemic detection method (the seasonal mixed model with day of week indicator and interpolated training data), this model captured one less epidemic giving it slightly a higher over- all FAR, however it had fewer false alarms and the ADD The 10% threshold was able to produce true alarms with low ADD when it was applied to absenteeism averaged across all secondary schools, although this was counter- acted by a high proportion of false alarms. This appears to contradict earlier findings that many schools would not reach 10% absenteeism even during an influenza sea- son [15]. Secondary school absenteeism in the WDG region was higher than elementary school absenteeism in most school years, with an overall average absenteeism of 7.76% for the secondary schools that reported during Ward et al. BMC Public Health (2019) 19:1232 Page 12 of 16 Fig. 4 Characteristics of regression models. Effects of different model factors and absenteeism data aggregations on FAR averaged over all regression model types with optimized parameters. Each row represents a different aggregation for absenteeism data and each column represents either a data handling method or whether an additional predictor aside from absenteeism was included in the model. The pairs of columns separated by spaces can be compared to view the effect on FAR across the different data aggregations, where a lighter shade indicates preferable (lower) FAR. The value within each cell is the mean FAR Fig. 4 Characteristics of regression models. Discussion In addition, most of the best-performing EWMA mod- els had a very small optimized λ value, indicating that a high degree of smoothing was required. A small λ also approximates a CUSUM model, which has been used in previous studies where absenteeism is used for influenza surveillance [17, 18]. One such study concluded that the CUSUM was not ideal for their data [17]. Finally, at a small value of λ EWMA models can accumulate “credit” [41]. If an increase in mean absenteeism were to occur following a day where the EWMA statistic had been calculated to be a value less than μ0, it would the study period. A 10% cut-off may therefore be too low for secondary schools while being too high for elemen- tary schools, and a more individualized approach such as that used in Mann et al. (2011) where thresholds are deter- mined by standard deviations for a rolling mean may work better for WDG [16]. In WDG aggregated absenteeism data performed better within the 10% threshold method than using an individual school’s absenteeism for the pur- poses of detecting a regional epidemic. However, an alarm for an individual school may still be useful for detecting within-school outbreaks. Data do not currently exist for the evaluation of this use of the 10% threshold, and so this could be an area for future investigation. The EWMA approach explored in this study is some- what similar to the adaptive thresholds of Mann et al. (2005), although all past observations (as opposed to only 30) were included and weighted in the calculation of the standard deviation [16]. The EWMA models improved FAR compared to the threshold method but did not do as well as many of the regression-type models. Although data transformations were attempted, normality of the absen- teeism data was not achieved, and the EWMA models may not have been able to sufficiently smooth out the day- to-day noise. Non-parametric modified EWMA models Ward et al. BMC Public Health (2019) 19:1232 Ward et al. BMC Public Health Page 13 of 16 Fig. 5 Alarms of the top-performing model. True and false alarms for the best performing model, faceted by school year: the seasonal logistic random intercept model using ES-allavg data with l = 7, = 0.20, interpolated missing values in the training data. Mean FAR = 0.299, mean ADD = 15.13 Discussion Averaged absenteeism is plotted as grey bars, with actual laboratory-confirmed influenza case counts overlaid as black bars, and the epidemic reference day is indicated by the dashed yellow line for each school year Fig. 5 Alarms of the top-performing model. True and false alarms for the best performing model, faceted by school year: the seasonal logistic random intercept model using ES-allavg data with l = 7, = 0.20, interpolated missing values in the training data. Averaged absenteeism is plotted as grey bars, with actual laboratory-confirmed influenza case counts overlaid as black bars, and the epidemic reference day is indicated by the dashed yellow line for each school year take several days for the EWMA statistic to reach the UCL because of the low weights assigned to recent val- ues, which may have limited the number of true alarms observed. The seasonality (trigonometric) covariates may act to counterbalance any unusually high values of absenteeism that occur during times of the year when influenza is not generally circulating yet, thus reducing the number of false alarms. Inclusion of a random intercept for school year (in the mixed and GEE models) also seemed to improve model performance in most cases. The random The regression-based models generally performed bet- ter than the EWMA models, particularly when covariates accounting for seasonality were included in the model. Discussion We tested models with up to 15 lagged school days of absenteeism, so this combined with the amount of data needed to estimate the intercept meant that in some years the models were not able to be used until nearly November. In the event of an unusually early seasonal epi- demic starting prior to November, it would not be able to be captured by the random intercept models. Alter- native solutions to estimating the random intercept for year-effect should be investigated. One possibility would be to use a previously estimated intercept for a new school year. For example, if information on the current influenza strain were available from earlier epidemics in a compa- rable nearby health region, an intercept could be used that was estimated on the WDG data from a previous year when there was a similar strain. Alternatively (and more realistically, given that WDGPH would not neces- sarily have access to strain information from other public health units), an intercept could be estimated using the most recent past year of WDG data and used with the current year’s data. (for example exceeding the total number reported absent), and incomplete and thus could not be used in this study. Increased adoption of this reporting feature by school administrators could greatly aid model performance as observations from schools that are experiencing an out- break or epidemic of an illness with non-respiratory symptoms could be excluded. Reporting of other reasons for unusual levels of absenteeism, such as field trips, would be similarly helpful. However, even if more detailed absen- teeism information were available, a school-absenteeism surveillance system has limitations due to the “missing” data from weekends and holidays as well as potentially differing effects of different influenza strains on children. For example, Cauchemez et al. (2008) found that influenza subtype B was more closely associated with children than subtype A\H3N2 [42]. One way to overcome these limi- tations would be to use a second surveillance system in addition to the absenteeism surveillance system. Possi- bilities could include an alternative form of syndromic surveillance that targets an older demographic (such as over-the-counter drug sales monitoring). We chose to define the start of an influenza epidemic by the first time two cases were reported to WDGPH within a week of each other. Discussion In the WDGPH region, there are gen- erally no laboratory-confirmed influenza cases observed outside of influenza season, however if influenza activ- ity became more sustained throughout the year in the region, or in a region with a larger population where more cases are seen overall, it may be necessary to explore alter- nate definitions for the beginning of the epidemic. The Moving Epidemic Method could be used to identify an influenza activity threshold above which the region would be defined as experiencing an epidemic [43]. Secondary school absenteeism was less consistent than elementary absenteeism, and the yearly mean averaged across all secondary schools decreased over the 2012– 2013 to 2015–2016 school years before increasing again (Fig. 1). This, along with the fact that fewer secondary schools than elementary schools report data, may explain why secondary school absenteeism generally performed less well than elementary school absenteeism in predicting influenza epidemics. Despite this, an Augmented-Dickey- Fuller test for non-stationarity found all absenteeism aggregations to be stationary, suggesting that the absen- teeism decrease was not enough to cause a significant change in temporal trend of the data. Preliminary analysis based only on the 2008–2014 data yielded better perfor- mance of the models using the secondary school data than was seen using the full dataset. If the predictive abilities of secondary school absenteeism have decreased in more recent years, it may not be worth continuing to collect secondary school absenteeism data. Another limitation of this study is that spatial infor- mation was not incorporated in any of the model-based epidemic detection methods. Approximately half of the WDG population live in rural areas, which accounts for 98% of the geographic area serviced by WDGPH [21, 22]. Since spatial identifiers were not provided with the absen- teeism data, we were unable to select the top or top three consistently reporting schools to be representative of a certain part of the region (ie. the City of Guelph) and so they were selected on the basis of most days reported. Therefore, some or all of the top schools chosen here could be in relatively remote locations. Depending on where the annual influenza season begins within the region in any particular year, influenza cases in Dufferin may precede cases in Guelph for example, resulting in absenteeism in a Guelph school raising an alarm too late for WDG as a whole. Discussion Table 6 Yearly alarms and ADD for the best performing model: the seasonal logistic random intercept model using ES-allavg data with l = 7, = 0.20, interpolated missing values in the training data School Year False Alarms True Alarms ADD 2010–2011 0 1 13 2011–2012 8 1 14 2012–2013 0 0 55 2013–2014 1 1 14 2014–2015 0 4 3 2015–2016 0 1 14 2016–2017 0 8 0 2017–2018 0 3 8 Mean FAR = 0.299, mean ADD = 15.13 Table 7 Yearly alarms and ADD for the seasonal random intercept model with 11 lags and = 0.25, using ES-allavg data with missing values deleted School Year False Alarms True Alarms ADD 2010–2011 0 2 13 2011–2012 0 1 14 2012–2013 0 0 55 2013–2014 1 1 12 2014–2015 0 2 7 2015–2016 0 0 70 2016–2017 0 5 5 2017–2018 0 1 13 Mean FAR = 0.313, mean ADD = 23.63 Table 7 Yearly alarms and ADD for the seasonal random intercept model with 11 lags and = 0.25, using ES-allavg data with missing values deleted School Year False Alarms True Alarms ADD 2010–2011 0 2 13 2011–2012 0 1 14 2012–2013 0 0 55 2013–2014 1 1 12 2014–2015 0 2 7 2015–2016 0 0 70 2016–2017 0 5 5 2017–2018 0 1 13 Mean FAR = 0.313, mean ADD = 23.63 Table 7 Yearly alarms and ADD for the seasonal random intercept model with 11 lags and = 0.25, using ES-allavg data with missing values deleted Ward et al. BMC Public Health (2019) 19:1232 Ward et al. BMC Public Health (2019) 19:1232 Page 14 of 16 Page 14 of 16 Ward et al. BMC Public Health (201 intercept models were considered to allow for possible differences in the relationship between absenteeism and odds of an influenza case from year to year. However, to be able to estimate the random intercept, the models must be trained on at least some data from a given school year in order to be used to predict outcomes for that same year. In this study we used data from September of each year to obtain the intercept estimate and then began prediction in October. Discussion Since WDGPH has access to school identifiers, it may be more useful for them to choose a few well-reporting schools from each of the larger towns in the region and run the chosen epidemic detection method on the separate locational averages in parallel. Absenteeism data is voluntarily reported to WDGPH daily by schools through an on-line system. The cur- rent system (introduced in 2017) features a field where school administrators can choose to enter the number of absences related to illness (in total, as well as categorized as respiratory, gastrointestinal and other). The illness and specific syndrome counts were observed to be unreliable, Ward et al. BMC Public Health (2019) 19:1232 Ward et al. BMC Public Health (2019) 19:1232 Page 15 of 16 Page 15 of 16 Another area for future work is the development of an improved metric or collection of metrics to use for epi- demic detection method evaluation. We chose to optimize model parameters based on the lowest FAR since select- ing based on the ADD would lead to too many false alarms (false alarms increase somewhat proportionately with true alarms). The FAR provides an idea of the balance between true and false alarms, but does not indicate where these alarms occur relative to the start of an epidemic. A low FAR can be misleading in certain cases; for example, a method that raised no false alarms and a single true alarm on the reference day each year would have a FAR of 0 when in reality the method is not giving an alarm any sooner than one that would be raised based on hospital reports alone. An improved metric for identifying optimal model parameters might define an ideal day (for example, 14 days prior to the reference day) for an alarm to be raised, and penalize both false and true alarms based on the differ- ence between the day on which they were raised and the ideal alarm day. This would also penalize models that raise alarms clearly unrelated to influenza activity more heavily than those whose alarm dates are only marginally outside the true alarm range. Although it produced some false alarms, those alarms did coincide with influenza cases, though those cases were spread too far apart to be classified within an epi- demic. Availability of data and materials Under a data sharing agreement, the data for this study were made available by, and used with permission from, Wellington-Dufferin-Guelph Public Health. Access to, and permission to use, the data must be obtained from Wellington-Dufferin-Guelph Public Health, Jennifer MacLeod, Manager, Health Analytics (jennifer.macleod@wdgpublichealth.ca). Funding g This work was funded in part by an NSERC Discovery Grant. p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. Comparison of the 10% threshold approach to several modelling-based methods showed that the 10% threshold approach can be improved upon to reduce the number of false alarms while still giving warning of influenza epi- demics ahead of laboratory-confirmed cases. Based on our findings, a seasonal logistic regression model with random intercept for school year is recommended for influenza surveillance in WDG. This approach was able to raise true alarms for almost every epidemic of influenza. Discussion In practice these alarms may still be useful in alerting WDGPH of when influenza or a similarly trans- mitted disease begins circulating in the region. Absen- teeism averaged over all reporting elementary schools is recommended as the model predictors, as it was found to give the best balance of true and false alarms for most of the epidemic detection methods. However, it is suggested that the school absenteeism surveillance system be used in conjunction with another influenza surveillance system, due to limitations caused by missing data and absenteeism patterns unrelated to influenza. Authors’ contributions LT-W formulated the research question. MAW, AS, LED, RD, and ZF contributed to the design of the study. MAW and AS conducted the statistical analyses and wrote the manuscript. LT-W facilitated data sharing and provided valuable information about the dataset. All authors edited the manuscript at several stages, and read and approved the final manuscript prior to submission. When using any of these epidemic detection methods in practice, it would be useful to test model performance as more years of data become available, and obtain updated optimized parameters on a yearly basis. Since linear inter- polation increases computation time for training models and was not found to significantly improve the results of methods compared to when the missing values were deleted, deleting missing values is recommended. The number of lagged days of absenteeism data included as predictors in the model and the probability thresholds for the regression-based models reported in this paper are optimized for the data currently available, but should be re-evaluated regularly to maintain the efficacy of influenza epidemic detection methods of this nature. The meth- ods identified in this study should also be evaluated with data from other public health regions prior to being used as epidemic detection method outside of WDG. Regional differences in weather, school attendance patterns, health care, and other factors could influence which method would be the most effective. Abbreviations l ADD: Accumulated days delay; CUSUM: Cumulative sum; DOW: Day of the week; ES: Elementary school; EWMA: Exponentially weighted moving average; FAR: False alarm rate; GEE: Generalized estimating equation; SS: Secondary school; UCL: Upper control limit; WDG: Wellington-Dufferin-Guelph; WDGPH: Wellington-Dufferin-Guelph Public Health Received: 22 May 2019 Accepted: 20 August 2019 Ethics approval and consent to participate Data used for this study is not publicly available, and was collected by Wellington-Dufferin-Guelph Public Health. 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Census Profile, Wellington County, Ontario. 2016 Census. 2016. https://www12.statcan.gc.ca/census-recensement/2016/ dp-pd/prof/details/page.cfm?Lang=E&Geo1=CD&Code1=3523&Geo2= PR&Code2=35&Data=Count&SearchText=Wellington&SearchType= Begins&SearchPR=01&B1=All&TABID=1.
https://openalex.org/W3014632520	https://vestnik.kemsu.ru/jour/article/download/4639/4116	Russian	null	Simple Sentence Information Structure in Teleut: Problem Statement	Vestnik Kemerovskogo gosudarstvennogo universiteta	2,020	cc-by	7,461	Информационная структура простого предложения в телеутском языке: к постановке проблемы* Денис М. Токмашев a, @, ID a Национальный исследовательский Томский политехнический университет, Россия, г. Томск @ kogutei@yandex.ru ID1 https://orcid.org/0000-0003-3941-043X Денис М. Токмашев a, @, ID a Национальный исследовательский Томский политехнический университет, Россия, г. Томск @ kogutei@yandex.ru ID1 https://orcid.org/0000-0003-3941-043X Денис М. Токмашев a, @, ID a Национальный исследовательский Томский политехнический университет, Россия, г. Томск @ kogutei@yandex.ru ID1 https://orcid.org/0000-0003-3941-043X Поступила в редакцию 22.01.2020. Принята к печати 12.02.2020. Аннотация: Рассматриваются основные подходы к изучению информационной структуры простого предложения и их при- менение к материалу тюркских языков. Цель – выявить и охарактеризовать различные типы информационных структур простого предложения в их взаимосвязи с формально-грамматическими и акцентными характеристиками на материале телеутского языка. Основными средствами выражения информационной структуры простого предложения, которую мы представляем в виде соответствующего функционально-семантического поля, в телеутском языке являются порядок синтагм и интонация, составляющие ядро средств управления информационной структурой, а также периферические средства – лексемы, частицы и аффиксы. Синтаксически информационная структура выражается порядком синтагм. Для повествовательных предложений характерно понижение частоты основного тона на синтагме, составляющей фокус- ную часть. Для прагматически нейтральных повествовательных предложений, не имеющих пресуппозиций, характерно прогрессивное расположение элементов информационной структуры «топик > фокус» с предикатом в крайней правой позиции. Поскольку топик и фокус являются шифтерными категориями, возможны как инверсии синтагм с сохранением прогрессивной информационной структуры, так и инверсии компонентов «фокус < топик» с сохранением порядка син- тагм. Инверсия линейных (синтагм) и нелинейных (топика и фокуса) элементов предложения обусловлена различными пресуппозициями. Лексико-грамматические средства управления информационной структурой находятся на периферии функционально-семантических полей. Их информационно-структурные характеристики совмещаются с основными функциями: выражением аспектуальности, модальности, эвиденциальности, (не)определенности. Тюркские языки обнаруживают значительное сходство в выборе средств управления информационной структурой. Выводы сделаны на основе полевого, сравнительно-исторического и описательного методов, структурного и компонентного анализа, методов моделирования семантики и визуализации спектрограмм. Ключевые слова: интонация, синтаксис, тюркские языки, актуальное членение предложения, акустическая фонетика Для цитирования: Токмашев Д. М. Информационная структура простого предложения в телеутском языке: к поста- новке проблемы // Вестник Кемеровского государственного университета. 2020. Т. 22. № 1. С. 268–277. DOI: https:// doi.org/10.21603/2078-8975-2020-22-1-268-277 Для цитирования: Токмашев Д. М. Информационная структура простого предложения в телеутском языке: к поста- новке проблемы // Вестник Кемеровского государственного университета. 2020. Т. 22. № 1. С. 268–277. DOI: https:// doi.org/10.21603/2078-8975-2020-22-1-268-277 высказывания и регулирующих соотношение в нем старой и новой информации, которая, в свою очередь, является планом содержания ИС. Вестник Кемеровского государственного университета, 2020, 22(1) Вестник Кемеровского государственного университета, 2020, 22(1) Языкознание Вестник Кемеровского государственного университета, 2020, 22(1) DOI: 10.21603/2078-8975-2020-22-1-268-277 * Работа выполнена при поддержке РФФИ, грант № 18-012-00775 А «Типология простого предложения в языках обско-енисейского языкового ареала: информационная и аргументная структуры». Вестник Кемеровского государственного университета, 2020, 22(1) DOI: 10.21603/2078-8975-2020-22-1-268-277 Вестник Кемеровского государственного университета, 2020, 22(1) Языкознание информация соотносится с представлением говорящего о ментальном состоянии адресата высказывания [1]. пропозиции, лежащей в основе высказывания, доступный обо- им собеседникам. «Topics are thus elements of the proposition that the utterance is construed about, and that are usually restricted to given, accessible elements» [5, р. 96]. р В качестве примера исследования ИС в отечественной лингвистике (называемой также коммуникативной струк- турой) можно привести работу Е. В. Падучевой, выполнен- ную на материале русского языка [2]; подробно история вопроса представлена у Я. Г. Тестельца [3]. В зарубежной лингвистике, как отмечает К. Ламбрехт, ИС впервые при- влекла внимание младограмматиков в конце XIX – начале XX в. (противопоставление психологического и логи- ческого субъектов и предикатов у Г. Пауля) [4]. Долгое время терминологическим эквивалентом ИС в советском и российском языкознании служило актуальное члене- ние предложения (В. Матезиус), затем получившее разви- тие у представителей Пражской лингвистической школы под названием функциональная перспектива предложения, а также у М. Хэллидэя, который и ввел в оборот понятие информационной структуры. М. Хэллидэй характеризовал ее как грамматическую категорию, отличную от синтак- сиса и семантики, но тесно взаимодействующую с ними. У. Л. Чейф предложил синонимичный термин information packaging. Проблема ИС затрагивается и в более поздних работах по дискурсивной прагматике. Из обзорных публи- каций по истории изучения, терминологии и проблематике ИС можно отметить работы Д. Матича [5] и М. Крифки [6]. g р Фокус высказывания (в прочих работах именуемый также новой информацией, ремой и комментарием), по К. Ламбрехту, – это информация, посредством кото- рой ассерция отличается от пресуппозиции [4, р. 213]. Другой подход к определению фокуса связан с теорией альтернативной семантики: фокус – это семантический оператор, порождающий альтернативу фокусной части высказывания, например, в предложении с интонационно выраженной частью (Он закрыл окно) фокусом является предикат закрыл, который допускает альтернативный вариант открыл, не выбранный говорящим как несоответ- ствующий его коммуникативному намерению. На сегодняшний день в лингвистике нет, таким образом, единого прототипического определения фокуса; выде- ляют два взаимно перпендикулярных подхода: топик vs. комментарий (фокус – часть высказывания, добавляемая к ментальной репрезентации слушающего) и фокус vs. фон (фокус – часть высказывания, допускающая альтернатив- ную переменную, а фон – не допускающая ее) [5, p. 97]. Как нетрудно заметить, комментарий в первом подходе в широком смысле соответствует фокусу, а фон во втором – топику, т. е. теме высказывания. ИС можно отметить работы Д. Матича [5] и М. Крифки [6]. В статье ИС понимается в смысле, предложенном К. Вестник Кемеровского государственного университета, 2020, 22(1) DOI: 10.21603/2078-8975-2020-22-1-268-277 Ламбрехтом, – как трихотомия взаимосвязанных оппо- зиций пресуппозиция – ассерция, узнаваемость – активация и топик – фокус. Противопоставление пресуппозиции и ассерции тесно связано с инкрементальной моделью (incremental model) дискурса, смысл которой в том, что изначально коммуниканты обладают различным объемом знаний, а цель коммуникации – минимизировать это разли- чие за счет расширения ментальной репрезентации (common ground) говорящего и слушателя [7]. Под пресуппозицией в этом контексте понимается информация, на момент начала речевого акта входящая, по мнению говорящего, в общую установку коммуникантов. Ассерция – информация, ранее не известная слушателю и добавленная в его ментальную репрезентацию; в последующем коммуникативном акте она переходит в разряд пресуппозитивных компонентов выска- зывания и может служить основанием для ассертивных. ИС на формально-языковом уровне реализуется с помо- щью различных средств, охватывающих фонетику (акцен- тология и просодия), синтаксис (порядок слов в клаузах и самих клауз, стратегия кодирования актантов и т. д.), мор- фологию (модальные и служебные слова, клитики, артикли, фокусные частицы и т. д.) и лексику (ремовыделительные наречия типа русского именно). Их разнообразие затрудняет выработку единых моделей предложения, заданных ИС, чему препятствует большой разброс типовых структур в языках мира. У. Л. Чейф предполагал, что каждый язык имеет свои средства управления ИС, а ведущую роль «по-видимому играют порядок слов и интонация, причем, возможно, во всех языках» [8, с. 266]. Закономерным представляется вывод Д. Матича, который он сделал в своей обзорной работе по ИС в лингвистике: информационная струк- тура предложения – универсальная категория дискурса, но необязательно универсальная категория языка [5, р. 99]. Топик высказывания может определяться двояко: как с позиции говорящего (aboutness, т. е. о чем сообщается новая информация), так и с позиции слушающего (giveness, т. е. что слушающему уже известно об объекте высказывания). Причем оба определения вынужденно пересекаются, так что несмотря на преобладающее в лингвистике определение топи- ка с позиции говорящего (что сообщаем слушающему об объ- екте?), говорящему неизбежно приходится учитывать, какой информацией об объекте высказывания владеет слушающий к началу коммуникативного акта (что слушающий должен знать для правильной интерпретации новой информации?). Таким образом, топик можно определить как компонент Перейдем к рассмотрению понятия ИС применитель- но к телеутскому языку и шире – к тюркским языкам. Исследователь караимского синтаксиса К. М. Мусаев пишет, что «вопрос о функции порядка слов в предложе- нии на материале тюркских языков изучен весьма слабо. Помимо общих замечаний отсутствуют специальные глубо- ко анализированные исследования большого фактического материала» [9, с. 253]. Одна из первых работ в российской тюркологии, кос- венно затрагивающих вопросы выражения «старого» и «нового» в высказывании ,– статья Л. Введение Под информационной структурой (ИС) в лингвистике принято понимать членение высказывания на информаци- онные сегменты, понимание которых является минимально достаточным для реализации коммуникативных намерений собеседников. Иными словами, ИС – это план выражения прагматики предложения, подобно тому как сегментные структуры предложения (лексемы и граммемы) являются планом выражения его семантики. ИС находится в тесном взаимодействии с сегментными структурами и управляет ими в акте коммуникации. Это позволяет рассматривать ИС предложения как глубинную структуру, на поверхнос- тном уровне реализуемую в виде набора грамматических и акцентных характеристик, соответствующих контексту В рамках исследования ИС в естественных языках сфор- мировался ряд подходов к ее изучению, а также разветвлен- ный терминологический аппарат, специфический для каждой школы – их подробный анализ выходит за рамки задач данной статьи. При многообразии подходов и терминов большин- ство работ по ИС учитывает речевую синхронию оппозиций говорящий – слушающий, известное – новое, семантика – прагматика и то, что некоторые формальные свойства предложения (например, порядок слов) могут быть поняты только с учетом контекста. Согласно У. Л. Чейфу, ИС отра- жает то, как закодированная с помощью языковых средств Cтатья распространяется на условиях лицензии Creative Commons Attribution 4.0 268 Cтатья распространяется на условиях лицензии Creative Commons Attribution 4.0 Вестник Кемеровского государственного университета, 2020, 22(1) DOI: 10.21603/2078-8975-2020-22-1-268-277 Языкознание «информативно более нагруженная часть – смысловое ядро высказывания», а основными средствами «актуализации коммуникативных частей высказывания являются поря- док слов, формально-грамматические средства: аффиксы, частицы, лексические элементы» [14, с. 23]. в которой упоминаются три основных функции порядка слов – грамматическая (расположение субъекта и объ- екта относительно предиката), коммуникативная (тема и рема) и стилистическая (стилистически мотивированное нарушение порядка слов) с сохранением грамматично- сти предложений, построенных с нарушением базового порядка слов, которые приобретают дополнительные характеристики. О влиянии порядка слов на ИС в тюркских языках автор пишет: «тюркские языки нельзя безогово- рочно отнести к категории языков со связанным порядком слов, в которых сугубо синтаксическая (грамматическая) функция порядка слов выдвигается на первый план в ущерб функции коммуникативной и стилистической» [10, с. 121]. Исследователь отмечает, что порядок слов в тюркских словосочетаниях выполняет только грамматическую и сти- листическую функции, а логико-грамматическая (т. е. информационно-структурная) функция порядка слов реа- лизуется в единицах более высокого порядка – синтагмах (единицах логико-грамматического членения). Уточняется, что ИС (в современном понимании) определяется не столь- ко порядком слов, сколько порядком синтагм, хотя линейно одна синтагма может соответствовать одному слову. Говоря о просодии как маркере ИС, автор считает ее сопут- ствующим и второстепенным по отношению к словопорядку явлением, отмечая тем не менее, что для прогрессивного (неинвертированного) порядка слов характерна окситония (постепенное нарастание интонации с пиком на фокусной части), а для регрессивного – баритония (резкое усиление интонации в начале с постепенным затуханием). Также характерно наличие пауз перед ремой и после нее [14, с. 22]. В кумыкском языке коммуникативные типы высказываний были изучены Н. К. Токтаровой, сделавшей аналогичный и универсальный для тюркских языков вывод: «В кумыкском языке в число средств, маркирующих актуальное членение, входит, прежде всего, порядок слов, интонация, лексические и грамматические средства» [15, с. 4]. Как и А. В. Емельянова, Н. К. Токтарова пишет о примате синтаксиса над фонетикой в формировании ИС кумыкского предложения: «логическое ударение выражается синтаксическим путем: употреблением логически выделенного слова перед сказуемым: Беш къо- накълай бардыкъ "Мы в гости ходили"… Роль фонетических средств при этом незначительна» [15, с. 18]. Автор приводит следующие примеры реализаций отмеченных стратегий кодирования ИС в кумыкском языке: Н. К. Дмитриев также обращает внимание на коммуни- кативно-прагматические функции порядка слов в тюркских языках (башкирском и кумыкском): «В башкирском синтаксисе (как и в синтаксисе других тюркских языков) существует такое положение: для того, чтобы логически оттенить какой-нибудь член предложения в целой фразе, необходимо изменить место этого члена» [11, с. 210]. Ср.: «Причины инверсии лежат вне грамматики, как таковой. 1 В действительности DA является частицей, в телеутском языке она также оттеняет фокус высказывания: Эмди меең-де палдарым тил пилбей jалар (Сейчас и мои-то дети языка не знают). Вестник Кемеровского государственного университета, 2020, 22(1) DOI: 10.21603/2078-8975-2020-22-1-268-277 Ю. Тугушевой [10], Cтатья распространяется на условиях лицензии Creative Commons Attribution 4.0 269 DOI: 10.21603/2078-8975-2020-22-1-268-277 Николай Сергеевич был, меня послал учиться в Кемеровский сельскохозяйственный техникум на агронома). к анализу ИС, так и из-за слабой изученности телеутского языка в целом2 и его синтаксиса в частности. Слово техникум формирует ассертивный компонент высказывания, который в последующем предложении редуци- руется до местоимения аны и, в свою очередь, формирует пре- суппозицию для синтагмы алтон пеш jылда, которая и является фокусом высказывания, а глагол и его аргументы образуют его топик, линейно включающий в себя и фокус. Схематически ИС примера (1) можно представить следующим образом: Мен аны алтон пеш jылда тÿгезип ийгем. Гипотезой исследования выступает утверждение, что ИС – обязательный элемент дискурса, речевая реализация которого обусловлена структурой языка. Ожидается, что генетически и / или типологически близкие языки должны использовать сходный инвентарь языковых средств выражения ИС. Мы предполагаем, что в данном аспекте телеутский язык (горно-алтайская группа) будет близок прочим тюркским язы- кам, например, чувашскому (булгарская группа), кумыкскому (половецко-кыпчакская группа) и др., а именно: использовать словопорядок, акцентные характеристики и лексико-граммати- ческие средства для организации ИС простого предложения. ФОКУС ТОПИК Как видно, сказуемое тÿгезип ийгем включается в топик, поскольку, хотя и является формально новой информацией для слушателя, относится к пресуппозиции, т. к. комму- никативное намерение говорящего – сообщить, в каком году он закончил обучение. Его «новизна» второстепенна по отношению к «истинному» фокусу. Непосредственно фокусная часть, в свою очередь, раскладывается на более мелкие смысловые компоненты алтон пеш и аффикс местно- временного падежа -да, но поскольку отдельное взятое сочетание алтон пеш jылда не образует пропозиции, оно не может иметь собственной ИС. Методы и материалы. Эмпирической основой иссле- дования служит аннотированный корпус устных бытовых и автобиографических текстов (наиболее нейтральных в сти- листическом отношении), записанных от носителей телеут- ского языка разного возраста и пола. В работе используются полевой, сравнительно-исторический и описательный методы, методы структурного и компонентного анализа, моделирования семантики и визуализации спектрограмм. Вестник Кемеровского государственного университета, 2020, 22(1) DOI: 10.21603/2078-8975-2020-22-1-268-277 Инверсия является отображением особых логических или психологических условий, под впечат- лением которых говорящий высказывает свою мысль. Прежде всего инверсия является средством усиления так называемого фразового ударения, или логического акцента» [12, с. 163]. Иными словами, инверсия в тюркских языках выступает как средство управления ИС. 1) порядок слов: Ахшам / кьар явду (Вечером / выпал снег) ~ Кьар явду / ахшам (Снег выпал / вечером). Обычная позиция ремы – перед глагольным сказуемым: Мен охумагъа тюшдюм (Я поступила учиться). Тематическая препози- ция ремы указывает на экспрессивность: Макътай адам / бийиклигин тавланы (Славит человек / высоту гор); 2) интонация с выделением ремы с помощью фразового ударения, представляющего интонационный центр пред- ложения, а темы – повышением тона: Ишине ону / анасы сююндю (Его делу / мать обрадовалась) ~ Ишине / ону анасы сююндю (Делу / его мать обрадовалась); Актуальное членение предложения в уйгурском языке выражается с помощью порядка слов, интонации и других средств [13, с. 378]. 3) лексико-грамматические средства: «в кумыкском языке аффикс да1, выполняющий функцию союза "и", слу- жит средством выделения ремы в предложении: Авруйгъан анасыны уьстюне / уланы да гирди "К болеющей матери / зашел и сын". С другой стороны, тема может быть выделена при помощи частицы чы "же": Шагьарда яшамагъа чы / тынч "В городе же / жить легко"» [15, с. 10]. Анализу коммуникативной структуры простого пред- ложения в чувашском языке посвящено исследование А. В. Емельяновой, во введении к которому автор отмечает, что «теория актуального членения агглютинативных язы- ков имеет совсем непродолжительную по научным меркам историю (Андреев И. А., Абдулаев К. М., Амиров Р. С., Сафиуллина Ф. С., Тугушева Л. Ю. и другие)» [14, с. 3]. Основным средством выражения коммуникативной струк- туры чувашского простого предложения автор считает порядок слов, а тема и рема соотносятся как определяющее и определяемое. В реме при этом дополнительно выделяется Рассмотрим различные стратегии кодирования ИС соб- ственно в телеутском языке – одном из миноритарных тюркских языков Саяно-Алтая. Необходимо отметить, что ИС телеутского простого предложения ранее не исследова- лась как из-за отсутствия единых определений и подходов Cтатья распространяется на условиях лицензии Creative Commons Attribution 4.0 270 Вестник Кемеровского государственного университета, 2020, 22(1) DOI: 10.21603/2078-8975-2020-22-1-268-277 Вестник Кемеровского государственного университета, 2020, 22(1) Языкознание Результаты Tone visualization of example (2) Пример (2) – ИС простого предложения, осложненного причастной конструкцией, образующей таксисную пару с главной частью: Пример (2) – ИС простого предложения, осложненного Пример (2) – ИС простого предложения, осложненного причастной конструкцией, образующей таксисную пару Пример (2) ИС простого предложения, осложненного причастной конструкцией, образующей таксисную пару par-ɣan-ča идти-PST2-DEL soldat-qa солдат-DAT олхоз-LOC работать-PST2.1SG Калкосто иштегем солдатқа парғанча – В колхозе (я) работал до ухода в армию. Рис. 2. Тональный рисунок примера (2) Fig. 2. Tone visualization of example (2) Рис. 2. Тональный рисунок примера (2) Fi 2 T i li ti f l (2) Экспрессивной реализацией данной ИС будет вариант с инверсией синтагм и, соответственно, закрепленных за ними компонентов ИС: г) Калкосто иштегем солдатқа парғанча. ФОКУС ТОПИК Экспрессивной реализацией данной ИС будет вариант с инверсией синтагм и, соответственно, закрепленных за ними компонентов ИС: Можно предположить, что в данном примере сдвиг сказуе- мого с ожидаемой терминальной позиции (Калкосто солдатқа парғанча иштегем) обусловлен его вхождением в топик, т. е. ИС. По мнению Л. Ю. Тугушевой, для ИС тюркского предложения важен порядок синтагм, а не слов. Схематично ИС примера (2) выглядит следующим образом: Таким образом, при сохранении прогрессивного поряд- ка компонентов ИС инверсия синтагм в предложении приводит к изменению ИС, а инверсия синтагм вместе с выраженными ими компонентами ИС (препозиция фоку- са) – к повышению экспрессивности (при этом на глубинном уровне все 4 варианта имеют одинаковую синтаксическую, но разную семантическую структуру). Схожее явление отмечено и в других тюркских языках, например, караим- ском и узбекском. «Когда мы говорим о порядке слов в про- стом распространенном предложении, в первую очередь оперируем материалами повествовательного предложения со спокойным равномерным тоном. В предложениях, эмо- ционально насыщенных, а также при особом подчеркивании или выделении какого-либо слова в предложении порядок слов может измениться» [9, с. 253]. «Инверсия помогает усилению содержания и эмоциональности речи. Такое положение особенно заметно в инверсии подлежащего и сказуемого» [18, с. 135]. ТОПИК ФОКУС a) Калкосто иштегем солдатқа парғанча. Тональный рисунок примера (2) (рис. 2) подтверждает, что фокусная часть высказывания находится после топи- кальной – на ней фиксируется максимальное понижение тона. Понижение тона отмечается и на главном сказуемом иштегем, поскольку оно располагается в конце синтагмы, а фокусной части предшествует длительная пауза. Как отме- чает Е. А. Шестера, в телеутском повествовательном пред- ложении «на слогах существительного… реализуется вос- ходящий тон, …на глаголе… – нисходящий, с различными вариациями мелодики отдельных слогов на фоне общего понижения основного тона» [17, с. 68]. в) Солдатқа парғанча калкосто иштегем. ТОПИК ФОКУС Результаты Базовым средством управления ИС в телеутском языке, как и в других тюркских языках, является порядок слов, в простом повествовательном предложении3 соответству- ющий модели SOV. Тональный рисунок примера (1) (рис. 1), выполненный в программе Praat, соответствует его линейной структуре. Частота основного тона (ЧОТ) падает по мере развертыва- ния фокусной части высказывания и достигает минимума на его последнем и коммуникативно наиболее нагруженном отрезке – аффиксе место-временного падежа, соответству- ющего русскому предлогу в. Пример (1) представляет собой экспрессивно- нейтральное простое предложение с типично тюркским SVO-синтаксисом. Для правильной интерпретации анафо- рического местоимения аны слушающему необходим кон- текст, понятный из предшествующего предложения: Алтон jылда паштық Фоменков Николай Сергеевич полғон, мени ийе перген ÿренеге, Кемеровский сельскохозяйственный техникумға, агрономғо (В шестидесятом году председателем Фоменков Повышение тона на сказуемом обусловлено следующим предложением, которое начинается с союза и придает при- меру (1) оттенок недосказанности. 2 Исследования отдельных уровней языка отражены в трудах кемеровской дериватологической школы, например в коллективной монографии [16] и пропозиционально-фреймовом словаре телеутского языка. 3 То есть содержащем одну предикативную единицу. (1) men anɨ alton peš jɨl-da tügez-ip iy-gem 1SG.PP 3SG.ACC шестьдесят пять год-LOC заканчивать-CVB AUX:посылать-PST2.1SG Мен аны алтон пеш jылда тÿгезип ийгем – Я окончил его в шестьдесят пятом году. Рис. 1. Тональный рисунок примера (1) Fig. 1. Tone visualization of example (1) (1) men anɨ alton peš jɨl-da tügez-ip iy-gem 1SG.PP 3SG.ACC шестьдесят пять год-LOC заканчивать-CVB AUX:посылать-PST2.1SG Мен аны алтон пеш jылда тÿгезип ийгем – Я окончил его в шестьдесят пятом году. tügez-ip iy-gem заканчивать-CVB AUX:посылать-PST2.1SG iy-gem AUX:посылать-PST2.1SG jɨl-da год-LOC p пять Мен аны алтон пеш jылда тÿгезип ийгем – Я окончил его в шестьдесят пятом году. Рис. 1. Тональный рисунок примера (1) Fig. 1. Tone visualization of example (1) 2 Исследования отдельных уровней языка отражены в трудах кемеровской дериватологической школы, например в коллективной монографии [16] и пропозиционально-фреймовом словаре телеутского языка. 3 То есть содержащем одну предикативную единицу. 2 Исследования отдельных уровней языка отражены в трудах кемеровской дериватологической школы, например в коллективной монографии [16] и пропозиционально-фреймовом словаре телеутского языка. 3 То есть содержащем одну предикативную единицу. 3 То есть содержащем одну предикативную единицу. Cтатья распространяется на условиях лицензии Creative Commons Attribution 4.0 271 271 Вестник Кемеровского государственного университета, 2020, 22(1) Вестник Кемеровского государственного университета, 2020, 22(1) Языкознание Языкознание DOI: 10.21603/2078-8975-2020-22-1-268-277 (2) kalkos-to ište-gem soldat-qa par-ɣan-ča колхоз-LOC работать-PST2.1SG солдат-DAT идти-PST2-DEL Калкосто иштегем солдатқа парғанча – В колхозе (я) работал до ухода в армию. Рис. 2. Тональный рисунок примера (2) Fig. 2. Результаты Поскольку топик и фокус являются шифтерными кате- гориями, возможны как инверсии синтагм с сохранением прогрессивной ИС (топик > фокус), так и инверсии компо- нентов ИС (фокус < топик) с сохранением порядка синтагм. Соответственно, пример (а) может иметь три дополнительных варианта реализации, отличающиеся порядком синтагм и / или компонентов ИС. Например, может быть инвертирован порядок компонентов ИС, фокус высказывания – «до каких пор говорящий работал в колхозе». ИС этого варианта совпа- дает с примером (а), но отличается экспрессивностью: Важным представляется предложенное К. Ламбрехтом разграничение прагматических и семантических субъектов и предикатов. Исследователь предлагает трехчастную струк- туру расположения фокуса в предложении в зависимости от его прагматики. Фокус может выражаться а) предика- том; б) аргументом; в) суждением в целом [4, р. 226–238]. Проиллюстрируем ее с помощью примера (2). б) Солдатқа парғанча калкосто иштегем. ФОКУС ТОПИК б) Солдатқа парғанча калкосто иштегем. ФОКУС ТОПИК ИС с фокусом предикат – ответ на вопрос: Солдатқа прағанча нени иштегең? (Чем ты занимался до ухода в армию?): Вариант с инвертированным относительно примера (а) порядком синтагм, фокус высказывания – «чем занимался говорящий до ухода в армию»: (2а) Солдатқа парғанча калкосто иштегем. в) Солдатқа парғанча калкосто иштегем. ТОПИК ФОКУС 272 Cтатья распространяется на условиях лицензии Creative Commons Attribution 4.0 Вестник Кемеровского государственного университета, 2020, 22(1) DOI: 10.21603/2078-8975-2020-22-1-268-277 Вестник Кемеровского государственного университета, 2020, 22(1) Языкознание DOI: 10.21603/2078-8975-2020-22-1-268-277 DOI: 10.21603/2078-8975-2020-22-1-268-277 относятся к сфере категории эвиденциальности. <…> Указание же на то, что информация об описываемой ситу- ации является неожиданной, "новой" для говорящего, относится к сфере категории миратива. В тюркских языках эвиденциальность и миративность выражается либо специ- ализированными средствами, либо совместно с другими значениями, модальными и аспектуальными» [19, с. 15]. К. В. Кичеева предлагает следующую типологию семантики эвиденциальных форм в хакасском языке: относятся к сфере категории эвиденциальности. <…> Указание же на то, что информация об описываемой ситу- ации является неожиданной, "новой" для говорящего, относится к сфере категории миратива. В тюркских языках эвиденциальность и миративность выражается либо специ- ализированными средствами, либо совместно с другими значениями, модальными и аспектуальными» [19, с. 15]. К. В. Кичеева предлагает следующую типологию семантики эвиденциальных форм в хакасском языке: Пресуппозиция: то, чем занимался говорящий до ухода в армию – топик для комментария Х Ассерция: Х = работал в колхозе Фокус: работал в колхозе Область фокуса: VP Пресуппозиция: то, чем занимался говорящий до ухода в армию – топик для комментария Х Ассерция: Х = работал в колхозе Фокус: работал в колхозе Область фокуса: VP Пресуппозиция: то, чем занимался говорящий до ухода в армию – топик для комментария Х Ассерция: Х = работал в колхозе Фокус: работал в колхозе Область фокуса: VP ФОКУС ИС с фокусом аргумент – ответ на вопрос: Қачанға jеттире калкосто иштегең? (До какого времени ты рабо- тал в колхозе?): (2б) Солдатқа парғанча калкосто иштегем. ИС с фокусом аргумент – ответ на вопрос: Қачанға jеттире калкосто иштегең? (До какого времени ты рабо- тал в колхозе?): ф р 1) пересказывательность (абсентив) – получение инфор- мации из косвенного источника; ФОКУС (2б) Солдатқа парғанча калкосто иштегем. ФОКУС (2б) Солдатқа парғанча калкосто иштегем. ИС с фокусом суждение – ответ на вопрос: Нени пол парды? (Что произошло?): ИС с фокусом суждение – ответ на вопрос: Нени пол парды? (Что произошло?): б) Солдатқа парғанча калкосто иштегем. ФОКУС ТОПИК ц ; 2) инференциальность – вывод говорящего о событии; Пресуппозиция: говорящий работал в колхозе до Х Пресуппозиция: говорящий работал в колхозе до Х Пресуппозиция: говорящий работал в колхозе до Х Ассерция: Х = уход в армию Фокус: до ухода в армию Область фокуса: NP 3) адмиративность (миративность) – констатация собы- тия в момент сообщения, неожиданность (удивление, восхищение, удовлетворенность) [20, с. 121]. Ассерция: Х = уход в армию Фокус: до ухода в армию Область фокуса: NP Ассерция: Х = уход в армию Фокус: до ухода в армию Область фокуса: NP Л. Йохансон выделяет в тюркских языках категорию индиректива (непрямой засвидетельствованности) в качестве эвиденциальной [21, р. 274], когда реципиентом коммуни- кативного акта выступает не только слушающий, но и сам говорящий, выражающий свое отношение к описываемой ситуации, ср. тел. Пу қорқышту пала тур! (Этот мальчик страшным, пожалуй, будет!), которое говорящий адресует самому себе и которое содержит фокусную (модально- эвиденциальную) частицу тур (пожалуй, однако же). Употребление этой частицы усиливает именное сказуемое қорқышту (страшный) и одновременно указывает на фокус высказывания. В ней совмещается как инференция (говоря- щий делает вывод, что мальчик – страшный), так и миратив (испуг и удивление говорящего фактом сообщения). р (2в) Солдатқа парғанча калкосто иштегем. ФОКУС (2в) Солдатқа парғанча калкосто иштегем. ФОКУС Пресуппозиция: – Ассерция: до ухода в армию говорящий работал в колхозе Фокус: до ухода в армию говорящий работал в колхозе Область фокуса: S В последнем примере отсутствие пресуппозиций приво- дит к совпадению ассерции и фокуса, которые распростра- няются на все высказывание в целом; оно, таким образом, приобретает событийный характер. В телеутском языке (как и в других тюркских языках Сибири) к ядерным способам выражения индиректива относится аффикс прошедшего т. н. «заглазного» времени -tIr, выражающий набор условно-эвиденциальных значений, приведенных К. В. Кичеевой для хакасского языка. К лексико-грамматическим способам управления ИС в телеутском языке можно отнести использование модальных слов и фокусных частиц, в меньшей степени – аспектуальных показателей. Как отмечает Л. А. Шамина, «значения, выражающие эксплицитное указание на источ- ник сведений говорящего о сообщаемой им ситуации, Индирективное значение абсентива реализуется в пере- сказе говорящим событий, свидетелем которых он не был (характерно для фольклорного нарратива): (3) abɨšqa ämegen-i-n čɨɣara sür-ip iy-tir старик старуха-POSS3-POSS.ACC PSTP: наружу гнать-CVB AUX-PST3 ФОКУС Абышқа эмегенин чығара сӱрип ийтир – Старик старуху свою прочь выгнал. ФОКУС Абам тÿрмеде отырған, ааң учун ол тирÿ jада қалтыр – Отец сидел в тюрьме, поэтому он остался жив. (4) aba-m türme-de otur-ɣan anɨŋ.üčün ol tirü jad-a qal-tɨr отец-POSS1 тюрьма-LOC сидеть-PST2 PSTP: поэтому 3SG живой жить-PTCP1 AUX-PST3 Индирективное значение инференциальности (говоря- щий делает умозаключение): (3) abɨšqa ämegen-i-n čɨɣara sür-ip iy-tir старик старуха-POSS3-POSS.ACC PSTP: наружу гнать-CVB AUX-PST3 Абышқа эмегенин чығара сӱрип ийтир – Старик старуху свою прочь выгнал. ФОКУС Абышқа эмегенин чығара сӱрип ийтир – Старик старуху свою прочь выгнал. Индирективное значение инференциальности (говоря- щий делает умозаключение): jad-a ФОКУС Абам тÿрмеде отырған, ааң учун ол тирÿ jада қалтыр – Отец сидел в тюрьме, поэтому он остался жив. ФОКУС Абам тÿрмеде отырған, ааң учун ол тирÿ jада қалтыр – Отец сидел в тюрьме, поэтому он остался жив. 273 273 Cтатья распространяется на условиях лицензии Creative Commons Attribution 4.0 Вестник Кемеровского государственного университета, 2020, 22(1) DOI: 10.21603/2078-8975-2020-22-1-268-277 Языкознание Индирективное значение миративности (модально- оценочная коннотация): Индирективное значение миративности (модально- оценочная коннотация): порядка слов, член предложения, выражающий рему, ста- новится в предсказуемную позицию. Например: Бовай // яр тувигә // барди… "Старик подошел к подножию обрыва". Здесь рема передается обстоятельством места яр тувигә» [13, с. 379]. «Любой член предложения, не исключая и субъекта, может быть поставлен непосредственно перед предикатом, если говорящий хочет обратить на него вни- мание своего собеседника» [23, с. 53]. И так апар албай қалтыр – И так и не смогли унести. (5) i tak apar al-bay qal-tɨr RUS.и так уносить AUX-CVB.NEG AUX-PST3 ФОКУС И так апар албай қалтыр – И так и не смогли унести. (5) i tak apar al-bay qal-tɨr RUS.и так уносить AUX-CVB.NEG AUX-PST3 ФОКУС В примерах (3)–(5) эвиденциальный показатель -tIr кос- венно указывает на то, что синтагма, в которой он употреблен, входит в фокус высказывания. В примере (4) на фокус также указывает сложный союз ааң учун (поэтому). Тем не менее ИС предложения в телеутском языке напрямую не зависит от эвиденциальности и формы на -tIr. Возможны ситуации, когда синтагма с -tIr будет входить в топик высказывания (в зависимости от его прагматики), как в примерах (6б)–(6г): (6а) Пир чанақ öлöң АРТЫҚ ПОЛ ҚАЛТЫР. Данная особенность отмечается как типологически характерная для всех тюркских языков: «Логическое уда- рение во фразе, как правило, находится непосредственно в позиции перед сказуемым» [24, с. 186]. «В состав ремы в тюркологии обычно выделяют смысловое ядро – пред- сказуемостный член или (при отсутствии такого члена) само сказуемое, на которое падает наибольшее логическое ударение» [25, с. 137]. Вернемся к рассмотрению гипотетической «фокусной» природы сказуемого на -tIr. По корпусным данным финитное сказуемое на -tIr более устойчиво в крайней правой позиции, в отличие от форм на -dI и -GAn. Это дает основание пред- положить, что синтагма, к которой относится форма на -tIr, будет чаще выступать в качестве фокуса высказывания, чем синтагмы с иными аспектуальными и модальными формами, хотя это требует корпусной верификации с учетом праг- матики предложений, поскольку в нейтральном контексте любое сказуемое будет попадать в фокус высказывания. ( ) р қ ң Одни сани сена ЛИШНИМИ ОКАЗАЛИСЬ. (6б) Пир чанақ ÖЛÖҢ артық пол қалтыр. Одни сани СЕНА лишними оказались. (6б) Пир чанақ ÖЛÖҢ артық пол қалтыр. Одни сани СЕНА лишними оказались. (6в) Пир ЧАНАҚ öлöң артық пол қалтыр. Одни САНИ сена лишними оказались. (6в) Пир ЧАНАҚ öлöң артық пол қалтыр. Одни САНИ сена лишними оказались. Список условных обозначений у (VP) – verb phrase, глагольная группа; (NP) – noun phrase, именная группа; (S) – sentence, предложение; (1) – 1 л.; (3) – 3 л.; (SG) – singular, единственное число; (POSS) – possessive, посессив; (PSTP) – postposition, послелог; (PP) – personal pronoun, личное местоимение; (ACC) – accusative, винительный падеж; (LOC) – locative, местно-временной падеж; (DAT) – dative, дательно-направительный падеж; (CVB) – converb, деепричастие; (AUX) – auxiliary, вспо- могательный глагол; (PST2) – past 2, давнопрошедшее время; (PST3) – past 3, неочевидное прошедшее время; (PTCP1) – participle 1, причастие настоящего длительного времени; (DEL) – delimitative, ограничительный падеж; (.) – кумуляция показателей. Интонационно фокусная часть простого повествова- тельного предложения характеризуется максимальным понижением частоты основного тона. Таким образом, акцентные и структурные характеристики телеутского взаимно связаны и обусловлены ИС предложения. В отличие Вестник Кемеровского государственного университета, 2020, 22(1) DOI: 10.21603/2078-8975-2020-22-1-268-277 Языкознание от чувашского языка, фокусная часть телеутского пред- ложения характеризуется понижением ЧОТ. Лексико- грамматические средства управления ИС в телеутском языке располагаются, в отличие от синтаксиса и просодии, на периферии функционально-семантических полей4. Они совмещают функции выражения элементов ИС с иными лексико-грамматическими категориями. ЧОТ на слове человек. В телеутском, кроме интонации, сигнальную функцию выполняет числительное пир (один), придающее слову кижи (человек) семантику неопределен- ности и неидентифицируемости. Соответственно, именно эта синтагма относится к «новой» информации, которая должна быть добавлена в ментальную репрезентацию слу- шающего, а числительное пир в ненумеративной функции указывает на фокус. Интерес для дальнейшего исследования представляет изучение лексико-грамматических средств телеутского языка с позиций выражения ИС (например, грамматиче- ских категорий перфектива и кунктатива, падежных форм, кондиционалиса и т. д.), изучение ИС предложений других типов (вопросительных и побудительных), а также возмож- ное влияние русского языка на стратегию кодирования ИС телеутского простого предложения. Заключение Телеутский язык обнаруживает изоморфизм способов и стра- тегий управления ИС простого предложения с другими тюркскими языками. Как в языке типа SOV, в котором сказуе- мое занимает терминальное правое положение, топикальные и фокусные части простого предложения в телеутском языке линейно не обособлены, фокусная часть (комментарий) и особенно его смысловое ядро тяготеет вправо и примы- кает к сказуемому, за исключением случаев, когда сказуемое выражает непосредственно фокус предложения. При вхож- дении сказуемого в топик предложения (превращения его в «прагматический субъект» по К. Ламбрехту), оно может менять свою традиционную терминальную позицию и при- мыкать к другому топикальному элементу в составе синтагмы. Таким образом, порядок слов (синтагм) играет определяю- щую роль в ИС телеутского простого предложения. (6г) ПИР чанақ öлöң артық пол қалтыр. ОДНИ сани сена лишними оказались. (6г) ПИР чанақ öлöң артық пол қалтыр. ОДНИ сани сена лишними оказались. (6г) ПИР чанақ öлöң артық пол қалтыр. ОДНИ сани сена лишними оказались. Ранее отмечалось, что, согласно К. Ламбрехту, ИС пред- ложения состоит из трех взаимно связанных оппозиций: пресуппозиция – ассерция, топик – фокус и узнаваемость – активация. Поэтому интересно также рассмотреть средства, кодирующие узнаваемость (identifiability) либо неопреде- ленность (unidentifiability) участника ситуации. В тюркских языках одним из таких средств является числительное bir (один) в функции неопределенного артикля. Пример оппозиций из турецкого языка: Ahmet oküzü aldi (Ахмет купил (этого) быка) – Ahmet bir oküz aldi (Ахмет купил (одного; некоторого) быка) [4, р. 85]. Пример (6а) иллюстрирует тот факт, что при неинверти- рованном порядке синтагм в тюркских языках фокус выска- зывания располагается на глагольной группе, а логический акцент – на той части, которая непосредственно примыкает к сказуемому. Это доказывается ровным тоном синтагмы подлежащего и его падением на синтагме сказуемого. Тон начинает понижаться на слове артық (лишний) и продолжа- ет – на сказуемом пол қалтыр (оказался). Понижающийся тон между синтагмами называется хезитацией (рис. 3). На казахском материале это показано Р. С. Амировым: «В казахском языке, так же как и в других тюркских языках, есть одна позиция, определяющая актуализацию, – пред- сказуемостная позиция. Эта позиция является актуали- зирующей для всех несказуемостных членов. Сказуемое не подвергается специальному приему актуализации. Оно по своей природе является тем членом, который всегда заключает в себе новое сообщение» [22, с. 38]. Это также характерно для уйгурского и туркменского языков: «Если актуальное членение предложения выражается с помощью Это же характерно и для телеутского, например, пир сӧс (какое-то слово), пир пеш-алты (примерно пять-шесть) и т. д. (7) Кöрзö – ағаштың тöзинде ПИР КИЖИ отур jат. Смотрит – под деревом ЧЕЛОВЕК сидит. Фокус высказывания приходится на именную группу пир кижи (какой-то человек), примыкающую к предикату. В без- артиклевом русском языке фокус выражается понижением 274 Cтатья распространяется на условиях лицензии Creative Commons Attribution 4 0 Рис. 3. Тональный рисунок примера (6а) Fig. 3. Tone visualization of example (6а) Рис. 3. Тональный рисунок примера (6а) Fig. 3. Tone visualization of example (6а) Cтатья распространяется на условиях лицензии Creative Commons Attribution 4.0 274 4 Термин функционально-семантическое поле носит условный характер из-за отсутствия единого мнения о том, является ли ИС грамматической либо прагматической категорией. Литература р ур 1. Chafe W. L. Givenness, contrastiveness, definiteness, subjects, topics, and point of view // Subject and Topic / ed. C. N. Li. N. Y.: Academic Press, 1976. P. 27–55. 2. Падучева Е. В. Коммуникативная структура и линейно-акцентные преобразования предложения (на материале русского языка) // Архитектура клаузы в параметрических моделях: синтаксис, информационная структура, порядок слов / под ред. А. В. Циммерлинга, Е. А. Лютиковой. М.: Издательский дом ЯСК, 2016. С. 25–75. 3. Тестелец Я. Г. Введение в общий синтаксис. М.: Рос. гос. гуманитар. ун-т, 2001. 796 с. у р у 4. Lambrecht K. Information structure and sentence form. Cambridge: Cambridge University Press, 1994. 388 p. 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Щура. 2-е изд., стер. М.: Едиториал УРСС, 2003. 426 с. ф ру ур р ур р 9. Мусаев К. М. Синтаксис караимского языка. М.: Academia, 2003. 347 с. 10. Тугушева Л. Ю. О порядке слов в тюркских языках (к постановке вопроса) // Тюркологический сборник / под ред. С. Г. Кляшторного. М.: Наука, ГРВЛ, 1966. С. 120–127. р у 11. Дмитриев Н. К. Грамматика башкирского языка. М.-Л.: АН СССР, 1948. 276 с. 12. Дмитриев Н. К. Грамматика кумыкского языка. М.-Л.: АН СССР, 1940. 206 с. р р у 13. Строй уйгурского языка / отв. ред. Г. С. Садвакасов. Алма-Ата: Наука КазССР, 1989. 470 с. Cтатья распространяется на условиях лицензии Creative Commons Attribution 4.0 275 Bulletin of Kemerovo State University, 2020, 22(1) Linguistics DOI: 10.21603/2078-8975-2020-22-1-268-277 14. Емельянова А. В. Литература Коммуникативная структура чувашского простого повествовательного предложения: автореф. дис.… канд. филол. наук. Чебоксары, 1999. 23 с. 15. Токтарова Н. К. 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DOI: https://doi.org/10.1075/tsl.54.15joh p jt p g j 22. Амиров Р. С. Способы актуального членения в казахском языке // Советская тюркология. 1970. № 6. р у // р 97 34 3 23. Поцелуевский А. П. Основы синтаксиса туркменского литературного языка. Ашхабад: ТуркменОГИЗ, 1943. 100 с. 23. Поцелуевский А. П. Основы синтаксиса туркменского литературного языка. Ашхабад: ТуркменОГ 23. Поцелуевский А. П. Основы синтаксиса туркменского литературного языка. Ашхабад: ТуркменОГИЗ, 1943. 100 с. 24. Баскаков Н. А. Историко-типологическая характеристика структуры тюркских языков: словосочетание и предло- жение. 2-е изд., стер. М.: URSS КомКнига, 2006. 285 c. 24. Баскаков Н. А. Историко-типологическая характеристика структуры тюркских языков: словосочетание и предло- жение. 2-е изд., стер. М.: URSS КомКнига, 2006. 285 c. р 25. Убрятова Е. А., Ефремов Н. Н., Неустроев Н. Н., Алексеев И. Е., Петров Н. Е., Васильев Ю. И. Грамматика совре- менного якутского литературного языка. Т. 2: Синтаксис. Новосибирск: Наука, 1995. 336 с. * This work was supported by the Russian Foundation for Basic Research, grant No 18-012-00775 A "Typology of a simple sentence in the languages of the Ob-Yenisei language range: information and argument structure". DOI: 10.21603/2078-8975-2020-22-1-268-277 DOI: 10.21603/2078-8975-2020-22-1-268-277 DOI: 10.21603/2078-8975-2020-22-1-268-277 For citation: Tokmashev D. M. Simple Sentence Information Structure in Teleut: Problem Statement. Vestnik Kemerovskogo gosudarstvennogo universiteta, 2020, 22(1): 268–277. (In Russ.) DOI: https://doi.org/10.21603/2078-8975-2020-22-1-268-277 For citation: Tokmashev D. M. Simple Sentence Information Structure in Teleut: Problem Statement. Vestnik Kemerovskogo gosudarstvennogo universiteta, 2020, 22(1): 268–277. (In Russ.) DOI: https://doi.org/10.21603/2078-8975-2020-22-1-268-277 References References 1. Chafe W. L. Givenness, contrastiveness, definiteness, subjects, topics, and point of view. Subject and Topic, ed. Li C. N. N. Y.: Academic Press, 1976, 27–55. 1. Chafe W. L. Givenness, contrastiveness, definiteness, subjects, topics, and point of view. Subject and Topic, ed. Li C. N. N. Y.: Academic Press, 1976, 27–55. 2. Paducheva E. V. Communicative structure and linear-accentual sentence transformations (based on Russian). Clause architecture in parametric models: syntax, information structure, word order, eds. Tsimmerling A. V., Liutikova E. A. Moscow: Izdatelskii dom IaSK, 2016, 25–75. (In Russ.) ( ) 3. Testelets Ia. G. An introduction to general syntax. Moscow: Ros. gos. gumanitar. un-t, 2001, 796. (In Russ.) 4. Lambrecht K. Information structure and sentence form. Cambridge: Cambridge University Press, 1994, 388. D CBO9780511620607 5. Matić D. Information structure in linguistics. International Encyclopedia of the Social and Behavioral Sciences, ed. Wright J. D. Amsterdam: Elsevier, 2015, vol. 12, 95–99. DOI: 10.1016/B978-0-08-097086-8.53013-X g J 6. Krifka M. Basic notions of information structure. Interdisciplinary Studies on Information Structure, eds. Fery C., Fanselow G., Krifka M. Potsdam: Potsdam University Press, 2007, vol. 6, 13–55. k y 7. Stalnaker R. C. Context and content: essays on intentionality in speech and thought. N. Y.: Oxford University Press, 1999, 294. 8. Chafe W. L. Meaning and the structure of language, tr. Shchur G. S., 2nd ed. Мoscow: Editorial URSS, 2003, 426. (In Russ.) 9. Musaev K. M. Karaim syntax. Мoscow: Academia, 2003, 347. (In Russ.) Meaning and the structure of language, tr. Shchur G. S., 2nd 8. Chafe W. L. Meaning and the structure of language, tr. Shchur G. S., 2nd ed. Мo g g g 9. Musaev K. M. Karaim syntax. Мoscow: Academia, 2003, 347. (In Russ.) 9. Musaev K. M. Karaim syntax. Мoscow: Academia, 2003, 347. (In Russ.) y 10. Tugusheva L. Iu. On the word order in Turkic languages (a problematic approach). Turkic collection, ed. Kliashtornyi S. G. Moscow: Nauka, GRVL, 1966, 120–127. (In Russ.) 11. Dmitriev N. K. Grammar of Bashkir. Moscow-Leningrad: Nauka, 1948, 276. (In Russ.) 12. Dmitriev N. K. Grammar of Qumyk. Moscow-Leningrad: Nauka, 1940, 206. (In Russ.) 13. Uighur language system, ed. Sadvakasov G. S. Alma-Ata: Nauka KazSSR, 1989, 470. (In Russ.) 14. Emelianova A. V. Communicative structure of the simple declarative sentence in Chuvash. Cand. Philol. Sci Cheboksary, 1999, 23. (In Russ.) 15. Toktarova N. K. Communicative sentence types in Qumyk. Dr. Philol. Sci. Diss. Simple Sentence Information Structure in Teleut: Problem Statement* Denis M. Tokmashev a, @, ID a National Research Tomsk Polytechnic University, Russia, Tomsk @ kogutei@yandex.ru ID1 https://orcid.org/0000-0003-3941-043X Received 22.01.2020. Accepted 12.02.2020. Received 22.01.2020. Accepted 12.02.2020. Abstract: The present research featured the main approaches to the study of the information structure of a simple sentence, as well as their application to the Turkic languages. The paper focuses on the case of the Teleut language. The research objective was to identify and characterize various types of information structure of a simple sentence in their relationship with formal- grammatical and prosodic characteristics. The study involved field, comparative-historical, and descriptive methods, structural and component analysis, methods of modeling semantics and visualization of spectrograms. The information structure of a simple sentence can be modeled as the corresponding functional-semantic field. In Teleut, it is represented mostly by syntagm order and intonation, which make the core of information structure management. The peripheral means are represented by lexemes, particles, and affixes. Syntactically, information structure is expressed by the phrase order. Narrative sentences are characterized by the decrease of the fundamental frequency of the phrase that makes up the focal part. Pragmatically neutral narrative sentences that do not have presuppositions are characterized by a progressive arrangement of topical and focal elements with a predicate in the terminal right position. Since topics and foci are shifter categories, syntax inversions with preservation of the progressive information packaging "topic > focus" are possible, as well as inversion of its components "focus < topic" while retaining the phrase order. The inversion of both linear (syntagms) and non-linear (topics and foci) elements of the sentence is due to various presuppositions. The lexical and grammatical means of information packaging management are on the periphery of the functional-semantic field. Their potential to control the information structure is combined with their other functions, namely the expression of aspectual, modal, evidential, definiteness, and other characteristics. Most Turkic languages share the means of information packaging management. Keywords: intonation, syntax, Turkic languages, actual division of the sentence, acoustic phonetics * This work was supported by the Russian Foundation for Basic Research, grant No 18-012-00775 A "Typology of a simple sentence in the languages of the Ob-Yenisei language range: information and argument structure". This article is distributed under the terms of the Creative Commons Attribution 4.0 International License 276 Linguistics Bulletin of Kemerovo State University, 2020, 22(1) This article is distributed under the terms of the Creative Commons Attribution 4.0 International License References Abstr. Makhachkala, 2011, 41. (In Russ.) 16. The language picture of the world of Teleuts, ed. Araeva L. A. Kemerovo: KemGU, 2016, 237. (In Russ.)t 17. Shestera E. A. Teleut: Intonation of declarative and interrogative utterances. Voprosy yazykoznaniya, 2014, (2): 61–75. (In Russ.) 18. Urinbaev B. U. Syntactic system of the Uzbek colloquial speech. Tashkent: Fan, 1978, 144. (In Russ.) 19. Shamina L. A. System of bipredicative constructions with non-finite verbal forms in the Turkic languages Dr. Philol. Sci. Diss. Abstr. Novosibirsk, 2004, 50. (In Russ.) 19. Shamina L. A. System of bipredicative constructions with non-f Dr. Philol. Sci. Diss. Abstr. Novosibirsk, 2004, 50. (In Russ.) 20. Kicheeva K. V. Verbal forms the meaning of evidentiality in the Khakass language. Filologicheskie nauki. Voprosy teorii i praktiki, 2018, (1-1): 121–124. (In Russ.) p 21. Johanson L. Evidentiality in Turkic. Studies in Evidentiality, eds. Aikhenvald A. Y., Dixon R. M. W. Amsterdam-Philadelphia: John Benjamins PC, 2003, 273–290. DOI: https://doi.org/10.1075/tsl.54.15joh J jt p g j 22. Amirov R. S. Ways of actual segmentation in the Gazakh language. Sovetskaya tyurkologiya, 1970, (6): 34–38. (In Russ.) 23. Potseluevskii A. P. Basics of Turkmen literary syntax. Ashgabad: TurkmenOGIZ, 1943, 100. (In Russ.) t 22. Amirov R. S. Ways of actual segmentation in the Gazakh language. Sovetskaya tyurkologiya, 1970, (6): 34–3 . S. Ways of actual segmentation in the Gazakh language. Sovetskaya tyurkologiya, 1970, (6): 34 38. (In Russ.) skii A. P. Basics of Turkmen literary syntax. Ashgabad: TurkmenOGIZ, 1943, 100. (In Russ.) 23. Potseluevskii A. P. Basics of Turkmen literary syntax. Ashgabad: TurkmenOGIZ, 1943, 100. (In Russ.) 24. Baskakov N. A. Historical and typological characteristics of Turkic languages' structure: phrase and sentence, 2nd ed. Moscow: URSS KomKniga, 2006, 285. (In Russ.) 25. Ubriatova E. A., Efremov N. N., Neustroev N. N., Alekseev I. E., Petrov N. E., Vasilev Iu. I. A grammar of contemporary literary Yakut. Vol. 2: Syntax. Novosibirsk: Nauka, 1995, 336. (In Russ.) 277 This article is distributed under the terms of the Creative Commons Attribution 4.0 International License 277
https://openalex.org/W4381890130	https://link.springer.com/content/pdf/10.1140/epja/s10050-023-01050-3.pdf	English	null	Method to evidence hypernuclear halos from a two-target interaction cross section measurement	European physical journal. A, Hadrons and nuclei	2,023	cc-by	7,715	Method to evidence hypernuclear halos from a two-targe interaction cross section measurement Simone Velardita1,a , Hector Alvarez-Pol2, Thomas Aumann1,3,4, Yassid Ayyad2, Meytal Duer1, Hans-Werner Hammer1,4, Liancheng Ji1,4, Alexandre Obertelli1,4, Yelei Sun1,4 1 Technische Universität Darmstadt, Fachbereich Physik, Darmstadt 64289, Germany 2 Universidade de Santiago de Compostela, Santiago de Compostela, 15782 Santiago, Spain 3 GSI Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt 64291, Germany 4 Helmholtz Forschungsakademie Hessen für FAIR, Frankfurt 60438, Germany Received: 26 January 2023 / Accepted: 6 June 2023 / Published online: 23 June 2023 © The Author(s) 2023 Communicated by Takashi Nakamura Received: 26 January 2023 / Accepted: 6 June 2023 / Published online: 23 June 2023 © The Author(s) 2023 Communicated by Takashi Nakamura Received: 26 January 2023 / Accepted: 6 June 2023 / Published online: 23 June 2023 © The Author(s) 2023 Communicated by Takashi Nakamura Abstract We present a two-target measurement method to determine the interaction cross section of hypernuclei with a target nucleus. The method allows to extract from two independent measurements the production cross section of a given hypernucleus as well as its interaction cross section on a speciﬁc target. The latter is then further analyzed to deduce the matter radius of the hypernucleus. The sensitivity of the method has been investigated for the speciﬁc case of the lightest hyperhalo candidate hypertriton (3 H) produced from 12C+12C collisions at 1.9 GeV/nucleon. Furthermore, its feasibility is demonstrated by detailed simulations for realistic experimental conditions at GSI/FAIR, using a dedi- cated HYDRA (HYpernuclei Decay at R3B Apparatus) time- projection chamber prototype. A precision of 15% or better in the interaction cross section can be achieved, allowing an extraction of the unknown 3 H matter radius and assessing its halo or non-halo character. with nucleons and create short-lived hypernuclei [5]. The extended exploration of the hypernuclear landscape opens a new area for nuclear structure and many-body baryonic interaction studies. Thanks to the absence of Pauli blocking with nucleons, a hyperon can occupy any orbital inside the nucleus and can be used as a probe of the inner nuclear den- sities [6], barely accessible otherwise with the exception of electromagnetic probes only applicable to stable nuclei. The measured binding energies and energy differences between spin doublet states in hypernuclei give precise information on the YN interaction [7]. Study of YY interactions at satura- tion density is also expected by measuring the ﬁne structure of double- hypernuclei [8]. Hypernuclei presenting a halo have been predicted (see e.g. [9–12]). Method to evidence hypernuclear halos from a two-targe interaction cross section measurement A nuclear halo is an intriguing quantum tun- nelling phenomenon observed in nuclei at the dripline. The notable feature of halo nuclei is the large nuclear size due to the extended distribution of the halo nucleon(s) outside the region authorized by classical mechanics, typically larger than 50% probability [13]. The hypertriton 3 H is an excellent candidate for a halo in its ground state since the is only bound by 130 ± 50(stat.) ± 40(sys.) keV [14] to a deuteron, a reference value from emulsion measurements. Meanwhile, a recent STAR measurement resulted in 410 ± 120(stat.) ± 110(sys.) keV [15], whereas a smaller value of 72 ± 63(stat.) ± 36(sys.) keV [16] was obtained by the ALICE collabora- tion. Due to the weak binding, the lifetime of 3 H is expected to be not signiﬁcantly different from that of the free which is still under debate due to the dispersion of different mea- surements [16–20], although the most recent experimental value of 253 ± 11(stat.) ± 6(sys.) ps [16] by the ALICE col- laboration is compatible with the free lifetime. Unlike the halo in exotic nuclei, the spatial extension of hypernuclear a e-mail: svelardita@ikp.tu-darmstadt.de (corresponding author) Eur. Phys. J. A (2023) 59:139 https://doi.org/10.1140/epja/s10050-023-01050-3 Eur. Phys. J. A (2023) 59:139 https://doi.org/10.1140/epja/s10050-023-01050-3 New Tools and Techniques 2.1 General description The method allows to measure the interaction cross sections of hypernuclei (A X) with a target nucleus and from these infer their matter radii. Hereby, the term ’interaction cross section’ refers to all reactions that lead to a ﬁnal state that is different from the initial hypernucleus in a bound state. The population of hypernuclei inside the target region depends on two main processes: (i) the beam interacts with the primary target and according to the production cross sec- tion σ of the hypernuclei (unknown, since only few data with large uncertainties exist) a certain yield of hypernuclei is produced, (ii) this yield is then attenuated by the interac- tion with the target nuclei, according to the interaction cross section σR of the hypernucleus with the target (unknown) or decay according to its lifetime τ. To obtain the two unknown cross sections, it is proposed to perform two separate mea- surements. In principle, there are several experimental con- ﬁgurations that can be considered to reach this objective. The ﬁrst possibility includes a single measurement using two tar- getsofthicknesses,d1 andd2,separatedbyaﬂightgapL.This conﬁguration requires less beam time since the measurement from both targets is done only once. However, distinguish- ing whether the primary vertex is allocated inside target 1 or 2 is technically challenging, and would require a high- granularity high-rate tracking detector placed in between the two targets. The second conﬁguration introduces two inde- pendent measurements using the same beam and two targets of the same material but with increasing thickness. Although it requires a longer beam time, reconstruction of the decay vertex distribution (DVD) can be obtained with high accu- racy as it is done with two independent measurements. The third conﬁguration includes two independent measurements using the same target but two different beams with differ- ent hypernuclei production cross-section. It requires a long beam time and since the hypernuclei production cross section is low (∼μb) such an option would require a measurement with a production cross section signiﬁcantly lower than the other, i.e., extremely beam-time consuming. For these rea- Measuring the matter radius of hypernuclei is challeng- ing for two main reasons: their low production cross section and their sub-nanosecond lifetime. 1 Introduction Hyperons (, , , ) are baryons with at least one strange valence quark. The lightest hyperon, , can only decay into a pion and a nucleon through the weak interaction, while the strong interaction conserves strangeness. The very short lifetime of hyperons (263 ps for the free [1]) makes it technically extremely difﬁcult to perform scattering or cap- ture experiments with hyperon beams to study the hyperon- nucleon (YN) and hyperon-hyperon (YY) interactions. Few p scattering data exist [2] and recent femtoscopy measure- ments provide new constraints on the bare YN and YY interac- tions [3,4]. Interestingly, hyperons can form bound systems 12 3 3 Eur. Phys. J. A (2023) 59 :139 139 Page 2 of 8 139 Page 2 of 8 halos has not been directly studied experimentally so far. It is expected that the glue-like role of a hyperon can facilitate the existence of hypernuclear neutron or proton halo state if the core nucleus is weakly unbound [10]. Two such inter- esting candidates are 6 He (Sn = 0.17 MeV) and 7 Be (S2p = 0.67 MeV) [10]. Exploring these weakly bound hypernuclear systems can provide a unique opportunity to study the fun- damental hyperon-nucleon interactions, which are not well constrained due to the lack of scattering data. New informa- tion on exotic hypernuclei would reveal facets of the YN and YNN interactions and would be an important benchmark for ab initio theories [21]. sity tail of the wavefunction is the electromagnetic response or Coulomb cross section, as well known from studies of halo nuclei [34]. A quantitative prediction of the effect for the case of the hypertriton has been made recently in Ref. [35]. In the following, we present a method adapting the interaction-cross section measurement to hypernuclei. We demonstrate that a reasonable sensitivity to the interaction cross section can be achieved and give quantitative constraint on the matter radius of hypertriton. 2 The two-target method Light hypernuclear halos might be a key probe to under- stand the formation of nuclei in relativistic heavy-ion col- lisions, as currently performed at LHC with ALICE and at FAIR with CBM in the future. Indeed, coalescence mod- els have been extensively used to describe the formation of composite objects (see e.g. [22–25]). Surprisingly, thermal- statistical models have been successful in describing the pro- duction of light (anti-)(hyper-)nuclei across a wide range of energies in A + A collisions [26,27]. The nucleosynthesis mechanism in relativistic ion collisions might be revealed by light halo hypernuclei under the condition that their basic fea- tures such as size and binding energy are known [28]. Indeed, thermal + blast-wave and coalescence models give very dif- ferent predictions for the production of 3 H as a function of the source size in the case 3 H has an extended wave function. Therefore, it is essential to determine the hypertriton matter radius experimentally to be used as an input, and not a free parameter, in coalescence models. Predictions for the matter radius of hyperhalo 3 H vary between 4–10 fm, depending on the binding energy. The structure of the hypertriton was stud- ied within the pionless effective ﬁeld theory (EFT), where a strong correlation between the binding-energy and its matter radius was found [29]. Considering the reference separation energy of 130 keV gives a matter radius of 4.9 fm. Lower binding-energy leads to higher values of the matter root mean square (rms) radius that can reach up to 10 fm [28]. 2.1 General description Although there are dif- ferent experimental methods to determine the matter radius of a nucleus, some of them require a high luminosity and are only applicable to stable nuclei, such as parity-violating elec- tron scattering [30], or coherent photo-pion production [31]. Proton elastic scattering is in principle applicable to short- lived nuclei [32] but still requires beam intensities close to or above 104 particles per second (pps). In the case of very low intensities, the measurement of interaction or reaction cross sections of a projectile with an ion target can lead to a quan- titative assessment on its matter radius. This was historically pioneered by Tannihata et al. for the two neutron-halo 11Li [33]. Another observable being sensitive to a halo-like den- 123 Eur. Phys. J. A (2023) 59 :139 Page 3 of 8 139 Fig. 1 Yield of hypernucleus A X along the ﬂight path assuming dif- ferent interaction cross sections (different curves), and a 12C beam at 1.9 GeV/nucleon with intensity of 106 pps impinging on a carbon target of thickness d = 5 cm (blue shaded band) nuclei can be formulated analytically: nuclei can be formulated analytically: nuclei can be formulated analytically: ⎧ ⎪⎨ ⎪⎩ dN(z)= −δ nσR N(z) dz , dN(z)= −N(z) γβcτ dz+δ nσN(z)−nσR N(z) dz , ( (2) where δ = 0, 1 (0 in free space and 1 inside a target), σR is the reaction cross section of the beam projectiles with the targets, n the number density of the targets, β the velocity of the A X in the laboratory frame, γ its Lorentz factor, τ its lifetime and c the speed of light. The boundary conditions are N(0) = 0 and N(0) = N0 = I · t, where I is the beam intensity and t is the total measurement time. Fig. 2.1 General description 1 Yield of hypernucleus A X along the ﬂight path assuming dif- ferent interaction cross sections (different curves), and a 12C beam at 1.9 GeV/nucleon with intensity of 106 pps impinging on a carbon target of thickness d = 5 cm (blue shaded band) Once the DVD of A X along the beam axis (z) has been extracted, it is of particular interest to extrapolate the popu- lations at the exit of the targets, where the contribution from their interaction is the largest, this corresponds to N(d1) from the ﬁrst measurement and N(d1 + L +d2) for the sec- ond one. These two quantities can be analytically calculated using the set of equations deﬁned in Eq. 2. By taking the ratio between the two populations, we eliminate the explicit dependence on σ. Re-arranging the terms one gets the fol- lowing analytical formulation of the A X interaction cross section: sons, we focus here on a method based on two separate mea- surements with identical beam and with two different target thicknesses of the same material, which will allow to access both σ and σR. For simplicity, the interaction cross section of projectile-like hypernucleus (A X) with a target (AY) can be expressed by the geometrical cross section σR = π[R(A X) + R(AY)]2 , (1) (1) where R(AY) = R0 A 1 3 and R0 = 1.25 fm. For the 3 H, the interaction cross section will be analysed in Sect. 2.3 within the eikonal formalism to obtain a microscopic connection to its matter radius. A (1 −e−B(σR) d1)e − L γβτc +B(σR) d2 −A e−B(σR) d2 + e−B(σR) d1 + A −1 = 0 , (3) Hypernuclei produced will be reconstructed via the invariant-mass method by measuring the weak decay prod- ucts in the ﬁnal state. The observable that will be used to determine the interaction cross section is the mesonic DVD along the ﬂight path downstream the target. The sensitivity of the DVD to σR (and consequently to the matter radius) is demonstrated in Fig. 1 for a generic hypernucleus, assum- ing: τ =200 ps, σ = 1.8 μb (see Sect. 2.3) and different interaction cross sections σR = 0 b, 1 b, 5 b. A sudden drop in the DVD can be seen due to the interaction downstream the target. 2.1 General description (3) where, to help the readability of the equation, the following substitutions have been used B(σR) = nσR + 1 γβcτ −nσR , A = N(d1) N0,d2 N(d1 + L + d2) N0,d1 · e−nσR d2 , Below, a description of the method is presented, as well as its sensitivity for the speciﬁc case of 3 H by an estimate of the uncertainties of its interaction cross section. N0,d1 = I ·t α is the number of beam projectiles that impinge on the ﬁrst target (z = 0) for the ﬁrst measurement, N0,d2 = N0,d1 = I ·t α is the number of beam projectiles that impinge on the ﬁrst target (z = 0) for the ﬁrst measurement, N0,d2 = I · t (1 −α) is that on the second target (z = d1 + L) for the second measurement and α represents the share of the total beam time among the two measurements, i.e., 0 < α ≤1. Note that the ratio between the two populations is contained in variable A, while the processes that affect the hypernuclei populations are contained in the function B(σR). N0,d1 = I ·t α is the number of beam projectiles that impinge on the ﬁrst target (z = 0) for the ﬁrst measurement, N0,d2 = I · t (1 −α) is that on the second target (z = d1 + L) for the second measurement and α represents the share of the total beam time among the two measurements, i.e., 0 < α ≤1. Note that the ratio between the two populations is contained in variable A, while the processes that affect the hypernuclei populations are contained in the function B(σR). 2.3 Sensitivity in the case of 3 H+12C at 1.9 GeV/nucleon 2.3 Sensitivity in the case of 3 H+12C at 1.9 GeV/nucleon The sensitivity of the method is investigated for the case of the hypertriton (3 H), where the considered production and decay channels are 12C + 12C →X + 3 H and 3 H → 3He + π−(branching ratio, BR = 26% [36]), respectively. As presented in Sect. 1, it is of particular interest to study the size of 3 H, the lightest predicted hyperhalo. In such a case, we cannot consider here the traditional geometrical interaction cross section of hypernucleus (Eq. 1), since the hypertriton is expected to be a dilute object for which the black-disk limit may not be suited. Instead, the correlation between the measured interaction cross section and the matter radius of 3 H will be analysed with microscopic wave functions and the eikonal formalism, valid at the considered incident energies, beyond the simplistic geometrical ansatz. The required inputs for the calculation are (i) the density distribution of 12C, (ii) the density distributions of the and the deuteron in the center of mass of 3 H, (iii) proton-neutron and proton-proton total cross sections, (iv) -nucleon total cross sections. Nucleon-nucleon cross sections at energies of ∼2 GeV have been measured [37], and the density distribution of 12C can be considered well known, in par- ticular, its charge density distribution from precision (e, e) measurements [38]. The neutron density distribution can be considered identical, as a good approximation. The - nucleon total cross sections have been measured [39,40] and show a ﬂat behaviour over a large range of energies. The measured values, ﬁtted over momentum p in GeV/c, of σ(p) = (34.3 ± 1.5) mb −p−1(−3.8 ± 17.6) mb GeV/c and σ(n) = (34.1 ± 3) mb −p−1(33 ± 35) mb GeV/c are close to the nucleon-nucleon total cross sections. Note that at the energies relevant in this paper, the total cross section is expected to reﬂect the size of the colliding baryons, and show little momentum dependence (less than 1%), consistent with the above mentioned measurements. The radial density distributions for the neutron, proton and in hypertriton are taken from theory. In the case of the pionless EFT, the rms radius of 3 H can be tuned by modifying the separation energy, as illustrated in [29]. 2.2 Analytical formulation In the following, the second experimental conﬁguration will be analyzed: the ﬁrst measurement is done using only one cylinder of thickness d1 and a second one with two cylin- ders of thicknesses, d1 and d2, separated by a ﬂight gap L. For L=0, this corresponds to two independent measurements with a target thickness d1 and a target of thickness d1 + d2. Assuming that all particles propagate along the beam axis (z), the population N(z) of beam particles and N(z) of hyper- In addition, for the case L = 0, Eq. 3 can be re-written as: e−B(σR) d1 −A e−B(σR) (d1+d2) −1 + A = 0 , (4) (4) where A = N(d1)N0,d2 N(d1 + d2)N0,d1 · e−nσR d2. 3 139 Page 4 of 8 Eur. Phys. J. A (2023) 59 :139 Thestatisticsoftheexperimentplaysafundamentalrolein the proposed method and determines the choice of the beam and targets. For the purpose of this sensitivity study, we con- sider realisticbeamconditions at GSI/FAIR[41]: beaminten- sity I = 106 pps, beam energy Ebeam = 1.9 GeV/nucleon, and a total beam time t = 1 day. At this energy the measured reaction cross section 12C+12C equals σR = 888 ± 19 mb [42]. The method is general and applicable to other beams and different energies under the condition that it is above the production threshold of 1.6 GeV/nucleon (elementary process N N →K N). In terms of the experimental setup, the parameters taken into consideration are: the target thick- nesses (d1 and d2), the ﬂight gap (L), and the share of total beam time among the two measurements (α). The mean free path of 12C in the target is calculated to be λ = 1/(σRn) = 10 cm. In the case of a pronounced halo, the mean free path of the hypertriton is expected to be also in the order of 10cm, which therefore, limits the maximum target thicknesses to avoid too many reactions. We analyze the contribution of these parameters to the uncertainty of the σR (δσR) considering a lifetime τ(3 H)= 223+12 −11 ps [43]. The trend of the ﬁgures presented in this section is not affected by the choice of the lifetime, therefore the conclusions drawn from the method will remain unchanged. Note that Eq. 3 cannot be solved analytically, therefore the numerical bisection method [44] was used. 2.3 Sensitivity in the case of 3 H+12C at 1.9 GeV/nucleon 2 Relative uncertainty of hypertriton interaction cross section (δσR/σR) with 12C for several matter radii as a function of the target thicknesses d1 and d2, for L = 0 and α = 50% Fig. 2 Relative uncertainty of hypertriton interaction cross section (δ thicknesses d1 and d2, for L = 0 and α = 50% Fig. 3 Top: relative uncertainty of hypertriton interaction cross section (δσR/σR), considering L = 0, d1 = 3 cm and d1 + d2 = 11 cm, for several hypertriton radii for a given amount of total beam time (1 day) as a function of α. Bottom: (δσR/σR) as a function of the ﬂight gap L, considering α = 50% Fig. 2 Relative uncertainty of hypertriton interaction cross section (δσR/σR) with 12C for several matter radii as a function of the target thicknesses d1 and d2, for L = 0 and α = 50% then, it has been exploited with light ions at several facilities [46–48]. The R3B (Reactions with Relativistic Radioactive Beams) setup at GSI/FAIR has the potential for a world- unique contribution to the study of -hypernuclei using rel- ativistic stable and radioactive beams [49,50]. Radioactive ion beams with kinetic energy above the production thresh- old of 1.6 GeV/nucleon can be transmitted by the current fragment separator FRS (Bρmax = 18 T · m) and the next- generation fragment separator Super-FRS (Bρmax = 20 T·m) in the future. In the example presented in this paper a stable beam of 12C is considered. Since light hypernuclei decay via pion (π−) emission, a dedicated setup providing sufﬁcient acceptance and efﬁ- ciency for low rigidity pions is required. Here, we introduce such a new experimental approach for kinematic-complete measurements, based on the detection of π−by a dedicated time-projection chamber (TPC) from the decay of hypernu- clei produced in heavy-ion collisions on a production tar- get. The TPC is named HYDRA, standing for HYpernuclei Decay at R3B Apparatus, where its prototype is shown in the bottom panel of Fig. 4: it is 1/3 the size of the full detec- tor, with an active area of 88 × 256 mm2 and has a drift region of 300mm long. In addition to the TPC, a plastic wall behind the exit window of the TPC is employed as a start of the drift time measurement and for triggering. 2.3 Sensitivity in the case of 3 H+12C at 1.9 GeV/nucleon We provide here the results for such calculations for hypertriton separation energies of 50 keV (rms radius = 7.9 fm from pionless EFT), 130 keV (rms radius = 4.9 fm) and 410 keV [15] (rms radius = 2.8 fm). The obtained cross sections are 1062 mb, 861 mb and 645 mb, respectively. In this section, the different predictions of the 3 H matter radius, and consequently interaction cross sec- tion (σR), will be analyzed considering detection efﬁciency εdet = 100% for the weak decay products, where in the next section realistic conditions are considered. Figure 2 shows the resulted relative uncertainty of σR (δσR/σR) as a function of the target thicknesses for dif- ferent matter radii. In the case of L = 0 and α = 50% a minimum is reached, where the lowest values of δσR/σR are obtained using a thin target for the ﬁrst measurement, d1 ∼3 cm, and a thicker target for the second one, d1 +d2 ∼ 8−11 cm. The values of d1 = 3 cm for the ﬁrst measurement and d1 + d2 = 11 cm for the second one will be used in this section. The top panel of Fig. 3 shows the dependence of δσR/σR as a function of α, considering L = 0, where a minimum is reached for 50% ≤α ≤70%. The bottom panel of Fig. 3 shows the trend of δσR/σR as a function of the ﬂight gap (L) for a ﬁxed α = 50%. The introduction of a gap between the two targets will only degrade the uncertainty as the yields of 3 H will be reduced due to its decay. To conclude, the optimal conﬁguration obtained with the proposed method is: no gap between the two targets (L = 0), same boundary conditions of the beam for the two measure- ments N (1) 0 = N (2) 0 , and a large difference between the two target thicknesses. Speciﬁcvalues for thelast parameters can- not be given as they strongly depend on the experiment, and therefore have to be evaluated accordingly. 12 123 Eur. Phys. J. A (2023) 59 :139 Page 5 of 8 139 R=2.8 fm R=4.9 fm R=7.9 fm 6 8 [cm] 2 d 0 2 4 [cm] 1 d 6 8 [cm] 2 d 5 10 15 6 8 [cm] 2 d [%] Fig. 2.3 Sensitivity in the case of 3 H+12C at 1.9 GeV/nucleon Decayed frag- ments (3He for 3 H) can be measured by scintillator ﬁbers and plastic array (TOFD) in R3B standard setup. The pro- duction targets, HYDRA prototype TPC and two additional ﬁber detectors, to determine the ion residue trajectories fol- lowing the decay of hypernuclei, will be installed inside the GLAD magnet [51] of the R3B setup, see Fig. 4 (top) for a schematic view of the experimental setup at R3B. Fig. 3 Top: relative uncertainty of hypertriton interaction cross section (δσR/σR), considering L = 0, d1 = 3 cm and d1 + d2 = 11 cm, for several hypertriton radii for a given amount of total beam time (1 day) as a function of α. Bottom: (δσR/σR) as a function of the ﬂight gap L, considering α = 50% 3 Realistic implementation of the method: HYDRA at R3B The ﬁrst hypernuclear experiment to be performed at R3B focuses on the determination of the interaction cross sec- tion (and consequently the matter radius) of the hypertriton, using the two-target method introduced in this paper. The production and decay channels are the same introduced in 3.2 Background estimate and measurement sensitivity The beam conﬁguration which will be used is: 12C with I = (1 −5) · 106 pps (trigger rate limitation, see below), Ebeam = 1.9 GeV/nucleon (maximum energy accessible currently at GSI), and t = 8 days (to ensure suf- ﬁcient statistics). Since the production cross section of the hypertritonis predictedtobeverylow1.8μb[50], highinten- sity beam, O(106 pps) is necessary. Combined with a thick target, this will lead to a high production rate of secondary particles and consequently a high trigger rate. Monte-Carlo simulations (using INCL++ [52] for fragmentation and the Dubna cascade model [53] for hypernuclei production) for a 6-cm thick 12C target and beam intensity of 106 pps result in a trigger rate (coincidence between the trigger wall and TOFD, see Fig. 4) of 30 kHz. This is at the limit of the possi- ble accepted rate by the R3B setup, and as a consequence, the maximum target thickness has to be ﬁxed to dmax = 6 cm. With this condition, the minimum uncertainty for the inter- action cross section is obtained using d1 = 1 cm and d2 = 5cm. the previous section, 12C + 12C →X + 3 H and 3 H →3He + π−, respectively. Realistic experimental conditions impose several limitations that cannot be neglected and are discussed in this section. The beam conﬁguration which will be used is: 12C with I = (1 −5) · 106 pps (trigger rate limitation, see below), Ebeam = 1.9 GeV/nucleon (maximum energy accessible currently at GSI), and t = 8 days (to ensure suf- ﬁcient statistics). Since the production cross section of the hypertritonis predictedtobeverylow1.8μb[50], highinten- sity beam, O(106 pps) is necessary. Combined with a thick target, this will lead to a high production rate of secondary particles and consequently a high trigger rate. Monte-Carlo simulations (using INCL++ [52] for fragmentation and the Dubna cascade model [53] for hypernuclei production) for a 6-cm thick 12C target and beam intensity of 106 pps result in a trigger rate (coincidence between the trigger wall and TOFD, see Fig. 4) of 30 kHz. This is at the limit of the possi- ble accepted rate by the R3B setup, and as a consequence, the maximum target thickness has to be ﬁxed to dmax = 6 cm. With this condition, the minimum uncertainty for the inter- action cross section is obtained using d1 = 1 cm and d2 = 5cm. 3.2 Background estimate and measurement sensitivity The produced hypertritons are tagged and identiﬁed by the invariant mass from their weak decay channel π−+3He. However, the interaction of the 12C beam with the two car- bon targets used for the measurement can produce a π−and a 3He ion which do not emerge from the decay of 3 H, and can therefore lead to background in the invariant-mass spectrum. The possible background contributions are: (1) the coinci- dence of π−and 3He both produced from the fragmentation of 12C, (2) the decay of a heavier hypernucleus which decays via pion emission together with a multi-ion ﬁnal state that includes 3He, and (3) a π−from the decay of a free , a K 0 S or a heavier hypernucleus and 3He produced in coincidence from the fragmentation of the 12C projectile. The following estimate does not take into account the two-step strangeness production, i.e., the production of hypertriton from fragments with A≥3 and Ekin >1.6 GeV/nucleon formed in the primary collision. The maximal number of 3 H produced from such a two-step process was estimated as 3% of the total amount of 3 H produced by the primary interaction and is therefore neglected. In order to estimate this upper limit the following assumptions have been made: (i) all fragments are produced at the entrance of the target, (ii) the fragments with A≥5 have a production cross section of 1 μb, while for A< 5 are esti- mated from Ref. [50] which gives values smaller than 1 μb. Fig. 4 Top: schematic of the experimental setup at R3B for invariant- mass spectroscopy of hypernuclei. Simulated trajectories correspond to weak decay events of 3 H into π−(green) and 3He (blue) after being produced from 12C+12C collisions at 1.9 GeV/nucleon. Positions of the detectors are optimized for the invariant-mass resolution and efﬁciency. Bottom: sketch of the HYDRA TPC prototype geometry and the exper- imental concept. The TPC aims at measuring π−from the mesonic decay of light hypernuclei. The trajectory of the π−is deﬂected in the GLAD magnetic ﬁeld of around 2 T the previous section, 12C + 12C →X + 3 H and 3 H →3He + π−, respectively. Realistic experimental conditions impose several limitations that cannot be neglected and are discussed in this section. 3.1 Proposed experimental setup 3.1 Proposed experimental setup 3.1 Proposed experimental setup The technique of using relativistic heavy-ion collisions to produce hypernuclei was ﬁrst introduced in 1973 [45]. Since 12 3 3 3 139 Page 6 of 8 Eur. Phys. J. A (2023) 59 :139 In the previous section, the detection efﬁciency has been neglected, i.e., assuming εdet = 100%. Here, several factors have to be taken into account: (i) detection efﬁciency of the π−in the TPC 29%, (ii) detection efﬁciency of the fragment in the tracking detectors 60%, (iii) dead time, spill structure and acceleration duty 40%, (iv) analysis loss 20%. Fig. 4 Top: schematic of the experimental setup at R3B for invariant- mass spectroscopy of hypernuclei. Simulated trajectories correspond to weak decay events of 3 H into π−(green) and 3He (blue) after being produced from 12C+12C collisions at 1.9 GeV/nucleon. Positions of the detectors are optimized for the invariant-mass resolution and efﬁciency. Bottom: sketch of the HYDRA TPC prototype geometry and the exper- imental concept. The TPC aims at measuring π−from the mesonic decay of light hypernuclei. The trajectory of the π−is deﬂected in the GLAD magnetic ﬁeld of around 2 T 3.2 Background estimate and measurement sensitivity Uncertainties from statistics and background subtraction are considered Radius (rms) [fm] σR [mb] δσR/σR [%] 2.8 (no halo) 645 ± 106 17 4.9 861 ± 129 15 7.9 1062 ± 134 13 Table 1 Interaction cross sections for 3 H with 12C using Eq. 4, assum- ing two independent measurements with 1-cm and 6-cm thick carbon targets. Uncertainties from statistics and background subtraction are considered Fig. 5 Simulated invariant-mass spectrum of 3 H for 8 days of beam time on target (see text for details) this is achieved, they can be ﬁtted to extract the kinemat- ics from the track curvature and the energy loss using the so-called Kalman ﬁlter [54]. An example of the application of the ﬁlter ﬁtting for charged particle tracks in the range of momentum of interest can be found in Ref. [55]. The algorithm provides a resolu- tion for the transverse momentum of (1-2)% for deuterons and tritons, close to the limits imposed by the spatial res- olution and multiple scattering. In addition, a Runge–Kutta representation of each track can be used to extrapolate the track back to the target position out of the TPC and recon- struct the vertex with an intrinsic resolution of about 2mm (standard deviation). should be less than 1.8 GeV/nucleon, determined through a comparison of the mixed events background and simulated signal distribution. The above cuts result in a signal over background of ∼3 see simulated spectrum in Fig. 5, while reducing the statistics of good events by 20%. The primary factors that contribute to the reduction are (i) and (ii), which are based on estimation of the vertex reconstruction precision described below. Yet, to test the sensitivity we varied the val- ues by a factor of 2, i.e., setting (i) to less than 10mm and (ii) to greater than 5mm. The resulted signal over background ratio will decrease by a factor of 2. The GENFIT package [56], which offers a complete Kalman ﬁlter ﬁtter has been implemented within the analysis ﬂow for HYDRA. Preliminary results for the reconstruction of simulated π−’s at 800 MeV/c with a homogeneous mag- netic ﬁeld of 2 T yield a momentum resolution of the order of 0.6%, adequate for inferring the kinematics of the reaction channel of interest. In the simulation, we estimate an invariant-mass resolu- tion of 2 MeV (sigma) based on the momenta of π−+3He. 3.2 Background estimate and measurement sensitivity In particular, the pion measurement in the TPC is challenging and requires a dedicated reconstruction algorithm, suited for non-homogeneous magnetic ﬁeld such as that of the GLAD magnet. Therefore, to be conservative, the simulation results presented in this section have been obtained assuming a ver- tex resolution of 5mm. A full simulation for precise recon- struction vertex algorithm is currently under development, and further details are provided below. Overall, using the HYDRA prototype, a precision of 15% or better for the interaction cross section, in the case of halo hypertriton, can be reached for d1 = 1 cm and d1+d2 = 6 cm, within 8 days of beam time at ∼(1−5)·106 pps, see Table 1 for details. In the HYDRA pioneering exper- iment, the main limitation arises from the reduced size of the TPC prototype. To compensate for this loss in efﬁciency, all other experimental conditions are maximized, in the limits of the experiment feasibility. In the future, more precise mea- surements can be achieved using the full-size TPC and an optimize setup accordingly. The reconstruction of kinematics from the tracks recorded by the HYDRA TPC can be realized using a sequential pro- cess that includes track ﬁnding and ﬁtting. Particles travers- ingtheTPCwill ionizethegas producingionizationelectrons that are drifted to the segmented TPC pad plane. Using the pixel (pad) position and the drift velocity of the electrons, tracks can be reconstructed in three dimensions obtaining a collection of points in the space representing the track (hit pattern). 3.2 Background estimate and measurement sensitivity The background from (1) can be mostly removed by selecting the decay vertex position upstream the target [50]. The other two sources of background involve a weak decay, and therefore the pion emission outside the target as in the case of hypertriton decay. The background from (2) was quantiﬁed assuming a mesonic decay of the heavier hypernu- cleus, followed by the Fermi breakup of the decayed heavy residue. The relative kinetic energy between π−and 3He from such background will be always few MeV smaller than 43 MeV, that is the Q-value for the decay of 3 H and therefore it is well separated from the invariant mass spectrum and the effect of background (2) can be considered as negligible. The main source of background in the invariant-mass spectrum comes from (3). These background events can be reduced by requiring that (i) the tracks of the detected decay pion and 3He intersect (5-mm minimum distance between the two tracks), (ii) the obtained decay vertex is outside the target by more than 10mm, (iii) the distance between the recon- structed hypertriton track and the beam trajectory is within 5mm, (iv) kinetic energy of the reconstructed hypertriton 3 3 Eur. Phys. J. A (2023) 59 :139 Page 7 of 8 139 Page 7 of 8 139 Fig. 5 Simulated invariant-mass spectrum of 3 H for 8 days of beam time on target (see text for details) Table 1 Interaction cross sections for 3 H with 12C using Eq. 4, assum- ing two independent measurements with 1-cm and 6-cm thick carbon targets. Uncertainties from statistics and background subtraction are considered Radius (rms) [fm] σR [mb] δσR/σR [%] 2.8 (no halo) 645 ± 106 17 4.9 861 ± 129 15 7.9 1062 ± 134 13 this is achieved, they can be ﬁtted to extract the kinemat- ics from the track curvature and the energy loss using the Fig. 5 Simulated invariant-mass spectrum of 3 H for 8 days of beam time on target (see text for details) Fig. 5 Simulated invariant-mass spectrum of 3 H for 8 days of beam time on target (see text for details) Table 1 Interaction cross sections for 3 H with 12C using Eq. 4, assum- ing two independent measurements with 1-cm and 6-cm thick carbon targets. Phys. Rev. C 102:039901, (2020), (erratum) Nield et al., Phys. Rev. C 13, 1263–1266 (1976) 47. S. Avramenko et al., Nucl. Phys. A 547(1), 95–100 (1992) 48. C. Rappold et al., Nucl. Phys. A 913, 170–184 (2013) 49. C. Rappold, J. López-Fidalgo, Phys. Rev. C 94, 044616 (2016) 49. C. Rappold, J. López-Fidalgo, Phys. Rev. C 94, 0 50. Y.L. Sun et al., Phys. 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https://openalex.org/W4362606739	https://www.researchsquare.com/article/rs-2728666/latest.pdf	English	null	Flexible surface-enhanced Raman scattering strips using colloidal ink of gold- silver alloyed nanoparticles	Research Square (Research Square)	2,023	cc-by	4,152	Flexible surface-enhanced Raman scattering strips using colloidal ink of gold- silver alloyed nanoparticles Honghao Tian Beijing University of Technology Youjian Qin Beijing University of Technology Hongmei Liu (  hmliu@bjut.edu.cn ) Beijing University of Technology Tian Li Beijing University of Technology Yuting Li Beijing University of Technology Xiaohui Fang Beijing University of Technology Xinping Zhang Beijing University of Technology Research Article Keywords: Surface-enhanced Raman scattering (SERS), Flexible SERS substrates, Au–Ag alloy nanoparticles；Localized surface plasmon resonance (LSPR) Posted Date: April 5th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2728666/v1 DOI: https://doi.org/10.21203/rs.3.rs-2728666/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Plasmonics on May 17th, 2023. See the published version at https://doi.org/10.1007/s11468-023-01872-3. Page 1/12 Abstract Surface-enhanced Raman scattering (SERS) has been used for trace detection at the single-molecule level. The low-cost preparation of high-performance test strips has enabled the development of SERS techniques. In this study, oil-dispersible metal or alloy nanoparticles prepared by the Brust-Schiffrin method were used as "inks" in a ballpoint pen to handwrite SERS test strips on polytetrafluoroethylene (PTFE) membranes. Because of the good PTFE lipophilicity, the flexible substrates had good uniformity. The large laser damage threshold of the PTFE membrane also enabled increased laser powers for SERS testing. The Au and Ag alloy nanoparticle inks exhibited increased performance with larger proportions of Ag. The Au1Ag8 nanoparticles had the best properties, and those strips could detect 10-11-M Rhodamine 6G dyes in a 5-µL volume with an enhancement factor of 5.4×108. The SERS strips were used to demonstrate detection of malachite green, the use of which is prohibited in aquaculture and fish tanks. Introduction Surface-enhanced Raman scattering (SERS) spectroscopy has been used for sensitive trace detection [1– 3] for chemical analysis, archaeological identification, and biological research. SERS substrates are usually rigid, such as glass or silicon. Electron-beam lithography [4], reactive-ion beam etching [5], and focused-ion beam etching [6] have been used to fabricate metallic nanostructures on substrates to obtain high-performance SERS. However, wipeable sampling with flexible SERS substrates is more useful. [7] Recently, Tay [8] et al. prepared flexible SERS substrates via inkjet printing of gold nanoparticles on cellulose-based filter paper to detect fentanyl aerosols for drug screening. By controlling the printing cycle, Ma[9] et al. used screen printing to load “ink” containing graphene oxide and silver nanoparticles on cellulose paper to detect pesticide residues on fruit. Flexible and disposable SERS substrates still usually require various laboratory facilities that increase costs. Polavarapu et al. [10] used a pen-on-paper approach to write nanoparticle sols on plain photocopy paper with a fountain pen to prepare low-cost SERS test strips. They reported that gold nanoparticles, gold nanorods, or silver nanoparticles prepared via aqueous chemical reductions could be used as inks, and those containing Ag nanoparticles exhibited high SERS activity over a broad range of excitation wavelengths. The handwriting method significantly reduced the cost of flexible SERS test strips. In 2017, Han [11] et al. used pen-writing to load aqueous silver nanoparticles on modified cellulose paper for flexible SERS arrays that were used to detect melamine in liquid milk at a 0.27 mg/mL detection limit. However, all the strips noted above reported low laser damage thresholds and were readily ablated during applications. Moreover, the nanoparticle inks were made via chemically reducing Au (III) or Ag(I) in aqueous solutions. These nanoparticles were protected by adsorbed ionic agents that were unstable, which resulted in aggregation, clogged pens, and non-uniform writing. Polytetrafluoroethylene (PTFE) membranes have high laser damage thresholds [12] and stable chemical properties, and thus could be ideal flexible substrates. However, the super- hydrophobicity of PTFE makes it difficult to load aqueous dispersions of metal nanoparticles. Page 2/12 Here, we used oil-dispersed gold nanoparticles synthesized via a modified Brust-Schiffrin method [13] to handwrite, via a ballpoint pen, SERS strips on a PTFE membrane. We also synthesized gold-silver nanoparticle alloys with various Au and Ag ratios to optimize the SERS sensitivity. In this way, the localized surface plasmon resonance (LSPR) and the oxidation resistance of the metal nanoparticles were significantly improved. Preparation and Characterization of metallic nanoparticles The metallic nanoparticles were prepared via a modified Brust-Schiffrin method [14]. A total of 1.5 g (2.75 mmol) of tetra-octylammonium bromide was added to a two-neck flask with 80 mL of toluene and magnetically stirred. Then 0.066 g (0.38 mmol) of AgNO3 dissolved in 2 mL of ultrapure water was added and stirred. The toluene solution became milky white with a pale-yellow precipitation. Then 0.088 g (1.52 mmol) of NaCl was added, and the pale-yellow flocculent precipitate disappeared within 5 min. A total of 0.16 g (0.38 mmol) of HAuCl4·4H2O was dissolved in 2 mL of ultrapure water and added to the solution, which then turned orange-red. After stirring for 5 min, 0.36 g (1.88 mmol) of hexanethiol was added, which caused the solution to become colorless. After stirring for 15 min, 0.28 g (7 mmol) NaBH4 dissolved in 20 ml of water was poured into the flask, and the solution became black. The reaction was finally stopped after stirring at room temperature for 4 h, and the aqueous phase was discarded. The black organic phase was distilled in a rotary evaporator at a temperature below 50°C to evaporate the solvent. The remaining black product was suspended in 30 mL of methanol, briefly sonicated to ensure complete dissolution of byproducts, collected via centrifugation, and then washed five times with 20 mL of methanol. Upon drying at room temperature in vacuo, alloy nanoparticles with a Au:Ag mole ratio of 1:1 were obtained. Various Au and Ag mole ratios, such as Au, Au1Ag1, Au1Ag2, Au1Ag4, Au1Ag8, and Ag, could be obtained by changing the AgNO3 and HAuCl4·4H2O stoichiometry. The metal nanoparticles synthesized by the Brust-Schiffrin method could be purified and dissolved in most organic solvents for their surfaces were modified by an alkylthiol monolayer through coordinate bond. Therefore, the metal nanoparticle ink concentrations could be precisely adjusted and the colloids did not aggregate. The gold or gold-silver alloy nanoparticles were dispersed in chloroform to prepare colloidal concentrations of 15 mg/ml, 35 mg/mL, and 55 mg/ml that were used to fill a ballpoint pen. A 0.5×0.5- cm SERS array was then written on the surface of the PTFE membrane. During the writing, the colloidal nanoparticles diffused along the membrane fibers via capillary effects and spread on the fiber surfaces as the solvent evaporated. Introduction Overall, the flexible SERS test strips on PTFE substrates exhibited high performance, good stability, and large laser damage thresholds. Preparation and Characterization of metallic nanoparticles Subsequently, the membrane was placed in a 200-°C muffle furnace for 20 min to remove the 1-hexanethiol ligands from the nanoparticle surfaces. At the same time, the nanoparticles fused into 10–20-nm diameter particles. TEM images of Au-Ag alloy nanoparticles are shown in Fig. 1(a-d). In Fig. 1(a, b), the particles are spherical with 1–5-nm diameters. Au and Ag both have face-centered cubic fcc crystal structures and have similar lattice constants; hence, they can form perfect alloys in any proportion. From the high- Page 3/12 Page 3/12 resolution TEM images in Fig. 1(c, d), we calculated approximately 0.243-nm interplanar spacings of adjacent lattice fringes, which corresponded to Au (111) and Ag (111) planes. The absorption maxima for nanoparticles with various proportions of gold and silver are plotted in Fig. 1(e). The wavelength of the LSPR extinction peak was linearly blue-shifted with increased Ag content. Specifically, the Au1Ag1 absorption peak was at 502 nm, the Au1Ag2 peak was at 482 nm, the Au1Ag4 peak was blue-shifted to 470 nm, the Au1Ag8 peak was centered at 458 nm, and the Ag peak was blue-shifted to 449 nm. Preparation of Handwritten Flexible SERS Strips The process of writing on the surface of the PTFE membrane with a ballpoint pen filled with colloidal ink is shown in Fig. 2(a). As noted above, the colloidal metal nanoparticles wet and spread on the PTFE surface. By maintaining the approximately 1-mm distance between pen strokes, the metal nanoparticles could be smoothly coated on the fiber surface. Hence, the method was simple and convenient. Distributions of nanoparticles (35 mg/mL) written on the PTFE membrane were characterized by SEM, as shown in Fig. 3(b). Compared with the blank PTFE membrane that had cracks caused by film stretching during fabrication [Fig. 3(a)], the surface of the written fiber was smoother in [Fig. 3(b)], due to the metal nanoparticles had filled these cracks. After annealing, the nanoparticles fused into larger particles with 5- 20-nm diameters. A large number of gaps less than 10 nm formed between adjacent nanoparticles, as shown in the inset of Fig. 3(b). These gaps were SERS "hot spots," where adsorbed analytes exhibited Raman spectra with significantly increased intensities. SERS Performances of Handwritten Flexible SERS characterization was performed with a WITec Alpha300A confocal Raman spectrometer. Vibrational Raman spectra of the analyte were acquired in "mapping" mode to characterize the properties of the flexible SERS substrate. The Raman spectrometer was equipped with a synapse charge-coupled device (CCD) detector and a power tunable 50-mW incident 532-nm laser. A 50× objective (0.8 numerical aperture) was used, and the laser focal spot was approximately 811 nm in diameter. The scanning area was 25×25 µm2, and 2500 spectra were collected from the mapping image. The integration time for each point was 0.5 s. An averaged spectrum over the entire mapping area was calculated for the final test result. We firstly investigated the effects of Au1Ag1 ink concentrations (15 mg/mL, 35 mg/mL, and 55 mg/mL) on SERS performance. In Fig. 4(a), the concentration has no significant effect on SERS performance; because of the good ink wettability, the PTFE fibers were like a reservoir. When the colloid concentration was low, the nanoparticles were mainly present on the surface after the solvent was volatilized. When the colloid concentration increased, there were particles both on the surface and inside the fiber. Hence, the SERS performance for the substrate prepared with 15 mg/mL ink was similar to that prepared with 55 mg/mL ink. Further increases in concentration (> 65 mg/mL) greatly decreased the performance because of excessive fusion of the metallic nanoparticles into solid blocks during annealing that prevented Page 4/12 Page 4/12 formation of hot-spots. Therefore, the optimal 35 mg/mL concentration was used to characterize the various Au-Ag alloys. SERS spectra of 10− 6-mol/L R6G analyte on SERS strips with various Au-Ag alloys are shown in Fig. 4. The SERS intensity increased with Ag content in the alloys. The intensity from the Au1Ag8 alloy strips was approximately ten times that from the Au strips. This was attributed to electromagnetic (EM) SERS spectra of 10− 6-mol/L R6G analyte on SERS strips with various Au-Ag alloys are shown in Fig. 4. The SERS intensity increased with Ag content in the alloys. The intensity from the Au1Ag8 alloy strips was approximately ten times that from the Au strips. This was attributed to electromagnetic (EM) enhancement and charge-transfer enhancement mechanisms. With the increased Ag content on the SERS substrate, the LSPR wavelength blue-shifted, which was better for 532-nm excitation. This generated a strong localized EM field between adjacent nanoparticles. SERS Performances of Handwritten Flexible Because the Raman intensity is proportional to the fourth power of the EM field intensity, the R6G Raman scattering was greatly enhanced. In the other mechanism, hot electrons from LSPR excitation decays enhanced charge-transfer between the plasmonic substrate and the adsorbed R6G, which changed the local electron density and increased R6G Raman scattering. Using the Au1Ag8 alloy, we characterized the SERS performance for various R6G concentrations (10− 6– 10− 12 M), as shown in Fig. 5. The SERS intensity decreased with decreasing of R6G concentration; characteristic peaks were still visible at 10− 11 M. However, when the concentration was 10− 12 M, the SERS spectrum was not uniformly detected at all mapping locations. Therefore, for the Au1Ag8 alloy and a 5-µL loading volume, 10− 11 M R6G was the detection limit. With Eq. (1), we calculated the number of R6G molecules loaded on a single mapping point at any concentration. = , Sl SA NE NA 1 where Sl was the area of the laser spot, SA was the distribution area of the solution to be detected, NE was the number of molecules detected in a single laser spot, and NA was the total number of molecules detected. For the 10− 11-M R6G concentration, there were 6 molecules in a single laser spot; and for 10− 12 M R6G, it was 0.6 molecules. This explained why the 10− 12-M solution could not be uniformly detected. The flexible SERS strips had a high R6G sensitivity. From Eq. (2), we can also calculate the enhancement factor (EF) of the substrate. where Sl was the area of the laser spot, SA was the distribution area of the solution to be detected, NE was the number of molecules detected in a single laser spot, and NA was the total number of molecules detected. For the 10− 11-M R6G concentration, there were 6 molecules in a single laser spot; and for 10− 12 M R6G, it was 0.6 molecules. This explained why the 10− 12-M solution could not be uniformly detected. The flexible SERS strips had a high R6G sensitivity. From Eq. (2), we can also calculate the enhancement factor (EF) of the substrate. Applications to On-Site Sampling and Detecting Malachite green (MG) was once widely used as a fungicide in aquaculture. However, because of its carcinogenic, teratogenic, and mutagenic hazards,[15] most countries have banned it since the 1990s. Nevertheless, because of its low cost and bactericidal effects, MG can still be found in aquaculture. Using Au1Ag8 SERS strips and beakers, we simulated MG sampling of a fish tank surface. We added various concentrations of aqueous MG solutions to the beakers, and let them stand for 4 h. We then poured out the solutions and wiped the beakers with SERS test strips that were wetted with ethanol, to mimic sampling of MG residues on the surface of fish tanks in a market. The test results for various MG concentrations are shown in Fig. 7. The laser wavelength was 532 nm, the power was 0.6 mW, and the integration time was 1s. Residual MG on the inner surface of the beaker was still clearly detected when the 10− 7-M MG solution was contained in the beaker. This demonstrated that the SERS substrates could be used as test strips for on-site sampling and detection. SERS Performances of Handwritten Flexible EF = ( ) / ( ) , ISERS NSERS IREF NREF EF = ( ) / ( ) , ISERS NSERS IREF NREF 2 where ISERS was the intensity of a specific analyte Raman band, and NSERS was the number of molecules contributing to ISERS. Similarly, IREF was the intensity of the same Raman band from a reference solution, and NREF was the number of molecules contributing to IREF. The EF was 5.4×108 for the 10− 11−M R6G where ISERS was the intensity of a specific analyte Raman band, and NSERS was the number of molecules contributing to ISERS. Similarly, IREF was the intensity of the same Raman band from a reference solution, and NREF was the number of molecules contributing to IREF. The EF was 5.4×108 for the 10− 11−M R6G Page 5/12 613-cm− 1 peak from Au1Ag8 alloy strips, which was one of the best reported enhancement factors for flexible SERS substrates. To demonstrate the good uniformity of the Au1Ag8 alloy SERS strips, spectra for more than 2500 points were collected in a 25×25-µm2 mapping area, with 0.5-s integration times at each point. The intensity of the 613-cm− 1 Raman peak is plotted in Fig. 6(a), and the average SERS spectrum over the entire mapping area is shown as red in Fig. 6(b). The maximum and minimum spectral intensities were plotted as blue and black curves, respectively, in Fig. 6(b). The high-intensity (bright) areas and the low-intensity (dark) areas were randomly distributed in the 2D mapping image, and the minimum-intensity spectrum still exhibited characteristic R6G peaks. Figure 6(c) shows cross-sections of the mapping image in Fig. 6(a), which reflect intensity fluctuations of 613-cm− 1 and 1362-cm− 1 peaks. From Fig. 6(c), we calculated 18% and 31% relative standard deviations for the 613-cm− 1 and 1362-cm− 1 peak intensities, respectively. The Raman signals were enhanced at all spots, indicating excellent uniformity in SERS activity. To test the laser damage threshold of the SERS strips, the 532-nm laser power was increased to 50 mW, and was focused on the strips with a 100× microscope objective (NA = 0.9), at which the laser power was attenuated to 5.14 mW. Both optical microscope and SEM images indicated no changes in the irradiated areas. Thus, the SERS strips had high laser damage thresholds. areas. Thus, the SERS strips had high laser damage thresholds. Conclusions We used plasmonic inks dispersed in oil to handwrite with a ballpoint pen high-performance SERS test strips on the surface of PTFE membranes. The test strips exhibited good reproducibility because the SERS nanoparticles were uniformly spread on the PTFE fibers. The membrane enabled flexible SERS strips with high chemical stabilities and large laser damage thresholds. The plasmonic nanoparticles of Au, Ag, and their alloys were prepared via a modified Brust-Schiffrin method. The substrates patterned Page 6/12 Page 6/12 with Au-Ag alloy nanoparticle inks exhibited increased SERS performance with Ag content, where the Au1Ag8 alloy had the best performance. The wipeable capability of the flexible SERS strips has potential applications for on-site sampling. As SERS functionalized membranes, they are also expected to be applied to lateral flow strips or gas detectors. with Au-Ag alloy nanoparticle inks exhibited increased SERS performance with Ag content, where the Au1Ag8 alloy had the best performance. The wipeable capability of the flexible SERS strips has potential applications for on-site sampling. As SERS functionalized membranes, they are also expected to be applied to lateral flow strips or gas detectors. Declarations Authors' Contributions Honghao Tian, Youjian Qin and Hongmei Liu completed the experiments of the thesis. Tian Li and Yuting Li characterized the metal nanoparticles. Honghao Tian and Hongmei Liu completed the data collation and the draft paper. Hongmei Liu, Xiaohui Fang and Xinping Zhang discussed and revised the paper. Funding This work was supported by the National Natural Science Foundation of China (61575007, 12074020, 62275005) and Beijing Municipal Education Commission (KZ202010005002). Data Availability The data presented in this study are available in the article. ata Availability The data presented in this study are available in the article. Conflict of Interest The author declares no competing interests. Conflict of Interest The author declares no competing interests. References J Chem Soc, Chem Commun 7:801–802 14. Li H, Liu HM, Qin YJ, Mu YY, Fang XH, Zhai TR, Zhang XP (2020) Gold-Stabilized Gold-Silver Alloy Nanostructures as High-Performance SERS Substrate. Plasmonics 15:2027–2032 14. Li H, Liu HM, Qin YJ, Mu YY, Fang XH, Zhai TR, Zhang XP (2020) Gold-Stabilized Gold-Silver Alloy Nanostructures as High-Performance SERS Substrate. Plasmonics 15:2027–2032 15. Henry S, Rodgers G, Willias D, Bosse G, Sullivan J (2008) Methemoglobinemia due to malachite green ingestion in a child. Clin Toxicol (Phila) 46:320–321 15. Henry S, Rodgers G, Willias D, Bosse G, Sullivan J (2008) Methemoglobinemia due to malachite green ingestion in a child. 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Han CQ, Li YQ, Jia Q, Bradley LH, Gan Y, Yao Y, et al (2017) On-demand fabrication of surface- enhanced Raman scattering arrays by pen writing, and their application to the determination of melamine in milk. Microchim Acta 184:2909–2917 12. Mitra A, Thareja R (1999) Determination of laser ablation threshold of Teflon at different harmonics of Nd:YAG laser using photothermal deflection technique. J Mater Sci 34:615–619 12. Mitra A, Thareja R (1999) Determination of laser ablation threshold of Teflon at different harmonics of Nd:YAG laser using photothermal deflection technique. J Mater Sci 34:615–619 13. Brust M, Walker M, Bethell D, Schiffrin DJ, Whyman R, (1994) Synthesis of thiol-derivatized gold nanoparticles in a two-phase Liquid-Liquid system. J Chem Soc, Chem Commun 7:801–802 13. Brust M, Walker M, Bethell D, Schiffrin DJ, Whyman R, (1994) Synthesis of thiol-derivatized gold nanoparticles in a two-phase Liquid-Liquid system. Figure 1 Transmission electron microscope (TEM) and high-resolution TEM images of Au-Ag alloys: (a, c) Au1Ag2 and (b, d) Au1Ag8. (e) Plot of absorbance maxima of nanoparticles with various Au-Ag atomic fractions. From left to right in the inset are photographs of colloidal solutions of Au, Au2Ag1, Au1Ag1, Au1Ag2, Au1Ag4, Au1Ag8, and Ag. Figures Figure 1 Transmission electron microscope (TEM) and high-resolution TEM images of Au-Ag alloys: (a, c) Au1Ag2 and (b, d) Au1Ag8. (e) Plot of absorbance maxima of nanoparticles with various Au-Ag atomic fractions. From left to right in the inset are photographs of colloidal solutions of Au, Au2Ag1, Au1Ag1, Au1Ag2, Au1Ag4, Au1Ag8, and Ag. Page 8/12 Page 8/12 Figure 2 (a) Photograph of a ballpoint pen filled with colloidal Au-Ag alloy nanoparticle “ink” used for handwriting on PTFE membranes to make SERS strips. (b) Optical microscope images of the ballpoint at different magnifications. Figure 2 Figure 2 (a) Photograph of a ballpoint pen filled with colloidal Au-Ag alloy nanoparticle “ink” used for handwriting on PTFE membranes to make SERS strips. (b) Optical microscope images of the ballpoint at different magnifications. (a) Photograph of a ballpoint pen filled with colloidal Au-Ag alloy nanoparticle “ink” used for handwriting on PTFE membranes to make SERS strips. (b) Optical microscope images of the ballpoint at different magnifications. Figure 3 Page 9/12 (a) Scanning electron microscope (SEM) images of the PTFE membrane before (a) and after (b) writing nanoparticle arrays. Insets in (a) and (b) are magnified SEM images. Figure 4 Figure 4 Surface-enhanced Raman scattering spectra of R6G (10-6 M) acquired with flexible PTFE substrates handwritten with plasmonic inks with (a) different sol concentrations, and with (b) different Au:Ag mole ratios. Spectra for Au:Ag molar ratios (from bottom to top) were Au, Au1Ag1, Au1Ag2, Au1Ag4, and Au1Ag8. The excitation light was 532-nm with a power of 0.6 mW. Page 10/12 Figure 5 Page 10/12 Figure 5 Figure 5 Figure 5 Page 10/12 Surface-enhanced Raman scattering spectra for various R6G concentrations (10-6–10-11 M) on Au1Ag8 alloy test strips. (Spectra obtained at 10-11 M were amplified 10-fold for clarity). All spectra were acquired with 532-nm excitation. Figure 6 (a) A map image from a 25×25-μm2 area for the 10-6-M R6G Raman vibration at 613 cm-1 on a Au1Ag8 alloy SERS strip. (b) The maximum (blue), average (red), and minimum (black) SERS intensities over the entire mapping area in (a); (c) Cross-sections [blue dashed line in (a)] for 613 cm-1 (top) and 1362 cm-1 (bottom) peak intensities of R6G. Figure 6 (a) A map image from a 25×25-μm2 area for the 10-6-M R6G Raman vibration at 613 cm-1 on a Au1Ag8 alloy SERS strip. (b) The maximum (blue), average (red), and minimum (black) SERS intensities over the entire mapping area in (a); (c) Cross-sections [blue dashed line in (a)] for 613 cm-1 (top) and 1362 cm-1 (bottom) peak intensities of R6G. Page 11/12 Page 11/12 Figure 7 SERS spectra acquired on flexible Au1Ag8 alloy test strips after wiping the surface of simulated fish tanks that had been filled with different MG concentrations (10-5–10-7 M). Figure 7 SERS spectra acquired on flexible Au1Ag8 alloy test strips after wiping the surface of simulated fish tanks that had been filled with different MG concentrations (10-5–10-7 M). Page 12/12 Page 12/12
https://openalex.org/W4392004796	https://www.mdpi.com/2673-3986/5/1/6/pdf?version=1708410910	English	null	The Molecular Epidemiology of Epizootic Hemorrhagic Disease Viruses Identified in Israel between 2015 and 2023	Epidemiologia	2,024	cc-by	9,337	Citation: Golender, N.; Hoffmann, B. The Molecular Epidemiology of Epizootic Hemorrhagic Disease Viruses Identified in Israel between 2015 and 2023. Epidemiologia 2024, 5, 90–105. https://doi.org/10.3390/ epidemiologia5010006 Natalia Golender 1,* and Bernd Hoffmann 2 Natalia Golender 1,* and Bernd Hoffmann 2 1 Department of Virology, Kimron Veterinary Institute, Bet Dagan 5025001, Israel 2 Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany; bernd.hoffmann@fli.de * Correspondence: golendern@moag.gov.il; Tel.: +972-3968-1668; Fax: +972-3968-1788 1 Department of Virology, Kimron Veterinary Institute, Bet Dagan 5025001, Israel 2 Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany; bernd.hoffmann@fli.de * Correspondence: golendern@moag.gov.il; Tel.: +972-3968-1668; Fax: +972-3968-1788 Abstract: Epizootic hemorrhagic disease (EHD) is an infectious, non-contagious viral disease seriously affecting cattle and some wild ruminants and has a worldwide distribution. All viruses can be subdivided into “Eastern” and “Western” topotypes according to geographic distribution via the phylogenetic analysis of internal genes. In Israel, during the last decade, three outbreaks were registered: caused by EHDV-6 in 2015, by EHDV-1 in 2016, and by EHDV-7 in 2020. Additionally, RNA of EHDV-8 was found in imported calves from Portugal in 2023. During the same period in other countries of the region, non-Israeli-like EHDV-6 and EHDV-8 were identified. Full genome sequencing, BLAST, and phylogenetic analyses of the locally and globally known EHDV genomes allowed us to presume the probable route and origin of these viruses detected in Israel. Thus, EHDV-6 has probably been circulating in the region for a long period when EHDV-1 and -8 appeared here for the last years, while their route of introduction into the new areas was probably natural; all of them belonged to the “Western” topotype. In contrast, EHDV-7 probably had the “Eastern”, anthropogenic origin. Data from the study can facilitate the evaluation of the appearance or reappearance of EHDVs in the Mediterranean area and enhance the planning of prevention measures. Keywords: cattle; arbovirus; Reoviridae; Orbivirus; ruminants; sequencing; phylogeny; outbreaks Article The Molecular Epidemiology of Epizootic Hemorrhagic Disease Viruses Identified in Israel between 2015 and 2023 Natalia Golender 1,* and Bernd Hoffmann 2 1. Introduction Epizootic hemorrhagic disease (EHD) is an infectious, non-contagious viral disease that is transmitted by blood-sucking insects of the genus Culicoides. This virus belongs to the genus Orbivirus of the family Sedoreoviridae [1,2]. The virus has a dsRNA linear genome of ten segments, which coded for seven structural (VP1-VP7) and three or four non-structural (NS1-NS3 and NS3a) proteins [3–5]. It shares many morphologic and structural characteristics with other members of the genus, such as the Bluetongue virus (BTV), African horse sickness virus, and equine encephalitis virus, and demonstrates immunological cross-reactivity with the Bluetongue virus group [6]. Like BTV, the primary determinant of serotype specificity is the outer capsid VP2 protein [7]. There are seven officially determined serotypes [8] and at least three putative strains representing new EHDV serotypes [9–11].i Academic Editors: Maria Luisa Marenzoni and Antoine Flahault Academic Editors: Maria Luisa Marenzoni and Antoine Flahault Received: 13 December 2023 Revised: 22 January 2024 Accepted: 14 February 2024 Published: 20 February 2024 EHD was first described in white-tailed deer (Odocoileus virginianus) in New Jersey in 1955 [12], which are especially susceptible and the most affected ruminant species by EHDV, with high morbidity and mortality rates. Although other ruminant species, such as pronghorn, mule deer, and black-tailed deer, may develop clinical signs, most EHDV infections of ruminants are mild or subclinical [13]. Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Previously, EHDV was thought to cause asymptomatic infection in cattle [14], except for the EHDV-2 Ibaraki strain, which was responsible for an extensive outbreak of the disease in cattle in Japan during 1959 [15–18]. However, EHDV-6 and 7 were responsible for the clinical manifestation of the disease in the Far and Middle East and North Africa. EHDV-6, which was observed in 2006 in https://www.mdpi.com/journal/epidemiologia Epidemiologia 2024, 5, 90–105. https://doi.org/10.3390/epidemiologia5010006 Epidemiologia 2024, 5 91 Tunisia, in 2007 in Turkey, and in 2015 in Japan and Israel. The infected cattle showed substantial clinical signs, which included fever, milk reduction, edema of the head, necrotic lesions of the oral mucosa, hypersalivation, hyperthermia, lesions of the teats, stiff gate, dysphagia and cessation of rumination, and redness of the lips and muzzle, accompanied by anorexia and respiratory distress [10,19–23]. Notably, epizootic diseases caused by EHDV-7 in 1997 in Japan were mainly characterized by abortion and stillbirth in addition to febrile illness [24], while for EHDV-7 in Israel, the clinical signs resembled those which were caused by EHDV-6 [22,25]. Successful virus isolation (VI) was also carried out from aborted fetuses and placentas [18], suggesting that the blood–placental barrier was crossed and the fetus was infected. Similarly, viral RNA was also detected in the aborted fetuses and placentas of Israeli cattle affected by EHDV-6 [22]. p y Comparing EHD caused by serotype 1, this serotype was not associated with clinical manifestation in ruminants in Japan [26], whereas Israeli EHDV-1 caused mostly asymp- tomatic or mild infection in cattle [27]. Considering other EHDV clinically described serotypes/strains, EHDV-10 was isolated from asymptomatic cattle in Japan [19], while novel Chinese EHDV was observed in a single febrile sentinel calf [11]. In contrast to the above-mentioned EHDV serotypes, recently identified EHDV-8 in the Mediterranean region is characterized by erosions on teats and mucosal membranes, cyanosis and edema of the tongue, submandibular edema, conjunctivitis, conjunctivitis and lacrimation, nasal discharge, respiratory distress, inappetence, and fever [28,29]. g p y pp EHDV has a worldwide distribution. In East Asia, at least seven serotypes of EHDV (EHDV-1, 2, 5, 6, 7, 8, and 10) have been discovered [10,19,30–32]. In Western Asia (the territory of Bahrain, Oman, and Israel), four serotypes of EHDV were identified (EHDV-1, -2, -6, and -7) [22,25,27,33,34]. In Turkey, which crosses Europe and Asia, an outbreak in cattle in goitered gazelle caused by EHDV-6 during 2007 was reported [21,35]. At least five eastern serotypes of EHDV (EHDV-2, -5, -6, -7, and -8) were isolated in Australia [4,33,36]. EHDV-1, -2, and -6 have wildly spread in the United States and Canada, where the disease was reported in deer and cattle [37–41]. In South America, EHDV-1 was isolated, and EHDV-6 was detected in both French Guiana and Ecuador [42,43]. Regarding the African continent, EHDV-1 and EHDV-6 were identified, but EHDV-6 and EHDV-8, which recently caused outbreaks in cattle, are more common [28,44–47]. The only strain of EHDV-4 in the world was discovered in Nigeria [33]. EHDV-8 was the first serotype reported on the territory of Europe (in Sardinia and Sicily, Italy, Spain, Portugal, and France) [29]. y p y y p g The novel history of EHDV in the “old world” began in 2003, when EHDV-6 was registered in Reunion Island [48]. A few years later, large outbreaks caused by EHDV-6 were reported in several countries of the Mediterranean Basin, including Morocco, Algeria, and Tunisia in 2006, and in the following year in Turkey [21,24,48]. Antibodies against EHDV- 6 were identified in sera samples collected in 2012–2013 in Tunisia [45], illustrating the continuous circulation of the virus in the region, which was confirmed by the evidence of a low seroprevalence among samples collected in 2015 in Libya [46]. Regarding other parts of Africa, RT-qPCR EHDV-positive samples were detected in the field samples collected from cattle between 2007 and 2010 in Kenya [49]. Recently, EHDV-6 was detected in asymptomatic cattle in 2016 in Mayotte [50] and caused an outbreak in 2015 in Japan [10,19] and Tunisia [51]. The latest EHD outbreaks caused by serotype 8 were reported during 2021–2023 in the Mediterranean region, including in Tunisia, Italy, Spain, Portugal, and France [28,52]. In Israel, EHD was suspected in 1951 [53] but was first confirmed as an EHDV-7 outbreak in 2006 [24]. The next outbreak, caused by EHDV-6, was observed in 2015 [23]. During the following year (2016), EHDV-1 was identified in Israeli cattle and a wild mountain gazelle [27]. Notably, in the same year, EHDV-1 also was detected in Egypt [54]. 2. Materials and Methods 2.1. Field Samples A total of 373 samples from 367 animals collected in 2020 from ill and dead cattle and wild/zoo ruminants, including aborted or malformed domestic or wild ruminant fetuses, were submitted for routine examination to the virology department of the Kimron Veterinary Institute, Israel (KVI). In addition, 635 whole blood samples from cattle tested in 2021 and 1178 tested samples collected in 2023 were included in this study. Since no EHDV-positive samples were identified in 2022, data on these samples were not included in this work. Clinical specimens collected in 2020 included the placenta, brain, and internal organs from aborted fetuses, whole blood from symptomatic ruminants, and spleen or lung from dead ruminants. Data on field samples tested for EHDV in 2020–2021 and 2023 are summarized in Table 1. Table 1. Information about Pan-EHDV RT-qPCR and EHDV isolation from cattle and different kinds of wild/zoo ill or dead animals tested in 2020–2023. Table 1. Information about Pan-EHDV RT-qPCR and EHDV isolation from cattle and different kinds of wild/zoo ill or dead animals tested in 2020–2023. Year/Organ Cattle Wild Ruminants Total VI w.b. s/l a.f w.b. s/l a.f 2020 No. tested samples 323 19 20 (14) 0 10 1 373 (367) No. of pos. samples 128 3 3 (2) 0 0 0 134 (133) 9 2021 No. tested samples 635 0 0 0 0 0 635 No. of pos. samples 149 0 0 0 0 0 149 2023 No. tested samples 1067 50 (44) 26 (19) 2 30 (26) 3 1178 (1163) No. of pos. samples 2 0 0 0 0 0 2 total No. tested samples 2025 69 (63) 46 (35) 2 40 (36) 4 2186 (2165) 9 No. of pos. samples 279 3 3 (2) 0 0 0 285 (284) w.b.—whole blood samples; s/l—spleen or lung samples; a.f.—aborted fetus and newborn animals; numbers in parentheses represent number of affected fetuses/newborn animals; VI—virus isolation. w.b.—whole blood samples; s/l—spleen or lung samples; a.f.—aborted fetus and newborn animals; numbers in parentheses represent number of affected fetuses/newborn animals; VI—virus isolation. 2.2. Nucleic Acid Extraction and Pan-EHDV Real-Time Polymerase Chain Reaction (RT-PCR) 2.2. Nucleic Acid Extraction and Pan-EHDV Real-Time Polymerase Chain Reaction (RT-PCR 2.2. The last outbreak of EHD in Israel was observed in 2020 when the Eastern genotype of serotype 7 was identified. The latest identification of the EHDV viral RNA was carried out in collected whole blood samples from imported Portuguese calves in September 2023. Due to the specific geographic situation of Israel, the local ruminant population has Epidemiologia 2024, 5 92 frequently been exposed to different serotypes and strains of EHDV, which are genetically and clinically different. The aim of the current work is a description of the probable routes of EHDV-7 introduction into Israel and its spread during 2020 outbreak based on phylogenetic analysis and collected epidemiological data. frequently been exposed to different serotypes and strains of EHDV, which are genetically and clinically different. The aim of the current work is a description of the probable routes of EHDV-7 introduction into Israel and its spread during 2020 outbreak based on phylogenetic analysis and collected epidemiological data. 2. Materials and Methods 2.1. Field Samples Nucleic Acid Extraction and Pan-EHDV Real-Time Polymerase Chain Reaction (RT-PCR) We extracted ribonucleic acid (RNA) from the tissue culture supernatant, chicken embryo homogenates, and field samples (whole blood, lung, and spleen) using the Invisorb Spin Virus RNA Mini Kit (STRATEC Molecular GmbH, Berlin, Germany), MagMAX™ CORE Nucleic Acid Purification Kit (Thermo Fisher Scientific, Austin, TX, USA), and IndiMag Pathogen Kit (Indical Bioscience, Leipzig, Germany). Viral RNA detection was performed using the VetMAX™EHDV kit (Applied Biosystems™, Thermo Fisher Scientific Inc., Lissieu, France). The pan-EHDV system described by Wernike et al. [55], which is based on the detection of Seg-5 fragment, was used as an alternative method. In accordance with the instructions of the authors and manufacturer of the RT-qPCR kit/system, the cut-off for all these methods was Cycle Threshold (Ct) 40. 2.3. Type-Specific RT-PCR and Sanger Sequencing For the identification of EHDV-8 by sequencing, the conventional RT-PCR was per- formed using a One-Step RT-PCR kit (Qiagen, Hilden, Germany); data on primers based on the Seg-2 detection of EHDV-6 and 8 are provided in Table 2. Primers were developed using the Genius 9.05 program (Biomatters Ltd., Auckland, New Zeeland) when EHD6/8-1F and EHD6/8-250R were designed to recognize EHDV-6 and EHDV-8; EHD8-S2-178F and EHD8-S2-522R were developed specifically to recognize a recently emerged EHDV-8; and EHD8-S2-447F and EHD8-S2-705R primers were developed to detect the Australian strain Epidemiologia 2024, 5 93 of EHDV-8 (Table 2). Primers for the partial sequencing of internal genes of EHDV-7 and primers used for covering missed regions from the sequence from whole genome sequenc- ing (WGS) of EHDV-7 are shown in Table S1. The cDNA fragments of positive samples were purified using the MEGAquick-spin Total Fragment DNA Purification Kit (iNtRON Biotechnology, Gyeonggi-do, South Korea) and subsequently sequenced by standard Sanger methods in both directions using an ABI 3730xl DNA Analyzer (Hylabs, Rehovot, Israel). Table 2. Information on used primers for identification of EHDV of serotype 8. Name of Oligo Oligo Sequence (5′ →3′) Source EHD6/8-1F GTT AAA TTR TTC CAG GAT GGA WA [22] EHD6/8-250R CAT CAT CAT AYC TCA TTA TYC CA EHD8-S2-178F AGA GGC GCG TAA TGT TTT C this study EHD8-S2-522R TGC TGA ATC ATA TCG TAA TGT A EHD8-S2-447F CCA AAT TTG TGG AAA GCT TG this study EHD8-S2-705R CGC ACT TTT GTT TGC TTA TCT TTA T Table 2. Information on used primers for identification of EHDV of serotype 8. 2.4. Whole Genome Sequencing and Phylogenetic Analysis Extracted RNA of the EHDV-7 strain ISR-2262/2/20 was submitted to Genotypic Tech- nology Pvt. Ltd., Bangalore, India. The whole procedure was described previously [56,57]. The resulting nucleotide (nt) sequences of the EHDV-7 and -8 were assembled, and nt sequences were aligned and pairwise compared using Geneious version 9.0.5 (Biomatters, Auckland, New Zealand) and/or BioEdit programs (https://bioedit.software.informer. com/7.2/ (accessed on 9 March 2017). / / ( ) Using HTS-SISPA technology [58] and the previously described procedure [59] (Ries 2020), the double-sense (ds) cDNA of EHDV-6 strain ISR-4487/15 and EHDV-1 strain ISR-2096/16 were prepared and submitted to Eurofins Genomic (Ebersberg, Germany) for genome sequencing on the Illumina platform. 2.3. Type-Specific RT-PCR and Sanger Sequencing The obtained fastq raw data were further processed using the Geneious Prime v2021.0.1 software (Biomatters Ltd., Auckland, New Zeeland) to construct complete genome sequences of the EHDV-6 strain ISR-4487/15 and EHDV-1 strain ISR-2096/16. Phylogenetic trees were constructed using the Mega X software [59]. For all phylo- genetic trees, the maximum-likelihood method (ML) and the Tamura–Nei models were applied. The sequences of the Israeli ISR-2096/15 (EHDV-6), ISR-4487/16 (EHDV-1), ISR- 2262/2/20 (EHDV-7), ISR-1692/2/23, and ISR-1692/9/23 (EHDV-8) strains were used as a representative strain for all phylogenetic trees of the phylogenetic analyses. As an outgroup BTV, different strains were chosen based on the BLAST NCBI analysis of one of the most closely related viral species to EHDV by internal genes, except Seg-6, where closely related BTV were not suitable as an outgroup due to clustering with EHDV-4 and -5 strains. For this purpose, the Warrego virus (WARV) was used. 2.5. Virus Isolation Attempts to isolate EHDV directly on Vero (African green monkey kidney) and BHK- 21 (baby hamster kidney) cells failed. Thus, red blood cells (RBS) were washed three times in PBS. The washed RBS were disrupted via dilution in sterile double-distilled water in a proportion of 1:10 (RBS to water). The resulting solution was used for inoculating Vero and BHK-21 cells, incubated in the cell incubator for one hour at a temperature of 37 ◦C, and washed two times with PBS. Eagle’s Minimum Essential Medium (EMEM), supplemented with 2% fetal bovine serum (FBS) and 1% penicillin–streptomycin (10,000 U/mL), was added to the cell monolayer. Cells were observed daily for the appearance of cytopathic effects. Three blind passages were performed. Sixty-two samples positive in RT-qPCR for EHDV samples were inoculated into embryonated chicken eggs (ECE) according to the method described by Komarov and Goldsmit [55]. Epidemiologia 2024, 5 94 3.3. Virus Isolation Nine EHDV-7 viruses were isolated in ECE during 2020–2021: eight from whole blood samples of viremic animals and one from a spleen sample. Five isolates from ECE were subsequently adapted to Vero or BHK-21 cells. Attempts to isolate EHDV-8 in tissue cultures and ECE were unsuccessful. 3. Results EHDV Detection by Pan-EHDV RT-qPCR from Field Samples Collected in 2020–2021 and 2023 In the 2020–2021 calendar year, 283 out of 1002 tested samples from 995 animals were positive for Pan-EHDV RT-qPCR (Table 1). In 2023, two calves, randomly selected for analysis from 15 imported calves from Portugal, were EHDV-positive and were identified by partial Sanger sequencing of Seg-2 as EHDV-8-positive. It is noteworthy that one of the imported calves was also positive in the pan-BTV-RT qPCR. Partial sequencing confirmed a high genetic homology with Spanish and Moroccan BTV-4 (data are not provided; primers for Sanger sequencing are provided in Table S1). All other samples tested in 2023 were EHDV-negative. 3. Results 3.1. Clinical Signs in Affected Animals and Geographic Distribution of Israeli EHDVs Clinical signs in affected animals and the geographic distribution of EHDV-6 and EHDV-1 were previously described. In brief, the main clinical signs observed in EHDV- 6-affected cattle included reduced milk production, weakness, drooling, lameness and recumbency, fever, slight erythema of nasal and oral mucosae, weight loss, and abortion. Dyspnea, cachexia, and death were observed less frequently [22]. Regarding EHDV-1- affected cattle, the clinical signs were milder, compared with those caused by EHDV-6. In many farms, EHDV-1 infection was asymptomatic or subclinical; milk yield reduction, fever, and recumbency were the only prominent clinical signs that were seen during the outbreak [27].i Considering EHDV-7, it was first detected at Hama’apil, located in the Central District of Israel, on 23 August 2020. The next registered case was identified three weeks later at Ma’ale Gamla, the settlement located in the Western part of the Golan Heights. From mid-September to mid-October 2020, EHDV-7 was found in only six settlements—five of them situated in the North of Israel and one in the center. In the second part of October 2020, EHDV was first identified in the Southern District of Israel. Since then, it has been found in areas from the North Golan Height to the Negev desert. The last registered EHDV detections occurred in the second part of April 2021, from the settlement located in the southern part of the Central District and Negev desert, where the weather is usually warmer than in the northern part of Israel. To summarize, the virus was detected in 80 settlements. Regarding clinical signs manifested by cattle affected by EHDV-7, they included recumbency, weakness, fever, lameness, hypersalivation and nasal discharge, sporadic death in a herd, milk reduction in milking cows, diarrhea, limb edema, and abortions. In mixed with BTV cases, clinical signs included a sharp decrease in body weight up to cachexia and anemia; cases of death in a herd were also reported.i On 15 September 2023, fifteen calves without prominent clinical signs were randomly chosen for testing for EHDV and BTV by RT-qPCR from the group of calves from Portugal imported on 31 August 2023, where EHDV-8 was reported since mid-July 2023. 3.2. EHDV Detection by Pan-EHDV RT-qPCR from Field Samples Collected in 2020–2021 and 2023 3.2. EHDV Detection by Pan-EHDV RT-qPCR from Field Samples Collected in 2020–2021 and 2023 3.2. 3.4. Sequence Analyses of EHDV-1, -6, -7, and -8 The coding regions of EHDV-1 strain ISR-2096/16, EHDV-6 strain ISR-4487/15, EHDV- 7 strain ISR-2262/2/20 were completely sequenced. Complete information about the strain and data on accession numbers, which were uploaded to the INSDC, are shown in Table S2. BLAST analysis based on a comparison of nt sequences of all completely sequenced Israeli EHDV strains with global EHDV is presented in Table 3. For revealing probable ancestors, more recent viruses were not included in the study. For this reason, Israeli EHDV-6 and Epidemiologia 2024, 5 95 EHDV-1 do not show any representation of Seg-1 and -4; however, Israeli EHDV-7 from 2006, in the case of Seg-1, and EHDV-4 from Nigeria from 1968, in the case of Seg-4, were shown to have the closest relationship via BLAST analysis. Both Seg-1 of Israeli EHDV-6 and EHDV-1 have more than a 99% identity with the Israeli EHDV-7 ISR2006/04/2006 strain from 2006. To sum up, six out of ten genome segments of Israeli EHDV-6 ISR-4487/15 had a high identity with Israeli EHDV-7 strains from 2006; four of them had more than a 99% identity, probably pointing out their local, regional origin. The last four segments have a high identity with “Western” strains from Africa and the Arabian Peninsula and surrounding islands, which can indicate the co-circulation of EHDVs of African and Middle East origin in the same area. Comparing EHDV-1 to the global EHDV strains, it was seen that only Seg-1 probably has a local origin when all other segments had an African origin. Looking at the BLAST analysis of Israeli EHDV-7 ISR-2262/2/20, only Japanese and Chinese EHDV strains have high homology with Israeli EHDV-7, illustrating its “Eastern” origin. The EHDV-8 strain identified in blood samples from calves imported from Portugal showed 99.70% identity with the last published Tunisian EHDV-8 strains identified in Culicoides sp. in 2022 (Table 3). Table 3. BLAST analysis of Israeli EHDV strains with global EHDV strains. Table 3. BLAST analysis of Israeli EHDV strains with global EHDV strains. Serotype/Israeli Strain Segment Identity (%) Accession Number/Serotype/Strain/Year Country of Isolation EHDV-6/ISR-4487/15 1 99.26 KM391733/EHDV-7/ISR2006/04/2006 Israel 2 96.02 HM156729/EHDV-6/ALG2006/02/2006 Algeria 3 99.37 KM391740/ EHDV-7/ISR2006/04/2006 Israel 4 92.71 AM745020/ EHDV-4/IbAr 33853/1968 Nigeria 5 99.46 JQ070181/EHDV-7/ISR2006/04/2006 Israel 6 96.28 AM745072/EHDV-6/318/1983 Bahrain 7 97.05 AY351653/EHDV/2003? The EHDV genome consists of 10 linear segments of double-stranded RNA. The viral genome encodes seven structural proteins (VP1 to VP7) and four different non-structural proteins (NS1, NS2, NS3/3a, and NS4). p ( ) Seg-1: Both Israeli EHDV-6 and EHDV-1 are most closely related to each other and to EHDV-7 from 2006. EHDV-7 from 2020 clustered with Japanese EHDV-6 strain HG-1E/15 3.5. Phylogenetic Analysis of Israeli EHDV Strains 3.5. Phylogenetic Analysis of Israeli EHDV Strains 3.4. Sequence Analyses of EHDV-1, -6, -7, and -8 France, Reunion 8 97.06 KM391730/EHDV-7/ISR2006/06/2006 Israel 9 98.89 KM391747/EHDV-7/ISR2006/06/2006 Israel 10 99.71 KM391724/ EHDV-7/ISR2006/06/2006 Israel EHDV-1/ISR-2096/16 1 99.57 KM391733/EHDV-7/ISR2006/04/2006 Israel 2 87.45 AM745008/EHDV-1/bAr22619/1967 Nigeria 3 96.22 AM745019/EHDV-4/IbAr 33853/1968 Nigeria 4 92.51 AM745020/EHDV-4/IbAr 33853/1968 Nigeria 5 97.52 AM745021/EHDV-4/IbAr 33853/1968 Nigeria 6 96.09 AM745012/EHDV-1/IbAr22619/1967 Nigeria 7 97.43 AM745013/ EHDV-1/IbAr22619/1967 Nigeria 8 95.63 AM745074/EHDV-6/318/1983 Bahrain 9 95.59 AM745025/EHDV-4/IbAr 33853/1968 Nigeria 10 95.05 EU928893 EHDV-3/Nigeria-ODV0001 Nigeria 10 95.05 AM745016/EHDV-1/ IbAr22619/1967 Nigeria EHDV-7/ISR-2262/2/20 1 96.30 LC552748/EHDV/ON-4/B/98/1998 Japan 2 98.13 LC202943/EHDV-7/KSB-14/E/97/1997 Japan 3 96.42 MK656455/EHDV-7/YN09-04/2013 China 4 92.48 AM745080/EHDV-2/Ibaraki/1959 Japan 5 97.09 MT013328/EHDV-10/JC13C644/2013 China 6 98.52 LC202954/EHDV-7/KSB-14/E/97/1997 Japan 7 98.60 LC552744/EHDV-7/KSB-14/E/97/1997 Japan 8 95.75 KM509057/EHDV-2/Ibaraki BK13/1997 Japan 9 95.20 LC552753/EHDV/ON-4/B/98/1998 Japan 10 97.87 LC552747/EHDV-7/KSB-14/E/97/1997 Japan EHDV-8/ISR-1692/2/23 2 99.70 OP937332/EHDV-8/Culicoides sp/2 TUN2022/2022 Tunisia Epidemiologia 2024, 5 96 from 2015, untyped Japanese strain ON-4/B/98 from 1998, and Chinese strain EHDV-10 strain JC13C644 from 2013 (Figure 1a). from 2015, untyped Japanese strain ON-4/B/98 from 1998, and Chinese strain EHDV-10 strain JC13C644 from 2013 (Figure 1a). g Seg-2: Israeli EHDV-6 strains clustered with EHDV-6 strains which were identified during 2006 outbreaks in North African countries and with EHDV-6 from Bahrain, Reunion Iceland, and South Africa. Israeli EHDV-1 ISR-2096/16 clustered with EHDV-1 E21/C identified in Egypt in the same year (2016) and with Nigerian EHDV-1 IbAr22619 from 1967. The Israeli EHDV-7 strain ISR-2262/2/20 from 2020 clustered with several Japanese EHDV-7 strains. EHDV-8, which was identified in imported from Portugal calves, clustered with Tunisian, and Italian strains isolated in 2021–2022 (Figure 1b). g Seg-2: Israeli EHDV-6 strains clustered with EHDV-6 strains which were identified during 2006 outbreaks in North African countries and with EHDV-6 from Bahrain, Reunion Iceland, and South Africa. Israeli EHDV-1 ISR-2096/16 clustered with EHDV-1 E21/C identified in Egypt in the same year (2016) and with Nigerian EHDV-1 IbAr22619 from 1967. The Israeli EHDV-7 strain ISR-2262/2/20 from 2020 clustered with several Japanese EHDV-7 strains. EHDV-8, which was identified in imported from Portugal calves, clustered with Tunisian, and Italian strains isolated in 2021–2022 (Figure 1b). Seg-3: The Israeli EHDV-6 ISR-4487/15 strain clustered with the Israeli EHDV-7 ISR2006/06 strain. The EHDV-1 strain ISR-2096/16 clustered with Tunisian EHDV-6 Tunisia 2577 from 2006, and Tunisian and Italian EHDV-8 strains from 2021 to 2022. Israeli EHDV-7 ISR-2262/2/20 strain clustered with Japanese EHDV-6 strain HG-1E/15 from 2015 (Figure 1c). Seg-4: Israeli EHDV-6 ISR-4487/15 strain clustered with Israeli EHDV-1 ISR-2096/16, forming a monophyletic group. 3.4. Sequence Analyses of EHDV-1, -6, -7, and -8 Israeli EHDV-7 ISR-2262/2/20 strain grouped with “East- ern” strains but made a separate branch (Figure 1d). strains but made a separate branch (Figure 1d). Seg-5: The Israeli EHDV-6 ISR-4487/15 strain clustered with the Israeli EHDV-7 ISR2006/04 strain; EHDV-1 ISR-2096/16 strain clustered with Nigerian EHDV-4 strains isolated in 1968. The Israeli EHDV-7 ISR-2262/2/20 strain grouped with the following Eastern strains: Japanese EHDV-6 HG-1E/15 from 2015, untyped Japanese strain ON- 4/B/98 from 1998, and Chinese strain EHDV-10 strain JC13C644 from 2013 (Figure 1e).i Seg-5: The Israeli EHDV-6 ISR-4487/15 strain clustered with the Israeli EHDV-7 ISR2006/04 strain; EHDV-1 ISR-2096/16 strain clustered with Nigerian EHDV-4 strains isolated in 1968. The Israeli EHDV-7 ISR-2262/2/20 strain grouped with the following Eastern strains: Japanese EHDV-6 HG-1E/15 from 2015, untyped Japanese strain ON- 4/B/98 from 1998, and Chinese strain EHDV-10 strain JC13C644 from 2013 (Figure 1e). g Seg-6: Israeli EHDV-6 strains clustered with EHDV-6 strains identified during 2006 outbreaks in North African countries and with EHDV-6 from Bahrain, Reunion Iceland, and South Africa. Israeli EHDV-1 ISR-2096/16 clustered with EHDV-1 E21/C identified in Egypt in the same year (2016) and with Nigerian EHDV-1 IbAr22619 from 1967. Israeli EHDV-7 strain ISR-2262/2/20 from 2020 clustered with several Japanese and Chinese EHDV-7 strains (Figure 1f). Seg-6: Israeli EHDV-6 strains clustered with EHDV-6 strains identified during 2006 outbreaks in North African countries and with EHDV-6 from Bahrain, Reunion Iceland, and South Africa. Israeli EHDV-1 ISR-2096/16 clustered with EHDV-1 E21/C identified in Egypt in the same year (2016) and with Nigerian EHDV-1 IbAr22619 from 1967. Israeli EHDV-7 strain ISR-2262/2/20 from 2020 clustered with several Japanese and Chinese EHDV-7 strains (Figure 1f). ( g ) Seg-7: Israeli EHDV-6 strains clustered with EHDV-6 and EHDV-8 “Western” regional strains, which originated from Bahrain, Tunisia, Italy, Reunion Iceland, and South Africa. Israeli EHDV-1 ISR-2096/16 clustered with Nigerian EHDV-1 IbAr22619 from 1967. Israeli EHDV-7 strain ISR-2262/2/20 from 2020 clustered with Japanese EHDV-2 strain KS-8/E/13, which is a slightly different result from BLAST analysis when the most closely related strain was EHDV-7 KSB-14/E/97 isolated in 1997. Notably, EHDVs cannot be strictly subdivided into “Eastern” and “Western” topotypes according to phylogenetic analysis of Seg-7 sequences (Figure 1g). g Seg-7: Israeli EHDV-6 strains clustered with EHDV-6 and EHDV-8 “Western” regional strains, which originated from Bahrain, Tunisia, Italy, Reunion Iceland, and South Africa. Israeli EHDV-1 ISR-2096/16 clustered with Nigerian EHDV-1 IbAr22619 from 1967. 3.4. Sequence Analyses of EHDV-1, -6, -7, and -8 Israeli EHDV-7 strain ISR-2262/2/20 from 2020 clustered with Japanese EHDV-2 strain KS-8/E/13, which is a slightly different result from BLAST analysis when the most closely related strain was EHDV-7 KSB-14/E/97 isolated in 1997. Notably, EHDVs cannot be strictly subdivided into “Eastern” and “Western” topotypes according to phylogenetic analysis of Seg-7 sequences (Figure 1g). g q g g Seg-8: The Israeli EHDV-6 ISR-4487/15 strain clustered with the Israeli EHDV-7 ISR2006/04 strain; the EHDV-1 ISR-2096/16 strain clustered with EHDV-6 318 strain from Bahrain. Israeli EHDV-7 ISR-2262/2/20 clustered with the Japanese EHDV-2 Ibaraki strain BK13 from 1997 (Figure 1h). g Seg-9: The Israeli EHDV-6 ISR-4487/15 strain clustered with the Israeli EHDV-7 ISR2006/06 strain; EHDV-1 ISR-2096/16 formed a separate branch with “Western” regional strains, belonged to serotypes 1, 4, 6, 7, and 8. Israeli EHDV-7 ISR-2262/2/20 clustered with “Eastern” strains belonged untyped Japanese strain ON-4/B/98 from 1998, Chinese strain EHDV-10 strain JC13C644 from 2013, and Australian EHDV-8 strain CPR 3961A isolated in 1982, but formed a separate branch (Figure 1i). p g Seg-10: The Israeli EHDV-6 ISR-4487/15 strain clustered with the Israeli EHDV-7 ISR2006/06 strain; EHDV-1 ISR-2096/16 clustered with Nigerian strains belonged to serotypes 1 and 3 (strains IbAr22619 and Nigeria-ODV0001, respectively), similarly to the results of BLAST analysis. Israeli EHDV-7 ISR-2262/2/20 clustered with Japanese EHDV-7 KSB-14/E/97 from 1997. As in the case of Seg-7, EHDVs cannot be divided into “Eastern” and “Western” topotypes by Seg-10. Additionally, some Japanese strains isolated in 2015–2017 that belong to serotypes 4 and 5 have significantly different sequences of the Epidemiologia 2024, 5 97 Seg-10 from all other global EHDV strains and are represented in Figure 1j by the EHDV-5 ON-11/E/16/2016 strain. m all other global EHDV strains and are represented in Figure 1j by the EHDV-5 ON- /E/16/2016 strain. Seg-10 from all other global EHDV strains and are represented in Figure 1j by the EHDV-5 ON-11/E/16/2016 strain. om all other global EHDV strains and are represented in Figure 1j by the EHDV-5 ON- 1/E/16/2016 strain. (a) Seg-1 (b) Seg-2 Figure 1. Cont. Figure 1. Cont. Epidemiologia 2024, 5 98 W (c) Seg-3 (d) Seg-4 Figure 1. Cont. (d) Seg-4 Figure 1. Cont. Epidemiologia 2024, 5 99 (d) Seg-4 (e) Seg-5 (f) Seg-6 Figure 1. Cont. (e) Seg-5 (f) Seg-6 Figure 1. Cont. (f) Seg-6 Figure 1. Cont. Epidemiologia 2024, 5 100 (f) Seg-6 (g) Seg-7 W (h) Seg-8 Figure 1. Cont. W W (g) Seg-7 (h) Seg-8 Figure 1. Cont. (g) Seg 7 (h) Seg-8 Figure 1. Cont. Epidemiologia 2024, 5 101 (i) Seg-9 13 (j) Seg-10 Phylogenetic trees of Israeli and global EHDV strains. (a–j) Phylogenetic trees of segments eli EHDVs used in the study are shown in bold and underlined. The phylogeny was using the maximum-likelihood method and the Tamura–Nei model method. The e of replicate trees in which the associated taxa clustered together in the bootstrap test plicates) are shown next to the branches. Viruses were identified by accession erotype/location/isolate/year. eneral, the phylogenetic analysis of internal genes showed mostly the same s BLAST analyses, which are presented in Table 3, except Seg-1 and -4, where due Figure 1. Phylogenetic trees of Israeli and global EHDV strains. (a–j) Phylogenetic trees of segments 1–10. Israeli EHDVs used in the study are shown in bold and underlined. The phy- logeny was inferred using the maximum-likelihood method and the Tamura–Nei model method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Viruses were identified by accession num- ber/serotype/location/isolate/year. 4. Discussion During the last fifteen years, Israeli cattle have been affected by many arboviruses for the first time. The appearance of new for the region pathogenic arboviruses has been higher than in all other countries in the Mediterranean region. Most of these viruses, which have segmented genomes, such as the Shuni virus and BTV-1, -3, -6, -8, and -9, were prominently of African origin and were seriously reassorted with the local strains [56,60–62], which points to their co-circulation with the local strains. p Considering BLAST and phylogenetic analysis of Israeli EHDV-6, when six out of ten viral segments have a close relationship with Israeli EHDV-7 identified in 2006, their ancestors probably circulated in the region for a prolonged period. Considering all viral genes of Israeli EHDV-1, identified in 2016, it probably originated in Africa since nine out of ten viral segments have a high identity with African strains. Interestingly, Israeli EHDV-1 and -6 have a very close identity to Seg-1, which indicates their probable common ancestor or reassortment with the common ancestor of both viruses, which can point to probable circulation in the region different EHDVs possessing the same sequence of Seg-1. This theory can be indirectly confirmed by the evidence of the closely related sequence of Seg-1 of recently identified EHDV-8 in the region (Figure 1a). Considering the published genetic analysis of EHDV-8 [28,29] and the analysis conducted in this study, it was found that EHDV-8, registered in the Mediterranean area from 2021 to 2023, is closely related by Seg-1 and -9 to Israeli EHDV-7 from 2006, EHDV-6 from 2015, and EHDV-1 from 2016. Meanwhile, all other viral segments probably originated from different EHDVs, which were previously identified in Africa. These facts point to the appearance of a non-identified EHDV-8 of African origin in the region. To sum up, EHDV-1, -6, and -8 identified in the Mediterranean region during the last decade had regional or/and African origin. In contrast, EHDV-7, which caused an outbreak in 2020 in Israel, possesses only “East- ern” segments and lacks “Western” segments. Therefore, it can serve as an indication of the route of introduction and timeframe of its circulation in the region. This provides us the op- portunity to presume that the introduction of this virus into the country was anthropogenic because Israel imported large amounts of livestock, which can be unexpectedly infected with EHDV. (i) Seg-9 (i) Seg-9 (i) Seg 9 (j) Seg-10 hylogenetic trees of Israeli and global EHDV strains. (a–j) Phylogenetic trees of segments i EHDVs used in the study are shown in bold and underlined. The phylogeny was sing the maximum-likelihood method and the Tamura–Nei model method. The of replicate trees in which the associated taxa clustered together in the bootstrap test cates) are shown next to the branches. Viruses were identified by accession otype/location/isolate/year. neral, the phylogenetic analysis of internal genes showed mostly the same Figure 1. Phylogenetic trees of Israeli and global EHDV strains. (a–j) Phylogenetic trees of segments 1–10. Israeli EHDVs used in the study are shown in bold and underlined. The phy- logeny was inferred using the maximum-likelihood method and the Tamura–Nei model method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Viruses were identified by accession num- ber/serotype/location/isolate/year. Epidemiologia 2024, 5 102 In general, the phylogenetic analysis of internal genes showed mostly the same results as BLAST analyses, which are presented in Table 3, except Seg-1 and -4, where due to very close identity one to another, we presented in Table 3 the next closely related sequences belonged to viruses from the global database. According to phylogenetic analysis, Israeli EHDV-6 and-1 belonged to the “Western” topotype, while EHDV-7 from 2020 belonged to the “Eastern” topotype. 4. Discussion 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. Acknowledgments: We thank Velizar Bumbarov for virus isolation and administration and Marisol Guini-Rubinstein, Anita Kovtunenko, and Olga Zalezski for technical assistance. Acknowledgments: We thank Velizar Bumbarov for virus isolation and administration and Marisol Guini-Rubinstein, Anita Kovtunenko, and Olga Zalezski for technical assistance. Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. my. Available online: https://ictv.global/report/chapter/sedoreoviridae/sedoreoviridae/orbivirus (accessed 3) 1. ICTV. Virus Taxonomy. Available online: https://ictv.global/report/chapter/sedoreoviridae/sedoreoviridae/orbivirus (accessed on 5 September 2023). 2. Matthijnssens, J.; Attoui, H.; Bányai, K.; Brussaard, C.P.D.; Danthi, P.; Del Vas, M.; Dermody, T.S.; Duncan, R.; F¯ang, Q.; Johne, R.; et al. ICTV Virus Taxonomy Profile: Sedoreoviridae 2022. J. Gen. Virol. 2022, 103, 001782. [CrossRef] [PubMed] 3. King, A.M.; Adams, M.J.; Carstens, E.B.; Lefkowitz, E.J. Virus Taxonomy, 9th ed.; Academic Press: San Die 3. King, A.M.; Adams, M.J.; Carstens, E.B.; Lefkowitz, E.J. 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Discussion This was seen in the example of imported calves from Portugal when calves were exposed to EHDV-8 (RNA of EHDV-8 was detected in the blood of these animals). Co-circulation of several serotypes in the region, processing different genotypes, can lead to the appearance of new strains with distinct clinical manifestations. When summarizing the data on the clinical symptoms of Israeli EHDVs, it was ob- served that these symptoms were similar to those seen in other EHDVs worldwide. Thus, Israeli and Japanese EHDV-1 had mild clinical manifestations [26,27]. At the same time, more severe clinical signs were caused by EHDV-6 and -7 in Japan and Israel, which in- cluded fever, milk reduction, edema of the head, hyperemia, hemorrhages and lesions of the mucosal membranes and teats, lameness and stiff gate, accompanied by anorexia and respiratory distress, and sporadic death, abortion, and stillbirth [19,20,22,25]. Since EHDV continues to spread to new areas, preventive measures such as a restric- tion of the transportation of infected animals, which demands wide systematic diagnostic tests both on the exporting and importing sides, strict quarantine procedures, developing and producing vaccines, and probable medical treatment of affected animals are of high importance. This will allow for not only a decrease in economic losses caused by direct effects from the disease as death of affected animals, milk losses, abortions, slaughtering Epidemiologia 2024, 5 103 of heavily diseased animals, and expenses for symptomatic treatment but also by indirect losses as extra expenses for veterinary services and trade restrictions. Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/epidemiologia5010006/s1. Table S1: List of primers used for partial sequencing of epizootic hemorrhagic disease virus serotype 7 and bluetongue virus serotype 4; Table S2: List of sequenced Israeli EHDV strains for the present study. Author Contributions: Methodology, N.G. and B.H.; writing—original draft preparation, N.G.; writing—review and editing, B.H.; visualization, N.G.; supervision, B.H. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: No human subjects were used in this study. Cattle field samples were collected as a part of routine diagnosis. Since no experiments were performed, approval from an ethics committee was not required. 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. p g 17. Kitano, Y. Ibaraki disease in cattle. 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https://openalex.org/W3094921256	https://painmedicine.org.ua/index.php/pnmdcn/article/download/233/205	Ukrainian	null	Therapeutic and diagnostic blockade of the knee joint as a component of pain management in gonarthrosis and a predictor of further successful radio-frequent denervation of the joint	Medicina bolû	2,020	cc-by	2,846	Медицина болю (Pain Medicine). – 2020. – Том 5, № 3 Оригінальна методика / Original method PainMedicine Journal Медицина Болю // Медицина Боли Том 5, № 3 • 2020 www. painmedicine.org.ua Міждисциплінарний • Науково-практичний журнал Оригінальна методика / Original method PainMedicine Journal Медицина Болю // Медицина Боли Том 5, № 3 • 2020 www. painmedicine.org.ua Міждисциплінарний • Науково-практичний журнал Оригінальна методика / Original method PainMedicine Journal Медицина Болю // Медицина Боли Том 5, № 3 • 2020 Оригінальна методика / Original method 0 Оригінальна методика / Original method Том 5, № 3 • 2020 р Том 5, № 3 • 2020 Медицина Болю // Медицина Боли www. painmedicine.org.ua DOI: 10.31636/pmjua.v5i3.5 Лікувально-діагностична блокада колінного суглоба як складова менеджменту больового синдрому при гонартрозі та предиктор подальшої успішної радіочастотної денервації суглоба ● ● медикаментозна терапія (фармакологічні пре- парати для системного та топічного застосу- вання – нестероїдні протизапальні препарати, анальгетики, хондропротектори, внутрішньосу глобові ін’єкції препаратів гіалуронової кислоти, стероїдів, PRP; Значний прогрес у розумінні патофізіології та ево- люції ОА привів не тільки до покращення діагностики патології, але й до перегляду методології та метрології захворювання. р , ; ● ● фізіотерапія; р , ; ● ● фізіотерапія; ● ● хірургічні методи лікування (артроскопічна са- нація, різні варіанти остеотомій, тотальне або часткове протезування суглоба). Це пов’язано з рядом факторів (4): Це пов’язано з рядом факторів (4): ● ● нерідко – безсимптомністю патології; ● ● дисоціацією між рентгенологічною картиною та клінічною маніфестацією; Фармакологічна підтримка при лікуванню болю, зумовленого ОА, має лімітовані переваги. Більше того, використання НПЗЗ пов’язане з високим ризиком та- ких побічних ефектів, як кровотечі та гастроінтести- нальні розлади (6, 7). ● ● частою невідповідністю даних артроскопії та рентгенографії; ● ● відсутністю надійних біомаркерів деградації хряща, які відображають прогресування ОА та мають прогностичне значення. Нехірургічні інтервенції, включаючи внутріш- ньосуглобові ін’єкції стероїдів, гіалуронової кислоти, акупунктура та періостальна стимулююча терапія, часто використовуються як допоміжні заходи, але не завжди допомагають адекватно контролювати больо- вий синдром (8, 9, 10, 11, 12). Використання шкал та опитувальників дозволяє оці- нити біль і функцію суглоба, зокрема перспективу па- цієнта та його працездатність шляхом оцінки таких дій, як ходьба, та інших стандартизованих рухів у контр- ольованих умовах. Зокрема, рутинно використову- ють візуально-аналогову шкалу (ВАШ), опитувальник Мак-Гілла, painDETECT. Серед ОА-специфічних інстру- ментів для вимірювання болю широко застосовується шкала “Індекс вираженості ОА Університетів Західного Онтаріо та Мак-Мастера” [Western Ontario McMaster Universities OA Index (WOMAC)]. За її допомогою ви- значають важкість болю при ОА кульшового та колін- ного суглобів під час виконання п’яти видів діяльно- сті – в положенні стоячи, під час ходьби, при підйомі сходами, у спокої та вночі. Індекс WOMAC – високоін- формативний показник, який можна використовувати для оцінки ефективності медикаментозного та неме- дикаментозного лікування. Хірургічне лікування є досить ефективним методом лікування остеоартрозу колінного суглоба (13). у р р у у Однак, враховуючи те, що значна частка пацієнтів є людьми похилого та старечого віку, із низкою су- путніх соматичних патологій, які перебувають у стадії суб- чи декомпенсації, вони не можуть розглядатись як кандидати на оперативне лікування взагалі або потре- бують компенсації вітальних показників, що дозволить безпечно виконати операцію та анестезіологічне забез- печення. Лікувально-діагностична блокада колінного суглоба як складова менеджменту больового синдрому при гонартрозі та предиктор подальшої успішної радіочастотної денервації суглоба Седлецький Р. Є. Седлецький Р. Є. ДУ “Інститут ортопедії та травматології НАМНУ”, Київ ДУ “Інститут ортопедії та травматології НАМНУ”, Київ Резюме. За даними ВООЗ, в останні десятиліття відбувається зміна структури захворюванності. Прогре- сивно збільшується чисельність людей з хронічним больовим синдромом. Зокрема, це пов’язано з постарін- ням населення та збільшенням питомої ваги дегенеративно-дистрофічних захворювань опорно-рухового апарату, в тому числі остеоартрозу колінного суглоба. Останній призводить не лише до тимчасової або постійної втрати працездатності, а й до значного зниження якості життя людини. За прогнозами ВООЗ, гонартроз у найближче десятиліття стане четвертою причиною інвалідності у жі- нок та восьмою – у чоловіків (1). За прогнозами ВООЗ, гонартроз у найближче десятиліття стане четвертою причиною інвалідності у жі- нок та восьмою – у чоловіків (1). Висновок: лікувально-діагностична блокада генікулярних нервів колінного суглоба є ефективною опцією терапії болю при гонартрозі та адекватним предиктором для наступного виконання радіочастотної де- нервації суглоба. Ключові слова: остеоартроз колінного суглоба (ОА), лікувально-діагностична блокада генікулярних нервів (ЛДБ), денервація (RFA), дерецепція, абляція, нейротомія Гонартроз за етіологією поділяють на первинний та вторинний. суглоб. Крім того, причиною можуть бути пошкоджен- ня стегнової та великогомілкової кісток, наколінка, що зрослися в неправильному положенні і призводять до зміни анатомічної та механічної осі нижньої кінців- ки, порушуючи конгруентність суглобових поверхонь і в подальшому призводячи до деформації та руйнуван- ня суглобового хряща. Рідко трапляються інші причини Первинний, або ідіопатичний, – причина виникнен- ня невідома. Коли причина пошкодження суглоба відома, йдеть- ся про вторинний остеоартрит. Найчастіше це внутріш- ньосуглобові переломи кісток, що утворюють колінний Медицина болю (Pain Medicine). – 2020. – Том 5, № 3 34 Оригінальна методика / Original method У стандарті лікування можна виділити кілька на- прямків: артрозу, наприклад, запальні захворювання різної еті- ології, що змінюють нормальний гомеостаз синовіаль- ної рідини (2). ● ● немедикаментозна терапія (бесіда з пацієнтом, спрямована на профілактику артрозу, – знижен- ня маси тіла, заняття лікувальною фізкультурою, профілактика травматизму, формування м’язо- вого корсета, використання ортезів та інших фіксаторів; У світі загальноприйнятими є класифікації морфо- функціональних змін у суглобі, засновані на рентгено- логічних змінах. Найпоширеніша з них – класифікація Кellgren – Lawrence, запропонована в 1963 році. За- пропоновано виділяти чотири ступені: І ст. – сумнів- ний; ІІ ст. – незначно виражені осифікати; ІІІ ст. – по- мірний, звуження суглобової щілини; ІV ст. – важкий, значне звуження суглобової щілини, зі склерозом суб- хондральної пластинки (3). Лікувально-діагностична блокада колінного суглоба як складова менеджменту больового синдрому при гонартрозі та предиктор подальшої успішної радіочастотної денервації суглоба Саме в таких випадках на допомогу приходять інтервенційні методи лікування болю, в тому числі лі- кувально-діагностична блокада генікулярних нервів та радіочастотна нейротомія колінного суглоба, яка по- лягає у перериванні больової імпульсації від джерела болю (в даному випадку колінного суглоба) до вищих нервових центрів. Відбувається це шляхом теплової деструкції так званих “генікулярних” нервів, що несуть сенсорні імпульси від колінного суглоба. Використан- ня методу нейронавігації (пошуку нервових структур та їх диференціювання на чутливі й моторні) робить процедуру абсолютно безпечною. Абляції в обов’язко- вому порядку передує діагностична (використовуєть- ся лише місцевий анестетик) або лікувально-діагнос- На сучасному етапі лікування переслідує наступні цілі (5): На сучасному етапі лікування переслідує наступні цілі (5): ● ● уповільнення прогресуючого перебігу захворю- вання; ● ● купірування больового синдрому; ● ● відновлення конгруентності та можливості осьового навантаження; осьового навантаження; ● ● досягнення ремісії; ● ● соціальна інтеграція пацієнта. ISSN 2414–3812 Оригінальна методика / Original method 35 тична блокада вищезазначених нервів. Лікувальний ефект останньої полягає у введенні, крім місцевого анестетику, стероїдного препарату, що активно зні- має запальний компонент болю. Оскільки процедура позасуглобова, кортикостероїд не чинить негативного ефекту на суглобовий хрящ та інші структури суглоба, не сприяє пришвидшенню подальшої деградації анато- мічних структур. Лише після того, як тестова блокада дасть позитивний результат (зменшення болю щонай- менше на 50 % протягом першої доби після процеду- ри), доцільно виконувати RF-абляцію. нартроз). Рентгенологічно ОА лівого колінного суглоба відповідає класифікації Kellgren – Lawrence-4. Ліку- ється під наглядом ортопеда-травматолога за місцем проживання, отримувала внутрішньосуглобові ін’єкцїї препаратів гіалуронової кислоти та PRP (Plasma rich platelet – терапія плазмою, збагаченою тромбоцита- ми) – без полегшення. Потребує ендопротезування суглоба, але з власних мотивів не готова до оператив- ного втручання. На момент звернення больовий син- дром за ВАШ – 7 балів, індекс WOMAC – 78. Останнім часом біль став абсолютно резистентним до медикаментозної терапії, значно порушує щоденну фізичну активність пацієнтки, унеможливлює повсяк- денні побутові справи. Колінний суглоб іннервується артикулярними гіл- ками різних нервів, серед яких основні – це стегновий, затульний, загальний малогомілковий та великогоміл- ковий (14, 15). Суглобові гілки колінного суглоба та- кож відомі як генікулярні нерви (рис. 1). Після остаточного узгодження всіх питань із паці- єнткою та її рідними й після отримання письмової зго- ди, успішно виконано лікувально-діагностичну блока- ду генікулярних нервів (фото додаються). Побічні ефекти Рис. 4. Бокова рентгенпроекція У переважній більшості випадків ЛДБ та RFA є без- печними методоми лікування болю з мінімальною кількістю побічних ефектів. Найпоширенішим з них є післяпроцедурний дискомфорт, що профілактується введенням місцевого анестетику в проекцію кожного цільового генікулярного нерва, а також призначенням анальгетиків у найближчі 3–5 днів після маніпуляції. В нашому випадку інших побічних ефектів, окрім ви- щезазначеного, не спостерігалось. Конфлікт інтересів – відсутній. Лікувально-діагностична блокада колінного суглоба як складова менеджменту больового синдрому при гонартрозі та предиктор подальшої успішної радіочастотної денервації суглоба Критерії відбору пацієнтів для ЛДБ та RFA колінно- го суглоба: ● ● біль за ВАШ 5 і більше балів; Процедуру виконано в умовах операційної, з до- триманням правил асептики та антисептики, під контролем електронно-оптичного перетворювача (С-дуга) під місцевою анестезією 2 % лідокаїном та в/в аналгоседацією (метамізол – 2 мл, декскетопрофен – 50 мг, парацетамол – 1 000 мг). ● ● тривалість больового синдрому не менше 3-х місяців; ● ● ІІ–ІV ст. за Kellgren – Lawrence; ● ● відсутність ефекту від медикаментозної терапії, фізіотерапії, внутрішньосуглобових ін’єкцій. фізіотерапії, внутрішньосуглобових ін’єкцій. Пацієнтка під час процедури перебувала в положен- ні супінації з подушкою в підколінній ямці для більшого комфорту. Після попередньої розмітки виконано оброб- ку операційного поля та місцеву анестезію. Після отри- мання передньо-заднього рентгензображення, спинно- мозкові голки Spinocan 22 G просунуто в місце проход- ження генікулярних нервів – верхніх медіального (рис. 2) та латерального, нижнього медіального (рис. 3) – місця переходу стегнової кістки в медіальний та латеральний надвиростки і переходу великогомілкової кістки в меді- альний надвиросток відповідно. Обов’язковий рентген- Пацієнтка О., 1949 р.н., зріст – 160 см, вага – 93 кг, звернулась зі скаргами на біль в обох колінних су глобах, переважно в лівому. Має ожиріння ІІ ступеня, цукровий діабет, ішемічну хворобу серця, гіпертонію. Тривалий час хворіє артрозом колінних суглобів (го- Рис. 1. Схематичне зображення генікулярних нервів Рис. 2. Передньо-задня рентгенпроекція, місце локації верхнього медіального генікулярного нерва Рис. 2. Передньо-задня рентгенпроекція, місце локації верхнього медіального генікулярного нерва Рис. 1. Схематичне зображення генікулярних нервів Медицина болю (Pain Medicine). – 2020. – Том 5, № 3 Оригінальна методика / Original method 36 Рис. 3. Передньо-задня проекція, локація нижнього медіального генікулярного нерва ситиме лише тимчасовий характер, однак жінка вирі- шила утриматись від радіочастотної абляції. Упродовж 5-ти місяців спостереження пацієнт- ка почуває себе задовільно, вільно пересувається по квартирі й за її межами, відмовилась від підтримуючої палиці, на фоні збільшення фізичної активності відзна- чила зменшення маси тіла. Безумовно, ЛДБ та RFA не є лікуванням артрозу як такого, але якщо процедура дозволяє людині тимчасо- во (в середньому 9–12 міс., інколи більше) позбави- тись болю, повернутись до звичних для неї побутових справ, досягнути стадії компенсації супутньої сома- тичної патології (зменшення маси тіла, нормалізація показників глюкози крові, артеріального тиску тощо), як етап передопераційної підготовки, то очевидно, що дана методика заслуговує на увагу. Рис. 3. Передньо-задня проекція, локація нижнього медіального генікулярного нерва ISSN 2414–3812 Література Рис. 4. Бокова рентгенпроекція 1. Kurys VN, Egorova SA, Misyukov VV. Sredstva i metody lechebnoj fizicheskoj kul’tury v processe reabilitacii bol’nyh pri osteoartrozah krupnyh sustavov. Sport Probleems; 2010; 1(14):46–49. контроль у боковій проекциї (рис. 4). Після досягнення цільових точок було введено 2 мл 2 % лідокаїну на кож- ну генікулярну гілку та 40 мг метилпреднізолону аце- тату сумарно на всі генікулярні нерви. Голки видалено, накладено асептичні пов’язки. Ускладнень не було. Па- цієнтці було рекомендовано дотримуватись свого звич- ного режиму активності та суворо зоборонено протягом першої доби з моменту виконання блокади приймати будь-які знеболювальні препарати з метою виключення хибнопозитивного результату. 2. Kosareva MA, Mikhaylov IN, Tishkov NV. Modern principles and approaches in the treatment of gonarthrosis. Euroasian Scientific and Industrial Chamber, Ltd.; 2018;(№6 2018). Available from: https://doi.org/10.17513/spno.28292 3. Kellgren JH. Atlas of standard radiographs. The epidemiol- ogy of chronic rheumatism. 1963;2. 4. Bellamy NI, Kirwan J, Boers M, Brooks P, Strand VI, Tugwell P, Altman R, Brandt K, Dougados MA, Lequesne MI. Recom- mendations for a core set of outcome measures for future phase III clinical trials in knee, hip, and hand osteoarthri- tis. Consensus development at OMERACT III. J Rheumatol. 1997 Apr 1;24(4):799–802. Через 24 год після блокади пацієнтка відзначила зменшення болю в колінному суглобі на 50 %, що слу- гувало підставою для наступної абляції. Враховуючи те, що блокада носила ще й лікувальний характер, було прийнято рішення відтермінувати денервацію та оці- нити ефект у подальшому. Через три тижні динаміка регресу больового синдрому зберігалась – зменшен- ня на 60 % від початкового, ВАШ – 2–3 бали, індекс WOMAC – 28. Пацієнтці було роз’яснено, що ефект но- Через 24 год після блокади пацієнтка відзначила зменшення болю в колінному суглобі на 50 %, що слу- гувало підставою для наступної абляції. Враховуючи те, що блокада носила ще й лікувальний характер, було прийнято рішення відтермінувати денервацію та оці- нити ефект у подальшому. Через три тижні динаміка регресу больового синдрому зберігалась – зменшен- ня на 60 % від початкового, ВАШ – 2–3 бали, індекс WOMAC – 28. Пацієнтці було роз’яснено, що ефект но- 5. Lapshina SA, Mukhina RG. Osteoarthritis: modern prob- lems of therapy. Russian medical journal. 2016;2:95–101. 6. Bjordal JM, Ljunggren AE, Klovning A, Slørdal L. Non-steroi- dal anti-inflammatory drugs, including cyclo-oxygenase-2 inhibitors, in osteoarthritic knee pain: meta-analysis of randomised placebo controlled trials. BMJ [Internet]. BMJ; 2004 Nov 23;329(7478):1317. Available from: https://doi. Література org/10.1136/bmj.38273.626655.63 ISSN 2414–3812 Оригінальна методика / Original method 37 treatment for chronic knee pain: a systematic review. Rheumatology [Internet]. Oxford University Press (OUP); 2007 Jan 25;46(3):384–90. Available from: https://doi. org/10.1093/rheumatology/kel413 7. Gutthann SP, GarcíaRodríguez LA, Raiford DS. Individual Nonsteroidal Antiinflammatory Drugs and Other Risk Fac- tors for Upper Gastrointestinal Bleeding and Perforation. Epidemiology [Internet]. Ovid Technologies (Wolters Klu- wer Health); 1997 Jan;8(1):18–24. Available from: https:// doi.org/10.1097/00001648–199701000–00003 13. g gy 13. American College of Rheumatology Subcommittee on Os- teoarthritis Guidelines. Recommendations for the medical management of osteoarthritis of the hip and knee: 2000 update. Arthritis Rheum 2000;43:1905–15 g 8. Brandt KD, Smith Jr GN, Simon LS. Intraarticular injection of hyaluronan as treatment for knee osteoarthritis: what is the evidence? Arthritis Rheum 2000;43:1192–203 14. Hirasawa Y, Okajima S, Ohta M, Tokioka T. Nerve distri- bution to the human knee joint: anatomical and immuno- histochemical study. International Orthopaedics [Inter- net]. Springer Science and Business Media LLC; 2000 Mar 10;24(1):1–4. Available from: https://doi.org/10.1007/ s002640050001 9. Dieppe PA, Sathapatayavongs B, Jones HE, Bacon PA, Ring EFI. Intra-articular steroids in osteoarthri- tis. Rheumatology [Internet]. Oxford University Press (OUP); 1980;19(4):212–7. Available from: https://doi. org/10.1093/rheumatology/19.4.212 15. Kennedy JC, Alexander IJ, Hayes KC. Nerve supply of the human knee and its functional importance. The American Journal of Sports Medicine [Internet]. SAGE Publications; 1982 Nov;10(6):329–35. Available from: https://doi. org/10.1177/036354658201000601 10. Felson DT, Anderson JJ. Hyaluronate Sodium Injections for Osteoarthritis. Archives of Internal Medicine [Inter- net]. American Medical Association (AMA); 2002 Feb 11;162(3):245. Available from: https://doi.org/10.1001/ archinte.162.3.245 g 16. Schiltenwolf M, Fischer C. Choi WJ et al. Radiofrequen- cy treatment relieves chronic knee osteoarthritis pain: a double-blind randomized controlled trial. Pain 2011; 152: 481–7. Pain [Internet]. Ovid Technologies (Wolters Kluwer Health); 2011 Aug;152(8):1933–4. Available from: https:// doi.org/10.1016/j.pain.2011.05.031 11. Weiner DK, Rudy TE, Morone N, Glick R, Kwoh CK. Efficacy of Periosteal Stimulation Therapy for the Treatment of Os- teoarthritis-Associated Chronic Knee Pain: An Initial Con- trolled Clinical Trial. Journal of the American Geriatrics So- ciety [Internet]. Wiley; 2007 Oct;55(10):1541–7. Available from: https://doi.org/10.1111/j.1532–5415.2007.01314.x p g 12. White A, Foster NE, Cummings M, Barlas P. Acupuncture Therapeutic and diagnostic blockade of the knee joint as a component of pain management in gonarthrosis and a predictor of further successful radio-frequent denerva- tion of the joint Лечебно-диагностическая блокада коленного сустава как составляющая менеджмента болевого синдрома при гонартрозе и предиктор успешной радиочастотной денервации сустава. Седлецкий Р. Е. Sedletskyi R. Ye. Sedletskyi R. Ye. Sedletskyi R. Ye. ГУ “Институт травматологии и ортопедии НАМНУ”, Киев State Institution “Institute of Traumatology and Orthopedics” of National Academy of Medical Sciences of Ukraine”, Kyiv Резюме. Согласно данным ВОЗ, в последнее десятиле- тие происходит изменение структуры заболеваемо- сти. Прогрессивно увеличивается количество людей с хроническим болевым синдромом. В частности, это связано со старением населения и возрастанием коли- чества заболеваний опорно-двигательного аппарата, в том числе остеоартроза коленного сустава. Послед- ний не только способствует временной и стойкой по- тере работоспособности, но и приводит к значитель- ному снижению качества жизни пациента. Резюме. Согласно данным ВОЗ, в последнее десятиле- тие происходит изменение структуры заболеваемо- сти. Прогрессивно увеличивается количество людей с хроническим болевым синдромом. В частности, это связано со старением населения и возрастанием коли- чества заболеваний опорно-двигательного аппарата, в том числе остеоартроза коленного сустава. Послед- ний не только способствует временной и стойкой по- тере работоспособности, но и приводит к значитель- ному снижению качества жизни пациента. Resume. According to WHO data, there has been a change in the morbidity structure in the past decades. The number of people with chronic pain syndrome (CPS) is increasing progressively. This is due to population aging and increases in the number of musculoskeletal disorders, including osteoarthritis of the knee joint, which causes not solely temporary or permanent loss of ability to work, but a significant decrease in quality of life in patients. According to WHO, gonarthrosis will be- come the fourth leading cause of disability among women, and eighth among men in the coming decade (1). По прогнозам ВОЗ, гонартроз в ближайшее десяти- летие станет четвертой причиной инвалидности у женщин и восьмой – у мужчин (1). Key words: osteoarthritis (OA) of the knee joint, diagnostic genicular nerve block, radiofrequency ablative denervation (RFA), dereception, ablation, neurotomy. Ключевые слова: остеоартроз коленного сустава (ОА), лечебно-диагностическая блокада геникулярных нервов (ЛДБ), денервация (RFA), дерецепция, абляция, нейротомия. Медицина болю (Pain Medicine). – 2020. – Том 5, № 3
https://openalex.org/W2050917730	https://link.springer.com/content/pdf/10.1007/s10645-012-9186-9.pdf	English	null	Editorial Report 2011	De Economist/Economist	2,012	cc-by	370	De Economist (2012) 160:81–82 DOI 10.1007/s10645-012-9186-9 De Economist (2012) 160:81–82 DOI 10.1007/s10645-012-9186-9 Editorial Report 2011 Jan van Ours Received: 4 January 2012 / Accepted: 5 January 2012 / Published online: 26 January 2012 © The Author(s) 2012. This article is published with open access at Springerlink.com The 4 issues of De Economist published in 2011 contain 21 papers of which 10 papers are published in regular issues and 11 papers are published in Special Issues. The arti- cles covers a wide range of issues from an analysis of the effect of manager turnover on ﬁrm performance (using data on Dutch football) to the impact of product market competition on training investments of ﬁrms. The second issue of 2011 was a Special Issue on Aging workforces (6 papers), the fourth issue of 2011 was a Special Issue on Productivity and internationalization (5 papers). In 2011 there were 54 submissions, the same number as in 2010. As shown in the table below, of these 54 submissions 13 were accepted, 29 were rejected, and 12 are still in the editorial process. Of the rejected papers 17 were desk-rejections, i.e. rejected within a week after submission. In 2011 61 decisions were taken, 28 papers were accepted while 33 were rejected. For reason of comparison the table below also shows the ﬁnal numbers for the 2010 submissions. The acceptance rate for the papers that were submitted in 2010 is 39%. Ignoring the desk-rejections, the acceptance rate is close to 50%. The average time to ﬁrst decision of the papers who got a revise and resubmit in the initial stage was 82days. Of the rejected papers this was 44days, which is substantially lower due to the desk rejections. Submitted in 2011 Decision in 2011 Submitted in 2010 Accepted 13 28 21 Submitted in 2010 – 15 – Rejected 29 33 33 Of which desk-rejections 17 17 10 In stock 12 – – Total 54 61 54 J. van Ours (B) Tilburg, The Netherlands e-mail: economist@uvt.nl 3 82 J. van Ours Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. 12 123
https://openalex.org/W4248528814	https://www.qeios.com/read/YE6PQU/pdf	English	null	Use_For	Definitions	2,020	cc-by	58	Use_For Use_For National Cancer Institute National Cancer Institute Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Qeios ID: YE6PQU · https://doi.org/10.32388/YE6PQU Source National Cancer Institute. Use_For. NCI Thesaurus. Code C62753. Indicates a term that had been used in a coding system and then subsumed by the given NCIt concept. Qeios ID: YE6PQU · https://doi.org/10.32388/YE6PQU 1/1
W4205549396.txt	https://bmcmusculoskeletdisord.biomedcentral.com/counter/pdf/10.1186/s12891-022-04996-5	en		Correction to: Are postoperative NLR and PLR associated with the magnitude of surgery-related trauma in young and middle-aged patients with bicondylar tibial plateau fractures? A retrospective study	BMC musculoskeletal disorders	2,022	cc-by	423	(2022) 23:62 Wang et al. BMC Musculoskeletal Disorders https://doi.org/10.1186/s12891-022-04996-5 Open Access CORRECTION Correction to: Are postoperative NLR and PLR associated with the magnitude of surgery-related trauma in young and middleaged patients with bicondylar tibial plateau fractures? A retrospective study Zhongzheng Wang1,2†, Yanwei Wang3†, Yuchuan Wang1,2, Wei Chen1,2,4 and Yingze Zhang1,2,4* Correction to: BMC Musculoskelet Disord 22, 816 (2021) https://doi.org/10.1186/s12891-021-04695-7 Following the publication of the original article [1] the authors noticed that the note for equal contributors “†Zhongzheng Wang and Yanwei Wang contributed equally to this work.” did not appear in the proof. The original article [1] has been updated. Reference 1. Wang Z, Wang Y, Wang Y, et al. Are postoperative NLR and PLR associated with the magnitude of surgery-related trauma in young and middleaged patients with bicondylar tibial plateau fractures? A retrospective study. BMC Musculoskelet Disord. 2021;22:816. https://doi.org/10.1186/ s12891-021-04695-7. Author details 1 Department of Orthopaedic Surgery, Third Hospital of Hebei Medical University, 050051 Shijiazhuang, Hebei, People’s Republic of China. 2 Key Laboratory of Biomechanics of Hebei Province, 050051 Shijiazhuang, Hebei, People’s Republic of China. 3 Department of Orthopaedic Surgery, North China Medical and Health Group Xingtai General Hospital, 054000 Xingtai, Hebei, People’s Republic of China. 4 NHC Key Laboratory of Intelligent Orthopaedic Equipment, 050051 Shijiazhuang, Hebei, People’s Republic of China. The original article can be found online at https://doi.org/10.1186/s12891- 021-04695-7. *Correspondence: drzyzhangyingze@163.com † Zhongzheng Wang and Yanwei Wang contributed equally to this work. 4 NHC Key Laboratory of Intelligent Orthopaedic Equipment, 050051 Shijiazhuang, Hebei, People’s Republic of China 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://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
https://openalex.org/W4223925854	https://hal.science/hal-03643617/document	English	null	The Internal Cranial Anatomy of a Female With Endocrine Disorders From a Mediaeval Population	Frontiers in endocrinology	2,022	cc-by	9,997	The Internal Cranial Anatomy of a Female With Endocrine Disorders From a Mediaeval Population Anna Maria Kubicka, Philippe Charlier, Antoine Balzeau To cite this version: Anna Maria Kubicka, Philippe Charlier, Antoine Balzeau. The Internal Cranial Anatomy of a Fe male With Endocrine Disorders From a Mediaeval Population. Frontiers in Endocrinology, 2022, 13 pp.862047. 10.3389/fendo.2022.862047. hal-03643617 The Internal Cranial Anatomy of a Female With Endocrine Disorders From a Mediaeval Population Anna Maria Kubicka, Philippe Charlier, Antoine Balzeau To cite this version: Anna Maria Kubicka, Philippe Charlier, Antoine Balzeau. The Internal Cranial Anatomy of a Fe- male With Endocrine Disorders From a Mediaeval Population. Frontiers in Endocrinology, 2022, 13, pp.862047. 10.3389/fendo.2022.862047. hal-03643617 The Internal Cranial Anatomy of a Female With Endocrine Disorders From a Mediaeval Population Anna Maria Kubicka 1,2, Philippe Charlier 3,4 and Antoine Balzeau 2,5 Anna Maria Kubicka 1,2, Philippe Charlier 3,4 and Antoine Balzeau 2,5 1 Department of Zoology, Poznan´ University of Life Sciences, Poznan´ , Poland, 2 PaleoFED Team, Unité Mixte de Recherche (UMR) 7194, Centre National de la Recherche Scientiﬁque (CNRS), Département Homme et Environnement, Muséum National d’Histoire Naturelle, Musée de l’Homme, Paris, France, 3 Laboratoire Anthropologie, Arche´ologie, Biologie (LAAB), Unité de Formation à la Recherche (UFR) des Sciences de la Sante´, Universite´ Paris-Saclay (UVSQ) & Muse´e du quai Branly - Jacques Chirac, Montigny- le-Bretonneux, France, 4 Direction, De´partement de la Recherche et de L’Enseignement Muse´e du quai Branly - Jacques Chirac, Paris, France, 5 Royal Museum for Central Africa, Department of African Zoology, Tervuren, Belgium Keywords: gigantism, acromegaly, computed-tomography, endocast, skull thickness, frontal sinuses, sella turcica HAL Id: hal-03643617 https://hal.science/hal-03643617v1 Submitted on 16 Apr 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. 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ORIGINAL RESEARCH published: 14 April 2022 doi: 10.3389/fendo.2022.862047 ORIGINAL RESEARCH published: 14 April 2022 doi: 10.3389/fendo.2022.862047 Edited by: D li Edited by: Francesco Doglietto, Agostino Gemelli University Polyclinic (IRCCS), Italy Edited by: Francesco Doglietto, Agostino Gemelli University Polyclinic (IRCCS), Italy Reviewed by: Elisabeth Eppler, University of Bern, Switzerland Sabrina Chiloiro, Catholic University of the Sacred Heart, Italy Reviewed by: Elisabeth Eppler, University of Bern, Switzerland Sabrina Chiloiro, Catholic University of the Sacred Heart, Italy Reviewed by: Elisabeth Eppler, University of Bern, Switzerland Sabrina Chiloiro, Catholic University of the Sacred Heart, Italy Correspondence: Anna Maria Kubicka amkkubicka@gmail.com Specialty section: This article was submitted to Bone Research, a section of the journal Frontiers in Endocrinology Received: 25 January 2022 Accepted: 17 March 2022 Published: 14 April 2022 Citation: Kubicka AM, Charlier P and Balzeau A (2022) The Internal Cranial Anatomy of a Female With Endocrine Disorders From a Mediaeval Population. Front. Endocrinol. 13:862047. doi: 10.3389/fendo.2022.862047 Specialty section: This article was submitted to Bone Research, a section of the journal Frontiers in Endocrinology Received: 25 January 2022 Accepted: 17 March 2022 Published: 14 April 2022 Specialty section: This article was submitted to Bone Research, a section of the journal Frontiers in Endocrinology Received: 25 January 2022 Accepted: 17 March 2022 Published: 14 April 2022 ORIGINAL RESEARCH published: 14 April 2022 doi: 10.3389/fendo.2022.862047 Gigantism and acromegaly have been observed in past populations; however, analyses usually focus on the morphological features of the post-cranial skeleton. The aim of this study is to characterize the internal anatomical features of the skull (brain endocast anatomy and asymmetry, frontal pneumatization, cranial thickness, sella turcica size) of an adult individual from the 11-14th centuries with these two diseases, in comparison with non-pathological individuals from the same population. The material consisted of 33 adult skulls from a mediaeval population, one of them belonging to an adult female with endocrine disorders (OL-23/77). Based on the CT scans, the internal cranial anatomy was analysed. The sella turcica of OL-23/77 is much larger than in the comparative sample. The endocast of the individual OL-23/77 shows a left frontal/left occipital petalia, while the comparative population mostly had right frontal/left occipital petalias. The asymmetry in petalia location in OL-23/77 comes within the range of variation observed in the comparative population. The individual has high values for cranial thickness. The frontal sinuses of the specimen analysed are similar in size and shape to the comparative sample only for data scaled to the skull length. Enlarged sella turcica is typical for individuals with acromegaly/gigantism. The pattern of the left frontal/left occipital petalia in the specimen OL-23/77 is quite rare. The position of the endocranial petalias has not inﬂuenced the degree of asymmetry in the specimen. Despite the large bone thickness values, skull of OL-23/77 does not show any abnormal features. The skull/endocast relationship in this individual shows some peculiarities in relation to its large size, while other internal anatomical features are within the normal range of variation of the comparative sample. 1 INTRODUCTION Therefore, the analysis of the morphometric data on the frontal pneumatization may improve the understanding of the effect of excessive secretion of GH on the skull growth factor. Several casesofgigantism and acromegaly have beenreportedin past human populations from the Third (2700 BC) (7), and Fifth Dynasties in Egypt (2494-2345 BC) (8), the Windmiller culture in California(2500-850BC)(9),MediaevalGreece(7thcentury)(10),a post-Mediaeval cemetery in Turkey (11) a Jewish necropolis in Spain (7th-12th centuries) (12), a late prehistoric village in Pottery Mound (14th-16th centuries) (13), and ruins in New Mexico (14th- 17th centuries) (14). These studies on osteological material focus mostly on skeletal morphology, body height and coexisting pathological conditions such as joint diseases, ossiﬁcation or periostitis (7–9, 15). Less often, research investigates the aspects of mortuary treatment and social perceptions (16). The medical literature describes more recent historical cases of patients with acromegalic and gigantism living in Europe, the USA and Russia in the 16th-19th centuries (17, 18). These clinical descriptions usually focusonlivingpatientsandcontain anthropometricdata[e.g.,body height and weight (19), information on physiological features (e.g., acceleratedpulse (20),deepvoice(19)],orcognitiveabilities(19).In turn, autopsy reports of the historical cases describe anatomical features of the pituitary area or internal organs (21, 22). All these medical data made a valuable contribution to the ﬁrst medical therapies of endocrine disorders (23). Excessive GH secretion may occur as a wide spectrum of heterogenous disorders therefore, each case of this endocrinological condition, even historical, provides new information on the pathological features. Nevertheless, very few cases of gigantism and acromegaly that is a more common endocrine disorder have been described in the palaeopathological literature. That is why this study presents a rare case of an adult individual with both gigantism and acromegalyfromamediaevalpopulationinPoland.Forthispurpose, we decided to focus on craniometric data that is rarely studied. The size and asymmetry of the endocast, frontal pneumatization, cranial thickness and sella turcica size of an adult female with endocrine disorderswerecomparedwithalargesampleofadultswithoutvisible pathological conditions from the same mediaeval group. CT images were used with modern anthropological methods such as three- dimensional reconstructions of endocasts and frontal sinuses and a tomographic map of variation in the total bone thickness. Since the pattern of cranial features is established early in ontogeny, this case study may show whether progressive endocrine disorders result in atypical features of the internal cranial anatomy. Moreover, documenting an individual from a pre-modern industrial society with acromegaly and gigantism using medicalfacilitiescontributes to our knowledge of normal human variation. 1 INTRODUCTION individuals with endocrine disorders is interesting in the following aspects. Gigantism and acromegaly are endocrine disorders whose differentiation has remained unclear long after its signs and symptoms were ﬁrst described (1). At present, gigantism is related to excessive growth hormone secretion (GH) during childhood, while acromegaly results in excess GH in adulthood (2). Most individuals with gigantism and acromegaly exhibit physical and anthropological symptoms such as accelerated growth, elongated facial features, disproportionately large hands, arthritis, frequent headaches and excessive sweating (2, 3). According to epidemiological data, these two diseases are not common (4), and their frequency differs in present-day societies. In Spain and Belgium, the prevalence of acromegaly ranges from 34 to 40 cases per million (c.p.m.); in the UK, it is much higher at around 86 c.p.m., and in Northern Finland, in turn, it is only 0.34 c.p.m (5). Slight more frequent prevalence was reported in women (4); however, it seems that both sexes are equally affected. In turn, only about 200 reported cases of gigantism are known worldwide so far (6). First of all, the endocast size of an individual with gigantism and acromegaly may provide new information in the context of the skull morphological integration, e.g. indicate whether changes in the facial bones may affect the cranial vault despite their ossiﬁed centres. Another important aspect is whether the larger endocranial volume and elongated facial features of a person with endocrine disorders result in a different pattern of cranial thickness to that in non-pathological individuals. Previous research shows increased thickness of the cranial vault, but investigated only the frontal and occipital bones (25). Other features of the internal cranial morphology, such as asymmetry is also worth investigation in order to check whether endocrine disorders are associated with an atypical pattern of endocast asymmetry. Lateralization of brain functions in humans is related to their high cognitive abilities as each hemisphere has a different specialization (29, 30). Therefore, considering that patients with acromegaly can exhibit mental disorders such as dementia, affective and mild cognitive disorders (31, 32), and gigantism can be related to poor mental development (33), we can suspect different endocast asymmetry than in healthy individuals. The last aspect concerns the frontal sinuses, which are characterized by considerable morphological variability (34, 35). There is a strong association between the brain asymmetry and the shape and extension of the frontal sinuses (35). Citation: Kubicka AM, Charlier P and Balzeau A (2022) The Internal Cranial Anatomy of a Female With Endocrine Disorders From a Mediaeval Population. Front. Endocrinol. 13:862047. doi: 10.3389/fendo.2022.862047 April 2022 \| Volume 13 \| Article 862047 1 Frontiers in Endocrinology \| www.frontiersin.org Internal Anatomy of a Giant Female Kubicka et al. 1 INTRODUCTION p Unfortunately, little attention is paid to craniometric analysis as well, as only a few studies have reported changes in the skull, and all havefocusedsolelyonlivingacromegalicpatients.Thesecasesshow extensive frontal sinus pneumatization, enlarged sella turcica (24), large sphenoidal and maxillary sinuses, increased thickness of the frontal and occipital cranial vault (25), and reduced posterior fossa (26). Acromegaly starts to develop after epiphyses fusion; therefore, the cranial cavity is usually not enlarged due to the already ossiﬁed centres, with simultaneous changes in the facial bones and mandibular lengthening (27). The human skull is an integrated structure where the relationship between the facial skeleton (i.e. the zygomatic processes, nasal, lacrimal and maxillary bones), basicranium and cranial vault show a uniform pattern of integration (28). Therefore, the analysis of the internal cranial anatomy (i.e. endocast morphology, frontal pneumatization) in Frontiers in Endocrinology \| www.frontiersin.org April 2022 \| Volume 13 \| Article 862047 2.1.3 Endocast The anatomy of the OL-23/77 endocast was analysed from the 3D reconstruction. In order to explore the relative position of the endocranial petalias, a tested and validated protocol was used (50). Initially, three anatomical points on each skull (i.e. glabella, inion and basion) were digitized. These in turn, were used to construct two lines, the ﬁrst (L1) connecting the glabella and inion, the second (L2) starting at the basion and passing perpendicularly through L1 (Figure 1). Then, four points were digitized in the following locations: two points on the most anterior part of the left and right frontal lobes, and two points on the most posterior part of the left and right occipital lobes (Figure 1). Six dimensions between the points located on the lobes and two lines (L1 and L2) were taken to quantify the location of the most protruding points on the frontal and occipital lobes in the antero-posterior, vertical and lateral views (Figure 1). The maximum length of the skull was then measured. The sex and age of 33 adult individuals from Ostrów Lednicki were assessed based on morphological changes in the skull and pelvis. The sex was assessed using the morphology of the supraorbital ridges, glabella region, orbits, mastoid processes, occipital condyles, greater sciatic notch, subpubic concavity, and ischiopubic ramus. Age at death was assessed based on the modiﬁcation of the pubic symphysis and cranial suture closure (40–43). The skeleton of an individual OL-23/77 was 208 cm long in situ and showed characteristic morphological features of an individual with endocrine disorders. Further examination showed that this was a female aged 25 to 30 at the time of death with a body stature estimated at 215.5 cm (44). Based on osteological, radiological and CT examinations, the skeleton was diagnosed as excessively long and massive but with normal bone proportions that indicate gigantism in the early years, and with an elongated and prognathic mandible, enlarged vertebrae and thick bones that indicate acromegaly in adulthood (16, 33). Furthermore, based on the X-rays and CT-scans, the OL-23/77 skeleton shows pituitary lesion, degenerative joint disease, Schmorls’s nodes [i.e. pathological condition of vertical herniation of intervertebral discs into the neighbouring vertebrae (45)] in almost all vertebrae and healed fractures of the humerus and tibia. Other pathological conditions indicating a developmental disorder of the external auditory canal and sclerotization of the left temporal bone were also found (46). 2.1.1 3D Reconstruction Theskullsof33adultindividualsfromOstrówLednickiwerescanned using 32-slice computed tomography (Siemens SOMATOM Sensation) in the cranio-caudal position with the standard protocol [0.625 slice thickness, 60 Vox energy, Balzeau et al. (47)]. Next, for each individual, a three-dimensional (3D) reconstruction of the skull was prepared using InVesalius software (ver. 3.1.1). Based on the reconstructedskulls,3Dendocastmodelswerethencreatedusingthe endomaker algorithm in R software (48). 2 MATERIAL AND METHODS The material consists of an adult individual with gigantism and acromegaly (OL-23/77) and 32 adults (16 females and 16 males) April 2022 \| Volume 13 \| Article 862047 Frontiers in Endocrinology \| www.frontiersin.org 2 Internal Anatomy of a Giant Female Kubicka et al. from a mediaeval population dated to a period from the end of 11th century to the 13-14th centuries (36, 37). The settlement is located in Ostrów Lednicki (Poland), near the eastern shore of Lake Lednica. According to archaeological and anthropological records, the subsistence economy of this population was based on agriculture, but other activities such as stockbreeding or hunting small mammals and birds were also present (38, 39). 2.1.4 Frontal Sinuses The 3D models of the frontal sinuses were manually reconstructed using multiple threshold values as a function of the modiﬁcation of the grey values of the tissues in the area of frontal pneumatization. The segmentation was performed using Avizo 7 software (FEI, Hillsboro, Oregon). Next, the 3D models of the skulls with the reconstructedfrontalsinuseswerepositionedintheFrankfurtplane (i.e.aplaneformedwithahorizontallinerunning fromthesuperior edgeoftheexternalauditorycanaltotheinferiorborderoftheorbit) to measure dimensions in the anterior, superior and vertical orientations (Table 1). As the frontal sinuses can be insufﬁciently preserved in the inferior extension, the variables were selected to ensure the measuring of parts that are usually well preserved. These measurements deﬁne the maximal extension of the frontal sinuses in 3D, including bilateral data for the two sinuses. The bilateral measurements (i.e. AL, AR, SL and SR) and two antero-posterior dimensions in the left lateral view (AP, AP2) were combined into three ﬁnal parameters: 2A, 2S and 2AP (Table 1). A relative value wasthencalculatedforeachﬁnalparameterofthefrontalsinusesby scaling measurements to the cube root of the volume. 2.2 Statistical Analysis 2.2.1 Sella Turcica For each individual, the volume [mm3] of the sella turcica was calculated using the formula: (width × length × height)/2 (49). Next, descriptive statistics were calculated separately for the comparative sample and OL-23/77. 2.1.3 Endocast The skeleton is preserved in good condition, with a few missing bones such as the 4th cervical vertebra, two metacarpal and 5 metatarsal bones (33). The comparative sample contains 32 adult individuals from Ostrów Lednicki that did not show any pathological changes (e.g. osteophytes, porosity, trauma) and were characterized by a good state of preservation of the skull. The average body stature of the comparative sample was 162.10 cm ( ± 7.37). The cranial vault thickness of OL-23/77 was computed across the whole vault using the Surface Thickness module in Avizo 7 software (FEI, Hillsboro, Oregon). A 3D tomographic map of variation in the total bone thickness was computed based on the 3D model of the exo- and endocranial skull surfaces. Next, the map was completed using a 2 to 22 mm colour scale from white to yellow, where white indicates bone thickness close to 2 mm and yellow indicates bone thickness close to 22 mm. 2.1.2 Measurements of the Sella Turcica Based on the 3D skull reconstruction, each sella turcica was measured using GomInspect software (ver. 2.0.1). According to Hawkins (49), three dimensions of this bone structure were measured: length (antero-posterior diameter), width (medio- lateral diameter) and height (supero-inferior). 2.2.2 Asymmetry of the Endocast In order to quantify asymmetry in petalia location, different indices were calculated in the study. Signed asymmetry (DA) is April 2022 \| Volume 13 \| Article 862047 Frontiers in Endocrinology \| www.frontiersin.org 3 Internal Anatomy of a Giant Female Kubicka et al. B OL-23/77). (A) lateral view with two projected lines (L1 and L2), points on the skull ital lobes (red dotted line and arrow). (B) superior view with a projected line (L1), f t l d i it l l b ( d d tt d li d ) A B FIGURE 1 \| The skull and endocast of an adult female with endocrine disorders (OL-23/77). (A) lateral view with two projected lines (L1 and L2), points on the skull (red) and the endocast (green), and the vertical dimensions of the frontal and occipital lobes (red dotted line and arrow). (B) superior view with a projected line (L1), points on the skull (red) and the endocast (green), and the lateral dimensions of the frontal and occipital lobes (red dotted line and arrow). B A A FIGURE 1 \| The skull and endocast of an adult female with endocrine disorders (OL-23/77). (A) lateral view with two projected lines (L1 and L2), points on the skull (red) and the endocast (green), and the vertical dimensions of the frontal and occipital lobes (red dotted line and arrow). (B) superior view with a projected line (L1), points on the skull (red) and the endocast (green), and the lateral dimensions of the frontal and occipital lobes (red dotted line and arrow). TABLE 1 \| Description of the measurements of the frontal sinuses in 3D. TABLE 1 \| Description of the measurements of the frontal sinuses in 3D. 2.2.4 Pneumatization of the Frontal Bone 2.2.4 Pneumatization of the Frontal Bone In order to analyse the pneumatization of the frontal bone, descriptive statistics were calculated separately for OL-23/77 and the rest of the sample. present when the difference between the left and right sides of the endocast is greater than zero. This is calculated using the following equation: (R – L), where R is a dimension of the right petalia, and L is a dimension of the left petalia. Positive values of this index indicate asymmetry towards the right side, while negative values mean asymmetry towards the left side of the endocast. The second index was in absolute asymmetry (AA), which indicates the degree of directional asymmetry but without the directional bias and is calculated using the equation (maximum value – minimum value). The indicator of directional asymmetry (AQ) was also calculated using the formula (R-L)/((R+L)/2) (51), Indicators of DA and AA were calculated for all six dimensions to explore the relative position of the endocranial petalias, while AQ was calculated only for the two dimensions quantifying frontal and occipital petalias in the lateral position. 2.2.2 Asymmetry of the Endocast Parameter View Description Final parameter W anterior the maximal lateral extension of the pneumatisation W H anterior the maximal height of the frontal sinus H AL anterior the maximal length of the left frontal sinus 2A (combined AL and AR AR anterior the maximal length of the right frontal sinus SL superior the maximal medio-lateral extension of the left frontal sinus 2S (combined SL and SR) SR superior the maximal medio-lateral extension of the right frontal sinus AP left lateral the length from the most anteriorly protruding point of the left sinus to the most posterior point in an horizontal direction 2AP (combined AP and AP2) AP2 left lateral the length from the most anterior point of the sinus to the maximal supero-posterior extension of the sinus RCV cube-root of the volume of both frontal sinuses RCV 3 RESULTS Length [mm] Width [mm] Height [mm] Volume [mm3] OL-23/77 (n=1) Giant 14.10 21.22 8.72 1304.52 Individuals from Ostrów Lednicki (n=32) Min 7.12 10.07 2.52 163.44 Mean-SD 8.81 11.70 3.57 218.66 Mean 10.17 13.61 5.27 361.56 Mean+SD 11.53 15.52 6.97 504.46 Max 12.90 17.75 8.61 650.62 SD 1.36 1.91 1.70 142.90 TABLE 2 \| Descriptive statistics of the sella turcica measurements. FIGURE 2 \| Left anterior views of the endocast of OL-23/77 showing the extension and position of the frontal lobes (pink), parietal lobes (yellow) temporal lobes (orange), occipital lobes (green), cerebellar lobes (blue), some aspects of the venous system in red (middle meningeal system, main venous sinuses and pacchionian granulations – noted PG), the position of the central and lateral sulci as well as several smaller sulci (in black) delimiting the three frontal convolutions (noted F1 to F3), the ﬁrst and second parietal convolutions, the three temporal convolutions and anatomo-functional areas: premotor cortex (pc), primary motor cortex (pmc), somatosensory cortex (sc). FIGURE 2 \| Left anterior views of the endocast of OL-23/77 showing the extension and position of the frontal lobes (pink), parietal lobes (yellow) temporal lobes (orange), occipital lobes (green), cerebellar lobes (blue), some aspects of the venous system in red (middle meningeal system, main venous sinuses and pacchionian granulations – noted PG), the position of the central and lateral sulci as well as several smaller sulci (in black) delimiting the three frontal convolutions (noted F1 to F3), the ﬁrst and second parietal convolutions, the three temporal convolutions and anatomo-functional areas: premotor cortex (pc), primary motor cortex (pmc), somatosensory cortex (sc). FIGURE 2 \| Left anterior views of the endocast of OL-23/77 showing the extension and position of the frontal lobes (pink), parietal lobes (yellow) temporal lobes (orange), occipital lobes (green), cerebellar lobes (blue), some aspects of the venous system in red (middle meningeal system, main venous sinuses and pacchionian granulations – noted PG), the position of the central and lateral sulci as well as several smaller sulci (in black) delimiting the three frontal convolutions (noted F1 to F3), the ﬁrst and second parietal convolutions, the three temporal convolutions and anatomo-functional areas: premotor cortex (pc), primary motor cortex (pmc), somatosensory cortex (sc). position of the occipital poles, the skull of the OL-23/77 is also large but only slightly continuing the regression line calculated for the comparative sample (Figure 3B). 3 RESULTS This means that the frontal poles are in a more posterior position than expected for an individual of this size. In contrast, the occipital poles are in a slightly more posterior position than expected. developmentisquitesimilaronbothsides,includingthepresenceof several anastomoses on the parietal lobes. We did not detect any potential alteration in the structure of the brain as identiﬁed by the position of the sulci (Figure 2). Important anatomical and functional areas such as the premotor cortex, the primary motor cortex and the somatosensory cortex exhibit normal morphology, disposition and extension. Similarly, the third frontal convolutions and the supramarginal gyrus are of normal size and shape. The glabella-inion length of OL-23/77 is 208.2 mm. The OL-23/77 skull has quite high values for bone thickness. The frontal bones are thicker overall than the parietal bones, but with thinner areas in the region of the temporal squama, at the third frontal convolution (Figure 4). Note the strong and thick reliefs in the anterior part of the frontal bone, related to the high bone thickness values as illustrated by the presence of a yellow area. This also corresponds to the area where the frontal poles are located on the endocranial surface. The occipital poles are located around the middle of the lambda-inion chord, where bone thickness is less pronounced than in the region of the inion. We also observe that the endinion is located below the inion, but this is a frequent pattern in Homo sapiens (53). Concerning the petalias, the endocast of OL-23/77 shows a left frontal/left occipital petalia. In the sample from Ostrów Lednicki comprising 32 individuals, the right frontal/left occipital is the most frequent, visible in 27 specimens. No left frontal/left occipital is visible in any specimen, but one specimen has a left frontal petalia. In terms of metric data (Table 3), the OL-23/77 skull values are within the range of variation observed for each parameter in the comparative sample. Values for the vertical petalia are higher than those for the antero-posterior petalia in the comparative sample, and values for the lateral petalia are even higher. Concerning pneumatization of the frontal bone, the OL-23/77 specimen exceeds the range of variation observed in the comparative sample for nearly all the variables analysed when absolute values are considered (Table 4). 3 RESULTS Table 2. shows the descriptive statistics of the sella turcica parameters. OL-23/77 is characterized by larger measurements (i.e. length, width, height and volume) of the sella turcica than the comparative sample. The endocast of OL-23/77 does not exhibit any abnormal features. There is no alteration of its global shape, no sign of pathologies on the endocranial surface or in the extension of the sinusdrainagepatternofthemiddlemeningealsystem.Theanalysis of the endocast anatomy shows that the superior sagittal sinus is visible nearly all along with its extension between the frontal and parietal lobes, continuing posteriorly into the right lateral sinus (Figure 2). The right lateral and sigmoid sinuses are larger than those visible on the left side. The middle meningeal system is well developedonbothsideswithaverywelldevelopedcoronalsegment and a well-marked posterior inferior branch (Figure 2). The 2.2.3 Regression Between Endocast and Skull Length The regression between the length of the skull and the position of the frontal and occipital poles was calculated using bivariate linear regression (RMA model) to summarize the relationship between variables. This model was calculated only with the comparative sample and is better than alternative regression models when outliers are expected (52). April 2022 \| Volume 13 \| Article 862047 Frontiers in Endocrinology \| www.frontiersin.org 4 Internal Anatomy of a Giant Female Kubicka et al. TABLE 2 \| Descriptive statistics of the sella turcica measurements. Length [mm] Width [mm] Height [mm] Volume [mm3] OL-23/77 (n=1) Giant 14.10 21.22 8.72 1304.52 Individuals from Ostrów Lednicki (n=32) Min 7.12 10.07 2.52 163.44 Mean-SD 8.81 11.70 3.57 218.66 Mean 10.17 13.61 5.27 361.56 Mean+SD 11.53 15.52 6.97 504.46 Max 12.90 17.75 8.61 650.62 SD 1.36 1.91 1.70 142.90 FIGURE 2 \| Left anterior views of the endocast of OL-23/77 showing the extension and position of the frontal lobes (pink), parietal lobes (yellow) temporal lobes (orange), occipital lobes (green), cerebellar lobes (blue), some aspects of the venous system in red (middle meningeal system, main venous sinuses and pacchionian granulations – noted PG), the position of the central and lateral sulci as well as several smaller sulci (in black) delimiting the three frontal convolutions (noted F1 to F3), the ﬁrst and second parietal convolutions, the three temporal convolutions and anatomo-functional areas: premotor cortex (pc), primary motor cortex (pmc), somatosensory cortex (sc). TABLE 2 \| Descriptive statistics of the sella turcica measurements. April 2022 \| Volume 13 \| Article 862047 Frontiers in Endocrinology \| www.frontiersin.org 3 RESULTS In the case of relative measurements (scaled to the cube root of the volume of pneumatization), the values for the specimen are comparable Figure 3A shows the regression (RMA) between the length of the skull (inion-glabella chord) and the position of the frontal poles. The OL-23/77 skull has higher values for both parameters and does not follow the regression observed in the comparative sample (32 adult individuals from Ostrów Lednicki). For the April 2022 \| Volume 13 \| Article 862047 Frontiers in Endocrinology \| www.frontiersin.org 5 Internal Anatomy of a Giant Female Kubicka et al. TABLE 3 \| Directional and absolute asymmetry of the frontal and occipital petalias. Frontal petalia Occipital petalia Antero-posterior Vertical Lateral Antero-posterior Vertical Lateral DA (AA) DA (AA) DA (AA) AQ DA (AA) DA (AA) DA (AA) AQ OL-23/77 (n=1) Giant -1.08 (1.08) -2.53 (2.53) 7.10 (7.10) 0.57 -0.36 (0.36) -5.55 (5.55) 13.85 (13.85) 0.25 Individuals from Ostrów Lednicki (n=32) Min -1.63 (0.01) -7.86 (0.53) -9.78 (0.03) -1.07 -4.58 (0.06) -8.42 (0.51) -15.20 (0.08) -0.43 Mean-SD -0.53 (0.23) -4.64 (1.23) -4.64 (0.84) -0.49 -3.05 (0.52) -5.01 (0.33) -5.02 (2.38) -0.12 Mean 0.43 (0.80) -0.10 (3.73) -0.09 (3.58) -0.04 -1.17 (1.79) -0.51 (3.34) 3.35 (7.40) 0.06 Mean+SD 1.39 (1.47) 4.44 (6.23) 4.46 (6.31) 0.40 0.71 (3.06) 4.00 (6.34) 11.72 (12.42) 0.24 Max 3.02 (3.02) 9.85 (9.85) 8.29 (9.78) 0.81 4.47 (4.58) 13.88 (13.88) 22.55 (22.55) 0.48 SD 0.96 (0.67) 4.54 (2.50) 4.55 (2.74) 0.45 1.88 (1.27) 4.50 (3.01) 8.37 (5.02) 0.18 DA, signed asymmetry; AA, absolute (unsigned) asymmetry; AQ, indicator of directional asymmetry of the lateral petalias; Giant, an individual with gigantism and acromegaly from Ostrów Lednicki; n, number of individuals; Min, minimal observed value; Max, maximal observed value; Mean - SD, mean value minus standard deviation, Mean - mean value, Mean + SD - mean value plus standard deviation; SD, standard deviation. TABLE 3 \| Directional and absolute asymmetry of the frontal and occipital petalias. DA, signed asymmetry; AA, absolute (unsigned) asymmetry; AQ, indicator of directional asymmetry of the lateral petalias; Giant, an individual with gigantism and acromegaly from Ostrów Lednicki; n, number of individuals; Min, minimal observed value; Max, maximal observed value; Mean - SD, mean value minus standard deviation, Mean - mean value, Mean + SD - mean value plus standard deviation; SD, standard deviation. 3 RESULTS A B FIGURE 3 \| Bivariate linear regression (RMA model) between the length of the skull (Y axis; Glabella-Inion length) and the position of the frontal (A) and occipital poles (B) on this axis (X axis, i.e. the position of the mean projection of the left and right poles on the Inion-Glabella chord). The blue dot shows the position of OL-23/77. A B B A FIGURE 3 \| Bivariate linear regression (RMA model) between the length of the skull (Y axis; Glabella-Inion length) and the position of the frontal (A) and occipital poles (B) on this axis (X axis, i.e. the position of the mean projection of the left and right poles on the Inion-Glabella chord). The blue dot shows the position of OL-23/77. ssion (RMA model) between the length of the skull (Y axis; Glabella-Inion length) and the position of the frontal (A) and occipital poles sition of the mean projection of the left and right poles on the Inion-Glabella chord). The blue dot shows the position of OL-23/77. GURE 3 \| Bivariate linear regression (RMA model) between the length of the skull (Y axis; Glabella-Inion length) and the position of the ) on this axis (X axis, i.e. the position of the mean projection of the left and right poles on the Inion-Glabella chord). The blue dot shows sample, which are used in palaeoanthropology for the reconstruction of cognitive abilities (47, 55). The endocast anatomy of an adult female with endocrine disorders (i.e. acromegaly and gigantism) exhibits no signs of pathology. Moreover, the middle meningealsystemandanatomo-functionalareas(premotor,primary motor and somatosensory cortexes) of this individual (OL-23/77) show normal development and morphology, as in other non- pathological individuals from the same population that were used for comparison. to the rest of the mediaeval population. This is informative because we observed that bone thickness in this area is higher than expected for an individual of this size, indicating that the frontal sinuses have not developed as much as the available space in the bone would have allowed. We also observed that the maxillary sinuses and sphenoid pneumatization in this specimen are of normal size and shape. Frontiers in Endocrinology \| www.frontiersin.org 4 DISCUSSION It was possible to measure and calculate the volume of the sella turcica in all collected individuals from the analysed mediaeval population. The sella turcica of OL-23/77 was enlarged (1304.52 mm3)comparedtothecomparativesample(mean:361.56mm3)and with an asymmetrically eroded ﬂoor of the bone. The large size and lyticdestructionofthesellaturcicaintheanalysedindividual(i.e.OL- 23/77) may result from the pressure erosion of an intrasellar tumour (49, 56). The calculated sella turcica volume of OL-23/77 falls within the range of other cases of gigantism and acromegaly (11, 49). The anatomical features of the skeleton and the large volume of the sella turcica conﬁrm the earlier anthropological assessment, the presence of endocrine disorder in OL-23/77. The timing of GH excess The morphological data of an adult individual with endocrine disorders (OL-23/77) were analysed in relation to non-pathological individuals from the same mediaeval group in Poland. The cranial length of OL-23/77 is similar to skull measurements reported in earlier archaeological studies and current patients with acromegaly (7–9, 11, 26). We were unable to analyse the cortical thickness of the cortex or variation in grey matter volume that is relevant for the cognitive abilities (54) as we investigated bone remains. Therefore we focus on the internal anatomy and its features, such as the size and asymmetry of the external endocast structures of the OL-23/77 April 2022 \| Volume 13 \| Article 862047 Frontiers in Endocrinology \| www.frontiersin.org 6 Internal Anatomy of a Giant Female Kubicka et al. A B C FIGURE 4 \| Graphic representation of the cranial thickness of the female with gigantism and acromegaly. (A) anterior view of the cranium, (B) lateral view of the cranium, (C) superior view of the cranium. Colours indicate the variation in bone thickness, from white (2mm) to dark red/yellow (22 mm). A B A C B FIGURE 4 \| Graphic representation of the cranial thickness of the female with gigantism and acromegaly. (A) anterior view of the cranium, (B) lateral view of the cranium, (C) superior view of the cranium. Colours indicate the variation in bone thickness, from white (2mm) to dark red/yellow (22 mm). TABLE 4 \| Morphometric data for the pneumatization of the frontal bone. 4 DISCUSSION W, maximal lateral extension of the frontal sinuses; H, maximal height of the frontal sinuses; 2A, the combined maximal length of the left and right frontal sinuses; 2S, the combined maximal length of the left and right frontal sinuses; 2AP, the combined length of the left and right frontal sinuses from the most anterior point to the most posterior point; RCV, cube-root of the volume of the frontal sinuses; Wr, relative maximal lateral extension of the frontal sinuses scaled to the cube root of the volume; Hr, relative maximal height of the frontal sinuses scaled to the cube root of the volume; 2Ar, the combined relative maximal length of the left and right frontal sinuses in anterior view scaled to the cube root of the volume; 2Sr, the combined relative maximal length of the left and right frontal sinuses in superior view scaled to the cube root of the volume; 2Apr, the combined relative length of the left and right frontal sinuses from the most anterior point to the most posterior point scaled to the cube root of the volume; n, number of individuals; Min, minimal observed value; Max, maximal observed value; Mean - SD, mean value minus standard deviation; Mean, mean value; Mean + SD, mean value plus standard deviation; SD, standard deviation; V, coefﬁcient of variation corrected for small sample size. determines the type of endocrine disorder, i.e. the occurrence of enlargement before and after the epiphyseal closure leads to gigantism or acromegaly, respectively. In turn, the onset secretion of GH in childhood and continuation into adulthood causes the coexistence of these two endocrine disorders (49, 56). The age at the time of death of the analysed female ranged from 25 and 30 years therefore gigantism is probable, as the body stature and elongated long bones suggest the excessive secretion of growth hormones early in life. Thick bones and enlarged and prognathic mandible of OL-23/ 77 might also indicate acromegaly. However, without genetic studies on gene mutations (e.g., AIP, PRKAR1A, GPR101, GNAS, MEN1, CDKN1B,SDHx,MAX(57),itisdifﬁculttoassesswhetherOL-23/77 was affected by gigantism and acromegaly or just one of these diseases. Especially, since some anatomical features such as elongated bones and high body stature, typical of gigantism, may also occur in individuals with acromegaly (56). Therefore, we suggest treating this case as an individual with gigantism/acromegaly. Frontiers in Endocrinology \| www.frontiersin.org 4 DISCUSSION W [mm] H [mm] 2A [mm] 2S [mm] 2AP [mm] RCV [mm] Wr [mm] Hr [mm] 2Ar [mm] 2Sr [mm] 2Apr [mm] OL-23/77 (n=1) Giant 80.3 41.4 88.9 90.2 75.8 30.2 265.8 137.0 294.3 298.4 250.9 Individuals from Ostrów Lednicki (n=32) Min 0 0 0 0 0 0 0 0 0 0 0 Mean-SD 24.8 13.0 23.8 23.4 12.8 9.2 195.2 113.3 197.1 196.5 118.7 Mean 41.4 22.2 41.1 42.3 25.7 14.6 269.6 146.3 265.3 269.9 164.7 Mean+SD 58.0 31.4 58.4 61.2 38.6 20.0 344.0 179.3 333.5 343.3 210.7 Max 72.3 50.4 79.4 87.7 70.6 26.2 453.3 200.5 366.2 410.3 269.5 SD 16.6 9.2 17.3 18.9 12.9 5.4 74.4 33.0 68.2 73.4 46.0 V* 40.1 41.6 42.2 44.8 50.6 36.7 27.7 22.6 25.8 27.3 28.1 W, maximal lateral extension of the frontal sinuses; H, maximal height of the frontal sinuses; 2A, the combined maximal length of the left and right frontal sinuses; 2S, the combined maximal length of the left and right frontal sinuses; 2AP, the combined length of the left and right frontal sinuses from the most anterior point to the most posterior point; RCV, cube-root of the volume of the frontal sinuses; Wr, relative maximal lateral extension of the frontal sinuses scaled to the cube root of the volume; Hr, relative maximal height of the frontal sinuses scaled to the cube root of the volume; 2Ar, the combined relative maximal length of the left and right frontal sinuses in anterior view scaled to the cube root of the volume; 2Sr, the combined relative maximal length of the left and right frontal sinuses in superior view scaled to the cube root of the volume; 2Apr, the combined relative length of the left and right frontal sinuses from the most anterior point to the most posterior point scaled to the cube root of the volume; n, number of individuals; Min, minimal observed value; Max, maximal observed value; Mean - SD, mean value minus standard deviation; Mean, mean value; Mean + SD, mean value plus standard deviation; SD, standard deviation; V*, coefﬁcient of variation corrected for small sample size. TABLE 4 \| Morphometric data for the pneumatization of the frontal bone. 4 DISCUSSION The topographic mapping of vault thickness we obtained shows, however, that the pattern observed in the sample (OL-23/ 77), where the frontal bones are thick and the temporal squama is thin, is the same as in non-pathological individuals (68, 69). Moreover, OL-23/77 shows an unusually high position of the orbits in the skull that is typical for patients with gigantism (70). Perhaps, the position and high thickness of the frontal bones limit the position of the frontal lobes. On the other hand, thick parietal bones may have a slight impact on the parietal lobes of OL-23/77 therefore the correlation between the skull length and the position of the occipital poles of the sample (OL-23/77) follows the regression line calculated for the comparative sample. endocrine disorders (i.e. gigantism and acromegaly) should be carried out with caution, especially when examining human remains. Nevertheless, this case study broadens our knowledge about the occurrence of gigantism and acromegaly in past populations, when the causes of these endocrine disorders were unknown, as well as provides information about variation in poorly investigated cranial traits. ETHICS STATEMENT Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. DATA AVAILABILITY STATEMENT The data supporting the conclusions of this article are available on request from the authors. g p p Medical studies focus on pneumatization of the skull but usually in the context of incomplete sphenoid sinuses, which can complicate transsphenoidal surgery performed on patients with acromegaly and gigantism (3, 70). In historical cases of these two endocrine disorders, the pneumatization of other bones of the skull is also analysed, but in the light of their presence and size. Pneumatization of the frontal sinuses was found to be present in two individuals with signs of gigantism and acromegaly from a Late Holocene site in Central California (9) and in historic New Mexico (13). Both of these studies emphasize the enlargement of the frontal sinuses, which is consistent with the measurements obtained in our study where the absolute values for the frontal sinuses of OL-23/77 exceed the range of variation observed in the comparative sample from Ostrów Lednicki. However, when the scaled frontal pneumatization measurements were taken into account, OL-23/77 falls within the range of variation observed in the comparative group. This may mean that the frontal sinuses follow the developmental trajectory of the skull so that their size is the result of allometric scaling. We observed that the frontal sinuses have not developed in the large areas available, which relates to the unexpected thickness of the frontal bone in these areas. This might be related to arrested propagation of pneumatization before the frontal superstructures had attained their full development. This kind of observation is of interest in view of the as yet poorly understood causes of development of the sinuses. FUNDING Research of the ﬁrst author (AMK) is ﬁnanced through the Polish Ministry of Science and Higher Education (506.511.09.00) and the Polish National Agency for Academic Exchange (PPB/ BEK/2018/1/00390/U/00001/02). Research of the last author (AB) on brain anatomy is ﬁnanced through the PaleoBRAIN project by the ANR (Agence nationale de la recherche, grant ANR-20-CE27-0009). Our study shows that the skull of OL-23/77 has the classic features observed in H. sapiens during previous studies in terms of endocastasymmetry,cranialvaultthicknessandfrontalsinuses(35, 71, 72).Therefore,the internalanatomyofthe adultfemale (OL-23/ 77) is similar to individuals without endocrine disorders. We did not observe any signs of pathology or abnormality of the brain, as the main anatomical and functional areas visible on the endocast exhibit a normal size, shape and expression. There is a relationship between brain torque and cognitive abilities (58); based on our results, it is not possible to conﬁrm the earlier assumption of cognitive impairment of the female examined (33). Although the previous research suggested the presence of cognitive dysfunctions in acromegaly patients (73, 74) possibly due to the positive correlation of GH and the severity of cognitive impairments (75), the latest results of the neuropsychological tests and brain cortical thickness do not indicate signiﬁcant differences between acromegaly patients and the general population (54). That is why the assessment of the cognitive abilities of individuals with AUTHOR CONTRIBUTIONS AMK, contributions to design, acquisition of data, data analysis and interpretation, writing of the manuscript, and approval of the article. PC, paleopathological analysis and interpretation, approval of the article. AB, contributions to design, data analysis and interpretation, writing of the manuscript, and approval of the article. All authors contributed to the article and approved the submitted version. Frontiers in Endocrinology \| www.frontiersin.org 4 DISCUSSION shows a left frontal/left occipital petalia, a pattern that is quite rare in Homo sapiens but occurs more often in females (58–60). In turn, 27 out of 32 specimens from the comparative sample exhibit a right frontal/left occipital petalia, which is the most common pattern in Homo sapiens (61–63). Based on the lateral and vertical frontal and occipital petalias, the orientation of the asymmetries observed in OL-23/77 does not follow the classic pattern, but it is not unexpected and is certainly not related to the large size of the specimen. Both asymmetry indices (DA and AA) are high in the sample (OL-23/77); however, the values are within the range (i.e. minimum and maximum values) of the comparative samples. This conﬁrms the previous ﬁndings that asymmetry is not the result of allometric scaling (59, 64). The high cranial thickness values of OL-23/77 conﬁrm the previous results indicating signiﬁcantly thicker cranial vaults in acromegalic patients than in non-pathological individuals (65, 66). This could therefore be a consequence of the effects of GH on bone growth and the large size of the skull. However, the frontal bones are much thicker than the parietal bones of OL-23/ 77, possibly due to the timing of an endocrine disorder and the faster growth of the neurocranial bones than of the facial skeleton Since the relationship between features of the brain torque (frontal/occipital petalia, bending and shift), cognitive function and mental health is signiﬁcant (58), the pattern of endocast asymmetry in this study was investigated. The OL-23/77 sample April 2022 \| Volume 13 \| Article 862047 Frontiers in Endocrinology \| www.frontiersin.org 7 Internal Anatomy of a Giant Female Kubicka et al. (67). The topographic mapping of vault thickness we obtained shows, however, that the pattern observed in the sample (OL-23/ 77), where the frontal bones are thick and the temporal squama is thin, is the same as in non-pathological individuals (68, 69). Moreover, OL-23/77 shows an unusually high position of the orbits in the skull that is typical for patients with gigantism (70). Perhaps, the position and high thickness of the frontal bones limit the position of the frontal lobes. On the other hand, thick parietal bones may have a slight impact on the parietal lobes of OL-23/77 therefore the correlation between the skull length and the position of the occipital poles of the sample (OL-23/77) follows the regression line calculated for the comparative sample. (67). REFERENCES 22. Symmers D. Acromegalic Giantism. Interstate Med J (1917) 24:1013–5. doi: 10.1159/000371808 23. De Herder WW. The History of Acromegaly. Neuroendocrinology (2016) 103:7–17. doi: 10.1159/000371808 1. 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April 2022 \| Volume 13 \| Article 862047 Frontiers in Endocrinology \| www.frontiersin.org 9 Internal Anatomy of a Giant Female Kubicka et al. 45. Trzciński D, Myszka A, Piontek J. High Stature and Body Mass Might Affect the Occurrence of Schmorl’s Nodes. Anthropol Rev (2017) 80:301–11. doi: 10.1515/anre-2017-0020 63. REFERENCES doi: 10.1016/j.biopsych.2021.11.002 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. 59. Xiang L, Crow T, Roberts N. Cerebral Torque is Human Speciﬁc and Unrelated to Brain Size. Brain Struct Funct (2019) 224:1141–50. doi: 10.1007/s00429-018-01818-0 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. 60. Balzeau A, Gilissen E, Grimaud-Hervé D. Shared Pattern of Endocranial Shape Asymmetries Among Great Apes, Anatomically Modern Humans, and Fossil Hominins. PloS One (2012) 7:1–10. doi: 10.1371/journal.pone. 0029581 61. Holloway RL, de la Costelareymondie MC. Brain Endocast Asymmetry in Pongids and Hominids: Some Preliminary Findings on the Paleontology of Cerebral Dominance. Am J Phys Anthropol (1982) 58:101–10. doi: 10.1002/ ajpa.1330580111 Copyright © 2022 Kubicka, Charlier and Balzeau. 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. 62. Barrick TR, Mackay CE, Prima S, Maes F, Vandermeulen D, Crow TJ, et al. Automatic Analysis of Cerebral Asymmetry: An Exploratory Study of the Relationship Between Brain Torque and Planum Temporale Asymmetry. Neuroimage (2005) 24:678–91. doi: 10.1016/j.neuroimage.2004.09.003 April 2022 \| Volume 13 \| Article 862047 Frontiers in Endocrinology \| www.frontiersin.org 10
https://openalex.org/W2526730386	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043012/pdf/fpsyg-07-01466.pdf	English	null	Cut-Off Points for Mild, Moderate, and Severe Pain on the Numeric Rating Scale for Pain in Patients with Chronic Musculoskeletal Pain: Variability and Influence of Sex and Catastrophizing	Frontiers in psychology	2,016	cc-by	7,676	ORIGINAL RESEARCH published: 30 September 2016 doi: 10.3389/fpsyg.2016.01466 1 ‘Revalidatie Friesland’ Centre for Rehabilitation, Beetsterzwaag, Netherlands, 2 Department of Health Sciences, Community and Occupational Medicine, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands, 3 Adelante Centre of Expertise in Rehabilitation and Audiology, Hoensbroek, Netherlands, 4 Department of Rehabilitation Medicine, CAPHRI Research School, Maastricht University, Maastricht, Netherlands, 5 Faculty of Health and Technology, Zuyd University for Applied Sciences, Heerlen, Netherlands, 6 Department of Rehabilitation Medicine, MGG Medical Centre Alkmaar and Gemini Hospital Den Helder, Alkmaar, Netherlands, 7 Rijndam Rehabilitation Institute, Rotterdam, Netherlands, 8 Roessingh Research and Development, University of Twente, Enschede, Netherlands, 9 Department of Rehabilitation, Centre for Rehabilitation, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands Edited by: Lorys Castelli, University of Turin, Italy Reviewed by: Diana M. E. Torta, Université catholique de Louvain, Belgium Gerrit Hirschfeld, Osnabrück University of Applied Sciences, Germany Objectives: The 0–10 Numeric Rating Scale (NRS) is often used in pain management. The aims of our study were to determine the cut-off points for mild, moderate, and severe pain in terms of pain-related interference with functioning in patients with chronic musculoskeletal pain, to measure the variability of the optimal cut-off points, and to determine the inﬂuence of patients’ catastrophizing and their sex on these cut-off points. Correspondence: Anne M. Boonstra a.m.boonstra@revalidatie-friesland.nl Specialty section: This article was submitted to Psychology for Clinical Settings, a section of the journal Frontiers in Psychology Methods: 2854 patients were included. Pain was assessed by the NRS, functioning by the Pain Disability Index (PDI) and catastrophizing by the Pain Catastrophizing Scale (PCS). Cut-off point schemes were tested using ANOVAs with and without using the PSC scores or sex as co-variates and with the interaction between CP scheme and PCS score and sex, respectively. The variability of the optimal cut-off point schemes was quantiﬁed using bootstrapping procedure. Received: 31 May 2016 Accepted: 12 September 2016 Published: 30 September 2016 Cut-Off Points for Mild, Moderate, and Severe Pain on the Numeric Rating Scale for Pain in Patients with Chronic Musculoskeletal Pain: Variability and Inﬂuence of Sex and Catastrophizing Anne M. Boonstra 1, Roy E. Stewart 2, Albère J. A. Köke 3, 4, 5, René F. A. Oosterwijk 6, Jeannette L. Swaan 7, Karlein M. G. Schreurs 8 and Henrica R. Schiphorst Preuper 9 INTRODUCTION understood, diﬀerences in pain perception between men and women (Rollman and Lautenbacher, 2001; Racine et al., 2012). Only Fejer et al. (2005) have studied the association between sex and the cut-oﬀpoints for interference with functioning in individuals with neck pain, and found a small diﬀerence between male and female patients. Assessment of pain intensity is considered one of the core outcome domains in clinical pain research (Dworkin et al., 2005), and is thus very commonly applied. The Numeric Rating Scale (NRS) is regarded as one of the best single-item methods available to estimate the intensity of pain (Jensen et al., 1999; Breivik et al., 2000). The NRS assesses pain intensity using a 0–10 ranking scale with 0 representing “no pain” and 10 “unbearable pain” or comparable statement. Clinicians, including psychologists, often use the categories of mild, moderate, and severe to simplify communication between patients and health care professionals. However, translating continuous measures such as NRS into discrete categories is not straightforward. Simply dividing an NRS into mild, moderate, and severe pain by dividing the scale into three equal parts is not a valid method (Serlin et al., 1995). Serlin et al. (1995) tried to solve this problem by correlating pain intensity to the level of interference of the pain with the daily functioning of patients with pain due to cancer, using a speciﬁc statistical technique, i.e., estimating how much of the variance in pain-related disability can be explained by diﬀerent possible pain intensity classiﬁcations. Their statistical approach has been repeated for the same patient population, i.e., cancer patients (Paul et al., 2005) as well as being applied to other patient populations (e.g., Zelman et al., 2005; Hirschfeld and Zernikow, 2013; Oldenmenger et al., 2013; Boonstra et al., 2014). Results from the literature (Hirschfeld and Zernikow, 2013; Oldenmenger et al., 2013) show that the cut-oﬀbetween mild and moderate pain, in terms of pain-related interference with functioning, is mostly placed between 3 and 4, and the cut-oﬀbetween moderate and severe pain between 6 and 8. The diﬀerences may be caused by diﬀerences in study samples, pain deﬁnitions, and/or measures of functioning. Diﬀerence in diagnoses is generally accepted as one of the main causes of diﬀerences in cut-oﬀpoints between studies (Zelman et al., 2003), while diﬀerences between study samples may also be explained by chance variation (Hirschfeld and Zernikow, 2013). Citation: Results and conclusion: The study showed that NRS scores ≤5 correspond to mild, scores of 6–7 to moderate and scores ≥8 to severe pain in terms of pain-related interference with functioning. Bootstrapping analysis identiﬁed this optimal NRS cut-off point scheme in 90% of the bootstrapping samples. The interpretation of the NRS is independent of sex, but seems to depend on catastrophizing. In patients with high catastrophizing tendency, the optimal cut-off point scheme equals that for the total study sample, but in patients with a low catastrophizing tendency, NRS scores ≤3 correspond to mild, scores of 4–6 to moderate and scores ≥7 to severe pain in terms of interference Boonstra AM, Stewart RE, Köke AJA, Oosterwijk RFA, Swaan JL, Schreurs KMG and Schiphorst Preuper HR (2016) Cut-Off Points for Mild, Moderate, and Severe Pain on the Numeric Rating Scale for Pain in Patients with Chronic Musculoskeletal Pain: Variability and Inﬂuence of Sex and Catastrophizing. Front. Psychol. 7:1466. doi: 10.3389/fpsyg.2016.01466 September 2016 \| Volume 7 \| Article 1466 Frontiers in Psychology \| www.frontiersin.org Boonstra et al. Cut-Off Points for Chronic Pain with functioning. In these optimal cut-off schemes, NRS scores of 4 and 5 correspond to moderate interference with functioning for patients with low catastrophizing tendency and to mild interference for patients with high catastrophizing tendency. Theoretically one would therefore expect that among the patients with NRS scores 4 and 5 there would be a higher average PDI score for those with low catastrophizing than for those with high catastrophizing. However, we found the opposite. The fact that we did not ﬁnd the same optimal CP scheme in the subgroups with lower and higher catastrophizing tendency may be due to chance variability. Keywords: musculoskeletal pain, numeric rating scale, pain interference, classiﬁcation, chronic pain INTRODUCTION Most studies have classiﬁed pain intensity using the statistical method described by Serlin et al. (1995) to estimate how much of the variance in pain-related disability can be explained by diﬀerent possible pain intensity classiﬁcations. The cut-oﬀpoint scheme explaining the highest proportion of the variance is then chosen as the optimal scheme. Although this method may have shortcomings, its use facilitates comparisons between studies. Hirschfeld and Zernikow (2013) used a bootstrap resampling procedure and found a very large variability in the cut-oﬀpoints in their sample of children and adolescents with chronic pain. They recommended that studies to deﬁne cut-oﬀpoints include measures of variability for the optimal cut-oﬀpoints. The aims of the present study were to determine the optimal cut-oﬀpoints for mild, moderate, and severe pain in terms of pain-related interference with functioning for patients with chronic musculoskeletal pain, as well as to measure the variability of the optimal cut-oﬀpoints, and to determine the association between these cut-oﬀpoints and patients’ catastrophizing tendency and their sex. Frontiers in Psychology \| www.frontiersin.org Study Design Cross-sectional study in the context of care as usual. Statistical Analysis Descriptive statistics were used to analyze the characteristics of the study sample. Marital status was dichotomized into living alone vs. being married or living with a partner. Ethics Statement All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. The data were collected in a setting of usual care, in order to measure the outcome of the treatment. The patients were asked to indicate if they did not allow their anonymous data to be used for the nationwide survey and/or for scientiﬁc studies. Because the data were collected during usual care, no approval of a Medical Ethics Committee was needed. Frontiers in Psychology \| www.frontiersin.org Procedure All data were collected prior to the start or in the ﬁrst 2 weeks of the rehabilitation program. Characteristics of the Sample Characteristics of the Sample Each patient’s pain intensity rating on the NRS was classiﬁed into three categories, viz. mild, moderate, and severe interference. We analyzed all 28 possible classiﬁcation schemes, ranging from 2,3 to 8,9. The cut-oﬀpoints in these classiﬁcation schemes were named after the upper values for the mild and moderate categories, in accordance with Serlin et al. (1995). For example, a 3,7 CP scheme means that the ﬁrst category ranges from 1 to 3, the second from 4 to 7 and the third from 8 to 10. The ﬁrst number, i.e., 3, is thus the upper value of the mild category and the second number, i.e., 7, the upper value of the moderate category. Other examples of schemes are: the 2,5 CP scheme with 1–2 classiﬁed as mild, 3–5 as moderate, and 6–10 as severe; the 3,5 CP scheme with 1–3 classiﬁed as mild, 4–5 as moderate, and 6–10 as severe; the 5,6 CP scheme with 1–5 classiﬁed as mild, 6 as moderate, and 7–10 as severe; and the 5,8 CP scheme with 1–5 classiﬁed as mild, 6–8 as moderate, and 9–10 as severe. The following background characteristics were assessed: age, sex, marital status, duration of current pain period, and localization of pain (mainly back pain, neck pain including cervicobrachialgia, widespread pain including ﬁbromyalgia, pain in an extremity including shoulder pain, other). Functioning I t f Functioning Interference with functioning was assessed with the Pain Disability Index, Dutch Version (PDI-DV; Soer et al., 2013). The PDI is a 7-item questionnaire to investigate the magnitude of self- reported disability in diﬀerent situations such as work, leisure time, self-care, and social activities. Each item is scored on an 11-item numeric rating scale in which 0 means no disability and 10 maximum disability. Three or less missing values per patient were replaced by the mean score of the other values. A higher score means greater disability and therefore greater interference with functioning. Patients The patients included in the study participated in a nationwide survey of patients with musculoskeletal pain, who were referred or admitted to rehabilitation treatment in one of the cooperating rehabilitation centers. The patients were included when they ﬁrst consulted their rehabilitation physician or started multidisciplinary inpatient or outpatient rehabilitation treatment. The study included patients from ﬁve rehabilitation centers, each with one (rehabilitation centers a, b, e), two (rehabilitation center d), or ﬁve (rehabilitation center c) treatment sites in the Netherlands. Some of these centers were departments of a university or general hospital, others were stand-alone rehabilitation centers. The centers are located in diﬀerent parts of the Netherlands, with patients from rural or semi-industrialized areas, living in villages or medium-sized An unresolved issue is the inﬂuence of psychological factors on cut-oﬀpoints. Catastrophizing (expecting or worrying about major negative consequences from a situation, even one of minor importance) is associated with pain severity and disability in patients with several chronic pain conditions (Wertli et al., 2014a,b). Another issue is the inﬂuence of the patient’s sex on the cut-oﬀpoints. There are clear, though incompletely September 2016 \| Volume 7 \| Article 1466 Frontiers in Psychology \| www.frontiersin.org 2 Cut-Off Points for Chronic Pain Boonstra et al. magnifying their pain (e.g., “I’m afraid that something serious might happen”), or feeling helpless to manage their pain (e.g., “There is nothing I can do to reduce the intensity of my pain”). A higher score means greater dominance of the subscale. The total score on the PCS was used in the analyses. to large towns and cities. Patients were included between the early months of 2012 and mid-2014; the exact time of inclusion diﬀered between the participating rehabilitation centers. Inclusion criteria were: age over 18 years and having had musculoskeletal pain for longer than 3 months. Exclusion criteria were inability to understand Dutch, current major psychiatric disorder (active psychosis, severe depression with risk of suicide attempt, addiction, etc.), unwillingness to provide data for research purposes, a score of “no pain” or missing data on the NRS and more than 3 missing values on the Pain Disability Index (PDI-DV, see measurements). Association of Catastrophizing and Patient’s Sex with the Cut-Off Points for Mild, Moderate, and Severe Pain in Terms of Pain-Related Interference with Functioning g The associations between the cut-oﬀpoint schemes and the patients’ catastrophizing tendency and sex were determined by once again conducting ANOVAs (using the Generalized Linear Model in SPSS, version 22) for each of the 28 CP schemes. In the two series of additional analyses (i.e., with PCS total score and sex), the NRS (recoded as 1–3) was again used as the independent variable and the PDI-DV score as the dependent variable, while the total score on the PSC and the patient’s sex were respectively included as co-variates, as was the interaction between CP scheme and PCS score and sex, respectively. In view of the results of the analyses with the PCS score, we decided to conduct separate analyses, ﬁrstly for the patients with a PCS score equal to or lower than the median of the PCS scores and the patients with a PCS score higher than the median of the PCS scores (dividing the population into two groups by the median split method), and secondly for patients in the lower and higher quartiles and the middle group of scores (dividing the population into three groups by the quartile split method). In total, therefore, 7 times 28 (196) ANOVAs were conducted. Again, the F-values of the CP schemes were used to determine which scheme ﬁtted best. In these two (median split method) and three (quartile split method) patient subgroups we also conducted the bootstrap resampling procedure described above. Secondly, we split the total group into patients with low, moderate, and high catastrophizing tendencies, and since the lower quartile of the PCS score was below 21 and the higher quartile was above 37, we performed the analyses separately for patients with a PCS score equal to or lower than 21, for PCS scores between 21 and 37, and for those with a PCS score higher than 37. For the patients with low catastrophizing tendency, i.e., a PCS score ≤21, the optimal CP scheme proved to be 3,6. For the patients with moderate catastrophizing tendency, i.e., > 21 and ≤37, and for those with high catastrophizing tendency, i.e., a PCS score > 37, the optimal CP scheme was 5,7 in both cases. Pain Intensity and Catastrophizing Bootstrapping analysis identiﬁed the optimal CP scheme (5,7) in 90.2% of the bootstrapping samples. The 3,6 scheme was identiﬁed as the optimal CP scheme in 3.4% of the samples and the 4,6 scheme in 3.3%. The variability of the optimal CP scheme was quantiﬁed using a bootstrap resampling procedure (STATA, version 13.1). In this procedure the distribution is estimated using the information based on a number of resamples from the total sample. One thousand (1000) repetitions of samples of the patients were used to yield suﬃciently stable estimates for the variability of the optimal cut-oﬀpoints. The optimal CP scheme for each of the 1000 randomly chosen samples was determined, using the above-mentioned method introduced by Serlin et al. (1995). The patients’ sex did not inﬂuence the optimal CP scheme: in the analyses in which sex and the interaction variable sex∗CP scheme were entered as co-variates, neither of these covariates contributed signiﬁcantly to the model. In the analyses in which the PCS score and the interaction variable PCS score∗CP scheme were entered as co-variates in catastrophizing, the PCS score contributed signiﬁcantly to the model in all analyses, while the interaction variable PCS score∗CP scheme contributed sometimes (i.e., in 2 of the 28 analyses). The latter ﬁnding was explained as chance variation because only 2 of the analyses found a signiﬁcant contribution. To explore the ﬁnding of the signiﬁcant contribution of the PCS scores to the models, we conducted more analyses, as described above. First we split the total group into patients with low and with high catastrophizing tendency, and since the median of the PCS score was 29, we performed the analyses separately for patients with a PCS score equal or lower than 29 and for those with a PCS score higher than 29. For the patients with low catastrophizing tendency, i.e., a PCS score ≤29, the optimal CP scheme proved to be 3,6 and for the patients with high catastrophizing tendency, i.e., a PCS score > 29, the optimal CP scheme was 5,7 (see Table 2). In the subgroup with low catastrophizing tendency, bootstrapping analysis identiﬁed the optimal CP scheme as 3,6 in 29% of the bootstrapping samples, while the 5,7 scheme was identiﬁed as the optimal CP scheme in 23% of the samples and the 4,6 scheme in 21%. Pain Intensity and Catastrophizing The NRS for pain is an 11-point numeric rating scale, with 0 representing “no pain” and 10 “unbearable pain.” The patients were asked to assign a number to their average pain in the last week. We decided to ask the patients to report their average pain, as two studies found no diﬀerences in the cut-oﬀpoint schemes of the NRS for average and worst pain (Paul et al., 2005; Zelman et al., 2005) and one study found only a small diﬀerence (Fejer et al., 2005). Zelman et al. (2003) also preferred the average pain measure for cut-oﬀpoint derivation, because in their view average pain better reﬂects the experiences regarding the interference of pain with daily activities and is more stable than worst pain. In order to determine which CP scheme best distinguished between mild, moderate and severe pain, we used the method introduced by Serlin et al. (1995). We conducted one-way ANOVAs (using the Generalized Linear Model in SPSS, version 22) for each of the 28 classiﬁcation schemes, using NRS scores recoded as 1, 2, or 3 (depending on the CP scheme) as the independent variable and PDI-DV scores as the dependent variables. A signiﬁcant F-value of the CP scheme indicated that there were signiﬁcant diﬀerences between the three pain severity categories in terms of pain-related interference. In accordance with Serlin et al. (1995), we interpreted the highest F-value as indicating the classiﬁcation scheme that maximized Catastrophizing was evaluated by the Pain Catastrophizing Scale (PCS; Osman et al., 1997). In this questionnaire the patients were asked to reﬂect on past painful experiences and indicate the degree to which they experienced each of 13 thoughts or feelings when in pain, on a 5-point scale from 0 (not at all) to 4 (all the time). Three or less missing values per patient were replaced by the mean score of the other values. Pain catastrophizing aﬀects how individuals experience pain: ruminating about their pain (e.g., “I can’t stop thinking about how much it hurts”), September 2016 \| Volume 7 \| Article 1466 3 Cut-Off Points for Chronic Pain Boonstra et al. the patients with NRS scores in the range of 1–5, 6–7, and 8–10 were 30.3 (SD 11.8), 39.7 (SD 10.6), and 45.4 (11.5), respectively. the diﬀerences between the groups and was therefore the most useful for distinguishing between mild, moderate, and severe pain-related interference. Association of Catastrophizing and Patient’s Sex with the Cut-Off Points for Mild, Moderate, and Severe Pain in Terms of Pain-Related Interference with Functioning In the subgroup with low catastrophizing tendency, bootstrap analysis identiﬁed the optimal CP scheme as 3,6 in 42% of the bootstrapping samples, while the 4,6 scheme was identiﬁed as the optimal CP scheme in 19% of the samples and the 5,7 scheme in 18%. In the subgroup with moderate catastrophizing tendency, bootstrapping analysis identiﬁed the optimal CP scheme as 5,7 in 87% of the bootstrapping samples, while the 4,6 scheme was identiﬁed as the optimal CP scheme in 3% of the samples and the 4,7 scheme also in 3%. In the subgroup with high catastrophizing tendency, bootstrapping analysis identiﬁed the Pain Intensity and Catastrophizing In the subgroup with high catastrophizing tendency, bootstrapping analysis identiﬁed the optimal CP scheme as 5,7 in 87% of the bootstrapping samples, while the 4,7 scheme was identiﬁed as the optimal CP scheme in 11% of the samples and the 4,6 scheme in 10%. Association of Catastrophizing and Patient’s Sex with the Cut-Off Points for Mild, Moderate, and Severe Pain in Terms of Pain-Related Interference with Functioning Association of Catastrophizing and Patient’s Sex with the Cut-Off Points for Mild, Moderate, and Severe Pain in Terms of Pain-Related Interference with Functioning RESULTS A total of 2854 patients enrolled in the study. Patient characteristics are presented in Table 1. The results of the ANOVAs for the total population are presented in Table 2, which lists only the mid-range of CP schemes. The F-values of the CP schemes not presented here were lower than the F-value with ranking 6 as indicated in Table 2. The 5,7 CP scheme had the highest F-value, indicating that this scheme provided the best ﬁt for distinguishing pain into three categories, i.e., mild, moderate, or severe pain, in terms of interference with functioning. This means that an NRS score in the 1–5 range corresponds to mild interference with functioning, while scores of 6 and 7 represent moderate interference and a score in the 8–10 range corresponds to severe interference with functioning. The mean PDI scores of September 2016 \| Volume 7 \| Article 1466 Frontiers in Psychology \| www.frontiersin.org 4 Cut-Off Points for Chronic Pain Boonstra et al. TABLE 1 \| Characteristics of patients with chronic musculoskeletal pain, Pain Disability Index (PDI) scores, numeric rating scale (NRS) for pain scores and Pain Catastrophizing Scale (PCS) scores, for total sample (n = 2854) and for each rehabilitation center (n total = a:435, b:539, c:840, d:683, e: 357). RESULTS All patients Rehab center a Rehab center b Rehab center c Rehab center d Rehab center e n n n n n n CHARACTERISTICS Age (years, mean (SD)) 2794 43 (12.5) 435 44 (11.5) 539 42 (12.2) 840 43 (12.8) 679 43 (13.1) 301 43 (12.2) Sex (% male) 2789 28 431 30 539 24 840 31 678 28 301 29 Marital status (% single) 2746 30 434 29 535 35 817 29 674 30 286 30 Work (%) 2657 319 531 835 673 299 • Employed or self-employed 51 47 62 50 50 43 • Student 4 2 6 5 4 4 • Without work, or homemaker 29 28 24 28 31 40 • Retired 4 4 2 4 5 4 • Other/mixed 12 19 7 14 10 10 Location of pain (%) 2854 435 539 840 683 357 • Widespread pain 18 10 36 26 7 • Neck pain 8 1 21 13 2 • Back pain 18 4 24 35 10 • Pain in extremity 7 0 9 18 2 • Others 4 1 4 7 4 • Unknown 45 84 6 1 75 100 Duration of complaints (%) 2503 154 533 836 679 301 • 3–6 months 5 1 8 4 5 3 • 6–12 months 11 9 12 12 12 10 • 1–2 years 20 20 18 24 18 19 • 2–5 years 25 19 25 23 27 27 • >5 years 39 52 37 38 38 42 FUNCTIONING PDI (mean, SD) 2854 39 (12.6) 435 37 (12.7) 539 41 (11.8) 840 37 (12.7) 683 36 (13.2) 357 40 (12.4) PAIN NRS (median, quartiles) 2854 7 (5–8) 435 6 (5–7) 539 7 (6–8) 840 6 (5–7) 683 6 (5–7) 357 7 (6–8) CATASTROPHIZING PCS 2846 435 535 840 679 357 • Total score Median, quartiles 29 (21–37) 22 (13–30) 21 (14–30) 31 (25–38) 33 (25–41) 35 (27–43) Mean, SD 30 (11.9) 22 (10.8) 22 (10.9) 32 (9.6) 34 (10.6) 36 (11.2) TABLE 1 \| Characteristics of patients with chronic musculoskeletal pain, Pain Disability Index (PDI) scores, numeric rating scale (NRS) for pain scores and Pain Catastrophizing Scale (PCS) scores, for total sample (n = 2854) and for each rehabilitation center (n total = a:435, b:539, c:840, d:683, e: 357). interference. The variability of the optimal CP scheme was low, as bootstrapping found the 5,7 CP scheme to be optimal in ∼90% of the samples. RESULTS This makes it unlikely that our ﬁndings were due to chance ﬂuctuations. optimal CP scheme as 5,7 in 35% of the bootstrapping samples, while the 2,6 scheme was identiﬁed as the optimal CP scheme in 22% of the samples and the 2,5 scheme in 12%. DISCUSSION No clear association was found between the cut-oﬀpoints and patients’ sex. In clinical practice, therefore, interpreting the NRS as mild, moderate or severe pain in terms of interference with functioning is independent of the patient’s sex. By contrast, the level of catastrophizing inﬂuenced the optimal CP scheme: the optimal scheme for patients with low catastrophizing tendency was 3,6, indicating that an NRS score ≤3 corresponds to mild interference of pain with functioning, 4–6 to moderate interference, and 7–10 to severe interference, whereas the optimal scheme for patients with high catastrophizing tendency was the same as for the total patient sample, i.e., 5,7, indicating that an NRS score ≤5 corresponds to mild interference of pain with The aim of the current study was to ﬁnd the optimal cut-oﬀ points for mild, moderate, and severe pain in terms of pain- related interference with functioning in patients with chronic musculoskeletal pain, as well as to measure the variability of the optimal cut-oﬀpoints and determine the association between these cut-oﬀpoints and patients’ catastrophizing tendency and sex. The NRS score cut-oﬀpoints (CPs) of 5 and 7 (i.e., a 5,7 CP scheme) were found to provide the best model ﬁt, indicating that an NRS score ≤5 corresponds to mild interference of pain with functioning, 6 and 7 to moderate interference and 8–10 to severe September 2016 \| Volume 7 \| Article 1466 Frontiers in Psychology \| www.frontiersin.org 5 Cut-Off Points for Chronic Pain Boonstra et al. TABLE 2 \| Comparison of different cut-off point (CP) schemes for classifying Numeric Rating Scale (NRS) scores as mild, moderate or severe pain in terms of interference with functioning: F-value in ANOVA using the CP scheme as independent variable and the Pain Disability Index (PDI) scores as dependent variables, for all patients and for the subgroups with low and high catastrophizing tendency (i.e., Pain Catastrophizing Scale (PCS) scores ≤ or > the median of the scores, 29). DISCUSSION In terms of the cut-oﬀpoints between mild and moderate, this ﬁnding implies the following: among patients with low catastrophizing tendency, the interpretation of an NRS score of 4 or 5 is that the patients with these scores experience moderate interference of their pain with functioning, while among patients with high catastrophizing tendency, the interpretation of the NRS score 4 or 5 is that the patients with these scores experience mild interference of their pain with functioning. Moderate interference with functioning would theoretically imply a higher PDI score than mild interference. However, as can be seen in Figure 1, the PDI scores of the patients with low catastrophizing tendency were lower for each NRS score than those of the patients with high catastrophizing tendency, thus including the group of patients with NRS scores 4 and 5. This contradicts the cut-oﬀpoint schemes and their interpretation. Two possible explanations may be given. Firstly, the optimal CP scheme for patients with a low catastrophizing tendency may actually also be 5,7 and our ﬁnding of the 3,6 scheme was a matter of chance variability. In the subgroup with lower catastrophizing tendency (both the subgroup with a PCS score lower than the median and the subgroup with PCS scores in the lower quartile), the variability was much higher than in the subgroup with higher catastrophizing tendency. The probability that the correct optimal CP scheme was not found is therefore rather high (type 1 error). Secondly, the statistical method introduced by Serlin et al. (1995), which uses the highest F-value to indicate the classiﬁcation scheme that maximizes the diﬀerences between the groups and is therefore the most useful for distinguishing between mild, moderate, and severe pain-related interference, may not be the best method for ﬁnding the optimal CP scheme. FIGURE 1 \| Boxplots of the Pain Disability Index (PDI) scores by Numeric Rating Scale (NRS) score for average pain during the last week for the patients with low and high catastrophizing tendency (i.e., lower or higher than the median of the total scorer on the Pain Catastrophizing Scale (PCS), viz. 29). Optimal cut-oﬀpoints of 5 and 7 were only mentioned in the literature by Zelman et al. (2003), for patients with osteoarthritis. That this particular CP scheme was found in only one other study may be due to the fact that it was not assessed by most other authors (see Table 3). DISCUSSION CP 3,6 CP 3,7 CP 4,5 CP 4,6 CP 4,7 CP 4,8 CP 5,6 CP 5,7 CP 5,8 CP 5,9 CP 6,7 ALL PATIENTS (N = 2854) CP scheme–PDI 332.63 306.15 317.17 337.60 334.67 253.48 337.63 369.65 324.08 306.96 291.35 Ranking 5 3 4 2 1 6 PATIENTS WITH PCS TOTAL SCORE ≤29 (N = 1461) CP scheme–PDI 173.14 140.30 163.10 172.20 152.79 122.50 170.00 172.34 157.38 154.46 136.35 Ranking 1 5 3 4 2 6 PATIENTS WITH PCS TOTAL SCORE > 29 (N = 1385) CP scheme–PDI 124,57 129.58 121.62 130.02 143.46 101.41 132.35 156.76 130.55 121.63 123.61 Ranking 6 5 2 3 1 4 Rankings are given below the F-values, from the highest (1) to the lowest (6) rank. CP: cut-off points, ﬁgures refer to highest scores in the ﬁrst and second categories, for example CP 4,7 means a CP scheme where the ﬁrst category includes the NRS scores 1–4, the second category NRS 5–7 and the third category NRS 8–10. TABLE 2 \| Comparison of different cut-off point (CP) schemes for classifying Numeric Rating Scale (NRS) scores as mild, moderate or severe pain in terms of interference with functioning: F-value in ANOVA using the CP scheme as independent variable and the Pain Disability Index (PDI) scores as dependent variables, for all patients and for the subgroups with low and high catastrophizing tendency (i.e., Pain Catastrophizing Scale (PCS) scores ≤ or > the median of the scores, 29). Rankings are given below the F-values, from the highest (1) to the lowest (6) rank. CP: cut-off points, ﬁgures refer to highest scores in the ﬁrst and second categories, for example CP 4,7 means a CP scheme where the ﬁrst category includes the NRS scores 1–4, the second category NRS 5–7 and the third category NRS 8–10. FIGURE 1 \| Boxplots of the Pain Disability Index (PDI) scores by Numeric Rating Scale (NRS) score for average pain during the last week for the patients with low and high catastrophizing tendency (i.e., lower or higher than the median of the total scorer on the Pain Catastrophizing Scale (PCS), viz. 29). functioning, 6 and 7 to moderate interference and 8–10 to severe interference. Frontiers in Psychology \| www.frontiersin.org DISCUSSION Our previous study (Boonstra et al., 2014) in a comparable population (not including patients of the present study), but with a smaller sample, found 3 and 6 to be the optimal cut-oﬀpoints between mild, moderate, and severe interference with functioning, whereas the present study found this 3,6 scheme to be only the ﬁfth best CP scheme. Our previous study used domains of the SF-36 (Aaronson et al., September 2016 \| Volume 7 \| Article 1466 6 Cut-Off Points for Chronic Pain Boonstra et al. TABLE 3 \| Published studies about optimal cut-off point schemes for mild, moderate, and severe pain in terms of interference with functioning. DISCUSSION Study, authors Type of pain/diagnosis Pain measurement n Optimal cut-off points found in the study Range of values studied by the authors for the lower cut-off point (between mild and moderate), and the higher cut-off point (between moderate and severe) Lower Higher Serlin et al., 1995 Cancer pain NRS, worst pain 470 4 6 Lower cut-off point: 3–4 Higher cut-off point: 6–7 Jensen et al., 1999 Leg amputation patients: NRS, average pain Phantom pain 74 4 7 Lower cut-off point: 3–4 Back pain 29 4 6 Higher cut-off point: 6–7 General pain 102 3 6 Zelman et al., 2003 Low back pain NRS, average pain 96 5 8 Lower cut-off point: 4–6 Osteoarthritis 98 5 7 Higher cut-off point: 6–8 Turner et al., 2004 CTS NRS, average pain No superior Lower cut-off point: 3–5 Low back injuries scheme 6 Higher cut-off point: 6–7 4 Zelman et al., 2005 Diabetic peripheral neuropathy NRS, worst and average pain 255 4 7 Lower cut-off point: 4–6 Higher cut-off point: 6–8 Paul et al., 2005 Cancer pain NRS, average pain 160 4 7 Lower cut-off point: 3–5 Higher cut-off point: 5–7 Fejer et al., 2005 Neck pain NRS, average, worst, and characteristic pain 1385 4 7 14 categories between 3 and 8 Hanley et al., 2006 Spinal cord injury NRS, (a) overall pain or (b) current pain at worst location a: 307 b: 174 a and b: 3 a: 7 Lower cut-off point: 3–4 b: 6 Higher cut-off point: 6–7 Li et al., 2007 Cancer pain, patients with bone metastases NRS, (a) worst, (b) average, and (c) current 199 a and b: 4, c: 2 a, b, and c: 6 Lower cut-off point: 2–8 Higher cut-off point: 3–9 Kapstad et al., 2008 Osteoarthritis of the hip NRS, average pain 224 4 6 Lower cut-off point: 3–5 Osteoarthritis of the knee 94 4 7 Higher cut-off point: 5–7 Kalyadina et al., 2008 Cancer pain, hematological malignancies or solid tumors NRS, worst pain 221 4 6 Lower cut-off point: 3–4 Higher cut-off point: 6–7 Ferreira et al., 2011 Cancer pain NRS, worst pain 143 4 7 Lower cut-off point: 3–5 Higher cut-off point: 5–7 Hoffman et al., 2010 Diabetic peripheral neuropathy NRS, average pain 401 3 6 Not mentioned Hirschfeld and Zernikow, 2013 Children and adolescents with chronic pain NRS, maximum pain Lower cut-off point: 2–7 Higher cut-off point: 3–8 Whole sample 2249 4 8 Constant pain 650 5 8 Chronic headache 430 4 8 Musculoskeletal pain 295 2 8 Boonstra et al., 2014 Musculoskeletal pain VAS, average pain 456 3 6 Lower cut-off point: 3–5 Higher cut-off point: 5–7 Brailo and Zakrzewska, 2015 Nondental orofacial pain NRS, average pain 245 4 7 Lower cut-off point: 3–5 Higher cut-off point: 5–9 Present study Musculoskeletal pain NRS, average pain 2854 5 7 Lower cut-off point: 2–8 Higher cut-off point: 3–9 Cut-off points (CP): ﬁgures refer to highest scores in the ﬁrst and second categories, for example CP lower 4, higher 7 means: ﬁrst category includes the NRS scores 1–4, second category NRS 5–7, third category NRS 8–10. Cut-off points (CP): ﬁgures refer to highest scores in the ﬁrst and second categories, for example CP lower 4, higher 7 means: ﬁrst category includes the NRS scores 1–4, second S S Conclusion In conclusion, we found that NRS scores ≤5 correspond to mild pain-related interference with functioning, scores of 6 and 7 to moderate interference and scores ≥8 to severe interference. This interpretation of the NRS in terms of mild, moderate and severe interference with functioning is independent of the patient’s sex, but seems to be inﬂuenced by their catastrophizing tendency. However, the diﬀerence in CP schemes we found for patients with lower and higher catastrophizing tendencies contradicts what is theoretically plausible. The reason why we did not ﬁnd the same optimal CP scheme in the subgroups of patients with lower and higher catastrophizing tendencies may be chance variability. The association between catastrophizing and cut-oﬀpoints has not been studied before, so no comparison with other studies is possible. As far as we are aware, only Fejer et al. (2005) studied the inﬂuence of patients’ sex on the cut-oﬀpoints for interference with functioning, and their analysis of CP schemes for average pain found a small diﬀerence between the sexes, viz. a lower cut-oﬀpoint between mild and moderate pain interference for women (4) than for men (6). Their other analyses, with the worst and what they called characteristic pain as independent variables, found no or other diﬀerences between women and men, and they ﬁnally concluded that the diﬀerences were small. LIMITATIONS One weakness of our study is the way the patients were included, i.e., using data from a nationwide survey, which meant that response rate and hence selection bias were unknown. In some rehabilitation centers, the localization of pain complaints was not recorded in the survey questionnaire for most patients (see Table 1). Moreover, none of the rehabilitation centers comprehensively recorded the diagnoses in the survey. Secondly, our study used the PDI to measure interference with functioning. It is possible that other instruments, such as the BPI, would have given diﬀerent results. Finally, we explored the eﬀect of catastrophizing by splitting the population using AUTHOR CONTRIBUTIONS The main strength of our study was the large study sample, the largest sample used until now in studies of this topic. It was also the ﬁrst study taking patient’s catastrophizing into account and the second to examine the inﬂuence of sex on the CP schemes. AB, contributed to the design of the work; and the acquisition, analysis, and interpretation of data; drafted the work, approves ﬁnal version to be published; agrees to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. RS contributed to design of the work; analysis, and interpretation of data for the work; revised the work critically for important intellectual content; approved ﬁnal version to be published; agrees to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. HS, AK, RO, JS, KS, contributed to design of the work; and interpretation of data for the work; revised the work critically for important intellectual content; approved ﬁnal version to be published; and agrees to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. DISCUSSION NRS: numeric rating scale; VAS: visual analog scale. s about optimal cut-off point schemes for mild, moderate, and severe pain in terms of interference with functioning. TABLE 3 \| Published studies about optimal cut-off point schemes for mild, moderate, and severe pain in terms of interference with functioning. Study, authors Type of pain/diagnosis Pain measurement n Optimal cut-off points found in the study Range of values studied by the authors for the lower cut-off point (between mild and moderate), and the higher cut-off point (between September 2016 \| Volume 7 \| Article 1466 7 Frontiers in Psychology \| www.frontiersin.org Cut-Off Points for Chronic Pain Boonstra et al. the median split and quartile split methods. Although these are common methods to split a population, they may have inﬂuenced the results. 1998) to measure interference with functioning, and the Visual Analogue Scale (VAS) for pain, instead of the PDI and NRS, respectively. These diﬀerent measures may be the reason why we found a diﬀerent CP scheme in the present study. 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https://openalex.org/W2146468353	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0142188&type=printable	English	null	Rasch Analysis of the Adult Strabismus Quality of Life Questionnaire (AS-20) among Chinese Adult Patients with Strabismus	PloS one	2,015	cc-by	7,253	RESEARCH ARTICLE Results Copyright: © 2015 Wang 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. The overall AS-20 did not demonstrate unidimensional; however, it was achieved sepa- rately in the two Rasch-revised subscales: the psychosocial subscale (11 items) and the function subscale (9 items). The features of good targeting, optimal item infit and outfit, and no notable local dependence were found for each of the subscales. The rating scale was appropriate for the psychosocial subscale but a reduction to four response categories was required for the function subscale. No significant DIF were revealed for any demographic and clinical factors (e.g., age, gender, and strabismus types). Data Availability Statement: The original datasheet of the participants in this study is within the paper and its Supporting Information files. Funding: This work was supported by cstc2013jcsf10014 and cstc2012jjA10145 (http:// www.ctin.ac.cn). The funder provided the analysis tool, and helped with data analysis and interpretation. Methods We evaluated the fitness of the AS-20 with Rasch model in Chinese population by assess- ing unidimensionality, infit and outfit, person and item separation index and reliability, response ordering, targeting and differential item functioning (DIF). Received: January 10, 2015 Accepted: October 19, 2015 Published: November 6, 2015 Received: January 10, 2015 Accepted: October 19, 2015 Published: November 6, 2015 Background Citation: Wang Z, Zhou J, Luo X, Xu Y, She X, Chen L, et al. (2015) Rasch Analysis of the Adult Strabismus Quality of Life Questionnaire (AS-20) among Chinese Adult Patients with Strabismus. PLoS ONE 10(11): e0142188. doi:10.1371/journal. pone.0142188 Citation: Wang Z, Zhou J, Luo X, Xu Y, She X, Chen L, et al. (2015) Rasch Analysis of the Adult Strabismus Quality of Life Questionnaire (AS-20) among Chinese Adult Patients with Strabismus. PLoS ONE 10(11): e0142188. doi:10.1371/journal. pone.0142188 The impact of strabismus on visual function, self-image, self-esteem, and social interactions decrease health-related quality of life (HRQoL).The purpose of this study was to evaluate and refine the adult strabismus quality of life questionnaire (AS-20) by using Rasch analysis among Chinese adult patients with strabismus. Editor: Jinhai Huang, School of Ophthalmology and Optometry and the Affiliated Eye Hospital, Wenzhou Medical University, CHINA Received: January 10, 2015 Accepted: October 19, 2015 Published: November 6, 2015 Editor: Jinhai Huang, School of Ophthalmology and Optometry and the Affiliated Eye Hospital, Wenzhou Medical University, CHINA Zonghua Wang1, Juan Zhou1, Xingli Luo2, Yan Xu3, Xi She4, Ling Chen5, Honghua Yin2, Xianyuan Wang1* 1 School of Nursing, Third Military Medical University, Chongqing, China, 2 Department of Ophthalmology, Daping Hospital, Third Military Medical University, Chongqing, China, 3 Department of Ophthalmology, Southwest Eye Hospital, Third Military Medical University, Chongqing, China, 4 Department of Ophthalmology, Xinqiao Hospital, Third Military Medical University, Chongqing, China, 5 Department of Gastroenterology, the 324th Hospital of PLA, Chongqing, China * xywang_tmmu@sina.cn Rasch Analysis of the Adult Strabismus Quality of Life Questionnaire (AS-20) among Chinese Adult Patients with Strabismus Zonghua Wang1, Juan Zhou1, Xingli Luo2, Yan Xu3, Xi She4, Ling Chen5, Honghua Yin2, Xianyuan Wang1* * xywang_tmmu@sina.cn Introduction Adult strabismus, with an estimated prevalence of 4% [1], is often accompanied by ocular mis- alignment, blurred vision, diplopia, and eyestrain. In addition to the functional complaints, Adult strabismus, with an estimated prevalence of 4% [1], is often accompanied by ocular mis- alignment, blurred vision, diplopia, and eyestrain. In addition to the functional complaints, strabismus could also decrease patients’ quality of life (QoL) by adversely affecting psychologi- cal and social functioning. Previous studies showed that adult strabismus patients have suffered from anxiety and depression, low self-esteem, lack of confidence, limited job opportunities and difficulties with interpersonal relationships [2–4]. Consequently, they are likely to avoid social contacts, abandon outdoor activities, and develop mannerisms to hide the defects. In recent years health-related QoL (HRQoL) among Chinese adult patients with strabismus have received increasing concerns from researchers and clinical professionals. Except for one study exploring improvements on psychosocial functioning after strabismus surgery [5], other studies have focused on translation and culturally adaptation of vision-related QoL (VRQoL) instruments [6–10] that may be applicable to strabismus patients. Our previous studies have translated the original adult strabismus-20 (AS-20) into Chinese. We chose the AS-20 since it had the advantages over other VRQoL scales for being developed specifically to strabismus and showed satisfactory reliability (Cronbach’s α = 0.94) and validity [11]. The Chinese version of the AS-20 was demonstrated by traditional psychometric methods (classical test theory, CTT) that it was a reliable (Cronbach’s α = 0.91) and valid tool in Chinese population [12] after some item deletions and re-categorization. Rasch model has been recently used to investigate psychometric properties of the AS-20 in American and India populations [13,14]. Both studies found the lack of unidimensionality in the full-length of the AS-20, and indicated the needs for reduction of response categories and for deletion of several misfitting items. However, no Rasch analysis of the AS-20 in Chinese set- tings has been reported. One benefit of Rasch analysis is that it considers both the difficulty of each item and the latent trait of subjects to provide interval-level estimates which increase sta- tistical power and minimize the cost of clinical research by significantly reducing the sample size required. In contrast, the traditional methods (CTT) has the limitation of assuming each item contributes equally to the overall assessment of the latent trait. These assumptions of uni- form weighting between response options for each item may not be true [14,15]. Conclusion The AS-20 was demonstrated by Rasch analysis to be a rigorous instrument for measuring health-related quality of life in Chinese strabismus patents if some revisions were made regarding the subscale construct and response options. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. 1 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 Quality of Life among Chinese Adult Patients with Strabismus Introduction Moreover, Rasch model provides a deeper insight into the underlying features, such as unidimensionality, items fit and targeting, and response ordering. These are essential for an instrument to make meaningful comparisons of latent trait between different groups, or to compare across time [15]. Given these advantages of Rasch model, this study aims to investigate the psychometric properties of the AS-20 by using Rasch analysis among adult strabismus patients in the context of Chinese culture. PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 Rasch Analysis In this study, we began with a test of dimensionality by performing the principal component analysis (PCA) of the residuals. The residuals are those parts of the data not explained by the Rasch model. Unidimensionality could be considered if the PCA results show raw unexplained variance by the first contrast with an eigenvalues smaller than 2.0 [16]. An assessment of local item independence and item fit mean square was conducted to fur- ther examine how each item fit the scale to measure the underlying trait. Intra-item standard- ized residual correlations of > 0.7 were used to identify high local dependence between items, which indicates that these items may duplicate some feature of each other or they both incor- porate some shared dimension. Such items should be considered of combining or eliminating. If a MnSq value of an item places between 0.7 and 1.3 [16], it indicates that the item contributes to a single underlying construct (unidimensionality). Otherwise, the patients may respond to the item erratically, i.e., it isn’t always ‘harder’ (or ‘easier’) for all patients. Generally such mis- fitting item should be removed. The standardized Z-score (ZSTD) is a t-statistic to report the statistical significance (probability) of the MnSq value; in this study, the ZSTD were presented but not considered for analyzing item fitness because these statistics were sample-dependent and may elevate as sample size increases [15]. The differential item functioning (DIF) provides an indicator to investigate whether items show different difficulty estimates across subgroups [17]. The demographic and clinical variables we selected for DIF analysis included: gender, age ( median age [29.69 years] vs. > median age), education level (below/ above high school), living areas (urban/ rural), social sup- port (always/ sometimes & none), strabismus type (exotrapia/ esotropia), diplopia (presence/ absence) and health insurance (presence/ absence). A DIF contrast < 0.50 logits was defined as small or absent, DIF 0.50 to 1.0 as minimal (inconsequential), and DIF > 1.0 as notable [17,18]. Then we inspected person and item separation and reliability (PSEP) and person-item maps to evaluate precision and targeting within each subscale. The minimum acceptable value of per- son separation is 2.0 in order to attain the desired level of reliability of at least 0.80. Item sepa- ration is used to confirm the item difficulty hierarchy (= construct validity). Statistical analysis Descriptive analysis of demography and clinical features were performed by SPSS software (IBM, version 21.0). Winsteps1 software (version 3.81.0) was adopted to conduct Rasch analysis. Methods Study cohort were 247 Chinese adult patients with strabismus attending three tertiary hospitals in Chongqing during April and September 2014. The strabismus patients who met the follow- ing criteria were invited to take part in the study [12]: 1) aged 18 years and over; 2) no history of any eye-related surgery or any diagnosed emotional disorders; 3) no other facial or ocular comorbidities or any acute eye diseases; and 4) the angle of deviation by prism at distance was no less than 15pd. All data were collected prior to any strabismus-related surgery. Information about demography and clinical features were also collected. Ethical approval was obtained from the human ethics committee of Third Military Medical University. The whole study was in accordance with the Declaration of Helsinki. No written consent were collected, and the consent to participate in this survey was assumed upon the completion of this questionnaire (i.e., completing the questionnaires implies giving consent to 2 / 13 Quality of Life among Chinese Adult Patients with Strabismus participate). The participants were left alone in a reception room to complete the Chinese ver- sion of the AS-20 after patient information sheet (PIS) and full introduction have been given. PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 Demographic and Clinical Characteristics A cohort of 247 adult patients with strabismus (mean age, 29.69 ± 11.07 years) completed the AS-20. One hundred twenty-two (49.4%) were female and 107 (43.3%) were living in rural areas. Less than a quarter of the patients (n = 54) hold a university certificate or above, while 104 graduated from high school, 69 from middle school and 20 from primary school. More than one-third of the patients (n = 95) reported that they had often received support from fam- ily members and friends. Almost two-thirds (n = 168) had no health insurance to cover strabis- mus-related treatment and they had to pay for themselves. Regarding clinical features, over a third (n = 90) complained a symptom of double vision. One hundred and seventy-three (70.0%) were diagnosed as exotropia, 74 (30.0%) were esotropia. All patients were in hospital for a strabismus-related corrective surgery for the first time. Rasch Analysis A desired value of item separation is 3 (reliability value 0.9), which implies that the sample size is large enough to confirm the item difficulty hierarchy of the instrument. The targeting refers to whether the latent trait (in this study, i.e., patients’ strabismus-related QoL) matched with the item discrimination (difficulty). For a well-targeted instrument, the difference between the per- son and item means on the person-item map should be less than 1.0 logits [19]. The category probability curves (CPC) were also examined to explore appropriateness of the response options [20]. The response options in the original AS-20 were a five-point Likert scale from 0 (always) to 100 (never). The CPC showed the range of QoL for which each of the five response categories were most likely to be chosen. Disordered categories indicate that neighboring categories may be indistinguishable to respondents and could be merged [21]. 3 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 Quality of Life among Chinese Adult Patients with Strabismus Unidimensionality The PCA results of the overall AS-20 revealed that 46.3% of the total raw variance was explained by the measures and the overall raw unexplained variance explained by the first con- trast had an eigenvalue of 3.9 (> 2.0) (Table 1), suggesting a second dimension. Mostly consis- tent with the original AS-20, the two subscales—the ‘psychosocial subscale’ and the ‘function subscale’- with the same containing items were presented (Fig 1), except for the item 17 ‘I feel stressed because of my eyes’ having been re-categorized into the psychosocial subscale instead of the function one. A second PCA was conducted separately in the newly identified subscales, both suggesting a feature of unidimensionality (Table 1): in the psychosocial subscale, 56.9% of the raw variance was explained by the measures, and 7.3% of the unexplained variance was explained by the first contrast, with an eigenvalue of 1.9; in the function subscale, 52.6% of the raw variance was explained by the measures and the unexplained variance by the first con- trast was 1.6 eigenvalue units. All the analyses afterwards were based on the Rasch-revised subscales. Local Item Dependence and Item Fit Differential Item Functioning All the DIF contrast were below 1.0 logits for all the selected variables, indicating that no items had notable DIF (S1 Dataset). Overall Performance of Psychosocial Subscale The revised psychosocial subscale consisted of the item 1–10 and the item 17. The person sepa- ration index (PSI) was 2.70, with a reliability value of 0.88. The item separation index (ISI) was 7.16, with the reliability of 0.98 (Table 4). The difference between the person and item means was less than 1.0 logits (0.45 ± 1.27 logits for mean person vs. 0.00 ± 0.57 logits for mean item; Fig 2A), which suggested good targeting that the item difficulty matched well with the partici- pants’ HRQoL. No disordered response categories were evident (Fig 3A), indicating each cate- gory properly represented stepwise increase in severity and the difference between categories on severity could be correctly endorsed by the respondents. Therefore the five-level Likert scal- ing was retained. Quality of Life among Chinese Adult Patients with Strabismus Fig 1. Plot of PCA of residuals analysis. Fig 1. Plot of PCA of residuals analysis. indicated that these items were locally independent (Corr. < 0.7). All items fit the Rasch model well since they have showed satisfactory infit and outfit MnSq values within the range of 0.71 and 1.30 (Table 3); therefore no item in both subscales was deleted. indicated that these items were locally independent (Corr. < 0.7). All items fit the Rasch model well since they have showed satisfactory infit and outfit MnSq values within the range of 0.71 and 1.30 (Table 3); therefore no item in both subscales was deleted. Local Item Dependence and Item Fit The intra-item correlations of standardized residuals were between -0.15 and -0.33 in the psy- chosocial subscale and between -0.11 and -0.28 in the function subscale (Table 2). These results The intra-item correlations of standardized residuals were between -0.15 and -0.33 in the psy- chosocial subscale and between -0.11 and -0.28 in the function subscale (Table 2). These results Table 1. Dimensionality analysis of the overall AS-20 and subscales. Overall scale Psychosocial (Item 1–10 & Item 17) Function (Item 11–16 & Item 18–20) Eigen % Eigen % Eigen % Total raw variance 37.2 100.0 25.7 100.0 19.0 100.0 explained by measures 17.2 46.3 14.7 56.9 10.0 52.6 explained by persons 6.1 16.5 7.3 28.2 3.9 20.4 explained by items 11.1 29.7 7.4 28.7 6.1 32.2 Raw unexplained variance (total) 20.0 53.7 11.0 42.7 9.0 47.4 1st contrast 3.9 10.6 1.9 7.3 1.6 8.3 2nd contrast 1.8 4.9 1.4 5.4 1.4 7.4 3rd contrast 1.6 4.2 1.3 5.0 1.2 6.3 4th contrast 1.2 3.3 1.1 4.4 1.1 5.9 5th contrast 1.1 3.0 1.1 4.1 1.0 5.2 doi:10.1371/journal.pone.0142188.t001 PLOS ONE \| DOI 10 1371/j l 0142188 N b 6 2015 4 / 13 Table 1. Dimensionality analysis of the overall AS-20 and subscales. PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 4 / 13 doi:10.1371/journal.pone.0142188.g001 We combined this category with either the category 5 (‘never’) and the cat- egory 3 (‘sometimes’) to find out which one could result in the best measurement precision by PSI and targeting. As shown in Fig 3C and 3D, both the revised four response categories were properly oriented; but the precision and targeting decreased after the combination of response 0.99 (Table 4). The item difficulty matched well with the participants’ HRQoL (0.67 ± 1.12 0.99 (Table 4). The item difficulty matched well with the participants’ HRQoL (0.67 ± 1.12 logits for mean person vs. 0.00 ± 0.72 logits for mean item; Fig 2B). For response ordering, the CPC illustrated that the category 4 ‘rarely’ response was underutilized (Fig 3B), resulting in dis- ordered thresholds. We combined this category with either the category 5 (‘never’) and the cat- egory 3 (‘sometimes’) to find out which one could result in the best measurement precision by PSI and targeting. As shown in Fig 3C and 3D, both the revised four response categories were properly oriented; but the precision and targeting decreased after the combination of response options, with the PSI of 1.53 (reliability = 0.70) and 1.85 (reliability = 0.77) separately and the targeting values over 1.0 logits (Table 4). The combination of the category 4 and the category 3 was chose considering the better PSR than the other combination. 0.99 (Table 4). The item difficulty matched well with the participants’ HRQoL (0.67 ± 1.12 logits for mean person vs. 0.00 ± 0.72 logits for mean item; Fig 2B). For response ordering, the CPC illustrated that the category 4 ‘rarely’ response was underutilized (Fig 3B), resulting in dis- ordered thresholds. We combined this category with either the category 5 (‘never’) and the cat- egory 3 (‘sometimes’) to find out which one could result in the best measurement precision by PSI and targeting. As shown in Fig 3C and 3D, both the revised four response categories were properly oriented; but the precision and targeting decreased after the combination of response options, with the PSI of 1.53 (reliability = 0.70) and 1.85 (reliability = 0.77) separately and the targeting values over 1.0 logits (Table 4). The combination of the category 4 and the category 3 was chose considering the better PSR than the other combination. Overall Performance of Function Subscale The revised function subscale consisted of the item 11–16 and the item 18–20. The PSI was 1.88 (< 2.0), with a reliability of 0.78 (< 0.80). The ISI was 9.74, with a reliability coefficient of 5 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 doi:10.1371/journal.pone.0142188.t002 Quality of Life among Chinese Adult Patients with Strabismus 0.99 (Table 4). The item difficulty matched well with the participants’ HRQoL (0.67 ± 1.12 logits for mean person vs. 0.00 ± 0.72 logits for mean item; Fig 2B). For response ordering, the CPC illustrated that the category 4 ‘rarely’ response was underutilized (Fig 3B), resulting in dis- ordered thresholds. We combined this category with either the category 5 (‘never’) and the cat- egory 3 (‘sometimes’) to find out which one could result in the best measurement precision by PSI and targeting. As shown in Fig 3C and 3D, both the revised four response categories were properly oriented; but the precision and targeting decreased after the combination of response options, with the PSI of 1.53 (reliability = 0.70) and 1.85 (reliability = 0.77) separately and the targeting values over 1.0 logits (Table 4). The combination of the category 4 and the category 3 was chose considering the better PSR than the other combination. Table 2. Local Item Dependence of Psychosocial and Function Subscales. Psychosocial subscale Function subscale Item No. Item No. Correlation of Residuals Item No. Item No. Correlation of Residuals 1 10 -0.33 19 20 -0.28 1 7 -0.26 13 18 -0.26 4 8 -0.23 15 19 -0.22 3 5 -0.22 12 18 -0.22 4 5 -0.22 14 16 -0.21 1 5 -0.21 16 19 -0.20 3 9 -0.21 13 15 -0.18 7 17 -0.21 12 15 -0.18 2 5 -0.20 12 19 -0.17 2 6 -0.20 13 19 -0.17 3 17 -0.20 11 18 -0.16 2 9 -0.20 14 18 -0.16 3 6 -0.19 14 15 -0.16 2 8 -0.19 16 18 -0.13 4 6 -0.18 15 18 -0.13 4 7 -0.17 11 19 -0.13 3 10 -0.16 13 20 -0.11 6 9 -0.16 11 20 -0.11 1 9 -0.16 11 15 -0.11 4 17 -0.15 18 20 -0.11 doi:10.1371/journal.pone.0142188.t002 Table 2. Local Item Dependence of Psychosocial and Function Subscales. 0.99 (Table 4). The item difficulty matched well with the participants’ HRQoL (0.67 ± 1.12 logits for mean person vs. 0.00 ± 0.72 logits for mean item; Fig 2B). For response ordering, the CPC illustrated that the category 4 ‘rarely’ response was underutilized (Fig 3B), resulting in dis- ordered thresholds. PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 Discussion This study was the first Rasch analysis to examine the psychometric properties of the full length of the AS-20 in Chinese adult patients with strabismus. Several findings were highlighted. First, no item was deleted in the overall AS-20 and subscales but a slight change has been made on the subscale form. Second, although the overall AS-20 failed to show unidimesionality, this was obtained in both Rasch-revised psychosocial subscale and function subscale. It is suggested to provide individual score of each subscale instead of obtaining a total score. Third, no misfit item and satisfactory targeting have been found; however, the function subscale did not fit the Rasch model well by showing inadequate measurement precision and underutilized response categories. This study indicated no item deletion but a form change that the item 17 should be re-cate- gorized into the psychosocial subscale. The re-categorization were also reported in our previous 6 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 Quality of Life among Chinese Adult Patients with Strabismus Table 3. Infit and Outfit MnSq of Psychosocial and Function Subscales. Item No. Inﬁt Outﬁt MnSq ZSTD* MnSq ZSTD* Psychosocial subscale 10 1.28 3.0 1.30 2.7 5 1.17 2.1 1.26 2.3 9 1.05 0.6 1.24 2.3 1 1.07 1.0 1.12 1.3 4 1.02 0.3 1.05 0.6 7 0.95 -0.7 1.06 0.6 3 0.98 -0.2 0.94 -0.6 8 0.93 -0.9 0.83 -1.7 17 0.88 -1.4 0.88 -1.3 6 0.87 -1.5 0.81 -2.0 2 0.74 -3.2 0.71 -3.3 Function subscale 19 1.21 2.4 1.23 2.2 14 1.19 1.6 1.02 0.2 13 1.15 1.4 1.05 0.4 11 1.01 0.1 1.14 0.9 12 1.14 1.1 1.06 0.4 16 1.00 0.0 0.82 -1.2 15 0.89 -1.4 0.99 -0.1 18 0.91 -1.0 0.97 -0.3 20 0.90 -1.0 0.81 -1.7 * the ZSTD were presented but not considered for analyzing item ﬁtness because these statistics were sample-dependent and may elevate as sample size increases. doi:10.1371/journal.pone.0142188.t003 Table 3. Infit and Outfit MnSq of Psychosocial and Function Subscales. * the ZSTD were presented but not considered for analyzing item ﬁtness because these statistics were sample-dependent and may elevate as sample size increases. doi:10.1371/journal.pone.0142188.t003 study of the AS-20 by CTT approach [12]: the factor analysis suggested deletion of two items since they revealed equivalent loading in two factors; and three items (including the item 17) originally from the function subscale were re-grouped into the psychosocial subscale consider- ing both factor loading and cultural difference. PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 * the ZSTD were presented but not considered for analyzing item ﬁtness because these statistics were sample-dependent and may elevate as sample size increases. Quality of Life among Chinese Adult Patients with Strabismus Table 4. Overall Performance of Psychosocial and Function Subscales. Ideal values Psychosocial Function* Function# Function§ Number of Items N/A 11 (items 1–10 and item 17) 9 (items 11–16 and items 18–20) 9 (items 11–16 and items 18–20) 9 (items 11–16 and items 18–20) PSI 2.0 2.70 1.88 1.53 1.85 PSI reliability 0.80 0.88 0.78 0.70 0.77 ISI 3.0 7.16 9.74 8.31 8.66 ISI reliability 0.90 0.98 0.99 0.99 0.99 Mean person N/A 0.45 ± 1.27 0.67 ± 1.12 1.25 ± 1.26 1.04 ± 1.43 Mean item N/A 0.00 ± 0.57 0.00 ± 0.72 0.00 ± 0.84 0.00 ± 0.90 the difference between the person and item means < 1.0 0.45 0.67 1.25 1.04 * the ‘rarely’ response option was not combined with the ‘never’ response option in the function subscale (the original 5-level response option) # the ‘rarely’ response option was combined with the ‘never’ response option in the function subscale (the combination of the category 4 and 5) § the ‘rarely’ response option was combined with the ‘sometimes’ response option in the function subscale (the combination of the category 3 and 4) Table 4. Overall Performance of Psychosocial and Function Subscales. * the ‘rarely’ response option was not combined with the ‘never’ response option in the function subscale (the original 5-level response option) # the ‘rarely’ response option was combined with the ‘never’ response option in the function subscale (the combination of the category 4 and 5) § the ‘rarely’ response option was combined with the ‘sometimes’ response option in the function subscale (the combination of the category 3 and 4) questionnaire; however, the inconsistency of the AS-20 item number and subscale form in dif- ferent cultures and ethnic groups suggested that the AS-20 in its current form was not perfect for its worldwide application. The culturally-appropriate translation and validation of the orig- inal AS-20 is required before performing in a new culture and ethnic population. q p g p p The reason for the need of deletion and re-categorization of the AS-20 may lie in that the HRQoL is a culture-dependent concept, thus the interpretation of HRQoL varies in different ethnic populations and cultures. Discussion In another study of the Chinese version of the AS-20, Yu et al. [10] have identified six principal factors without deleting an item, but these fac- tors could be regrouped into two subscales, with all of the 20 items loading in the same subscale as the original AS-20. Each item was correlative with their respective subscale and the Cron- bach’s α ranging from 0.819 to 0.883 for the overall AS-20 and the two subscales. Additionally, it was also reported by Rasch analysis that deletion of items and re-construction could improve the AS-20 rigorousness. The earliest Rasch study of the AS-20 was conducted by Leske and col- leagues in American strabismus patients [14]. They found both the subscales lacked unidimen- sionality and recommended formation of four new subscales; but two of these showed low measurement precision and did not function well. A new component of self-perception was divided from the psychosocial subscale, while a second dimension of reading function was divided from the function subscale. Recently Gothwal et al. have conducted Rasch analysis of the Indian version of the AS-20 in 584 Indian adult patients with strabismus [13]. They found that the AS-20 had adequate precision (PSR = 0.87) and targeting but failed to achieve unidi- mensional unless they deleted six multi-dimensionality causing items and an additional three misfitting items. Overall, these studies indicated that the AS-20 was a reliable and valid PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 7 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 In the development of the original AS-20, the function sub- scale included items relating to physical and emotional functions, while the psychosocial sub- scale were related to psychosocial functioning and self-awareness [11]. Emotional functions represented a health status without the expression of anxiety and depression; while psychoso- cial functioning was a broader concept, which stand for not only good psychological health, but also harmonious relationship with nature and social environment [22]. The re-categoriza- tion of the item 17 (‘I feel stressed because of my eyes’) may suggest the stress associated with strabismus could go beyond emotional functions and extend to psychological and social func- tioning in Chinese strabismus patients. Therefore they interpreted the item 17 more relevant to psychosocial functions rather than emotional complaints. y Another reason we assume is that HRQoL is a complicated term covering six aspects of physical, social, and psychological functioning, role activities, overall life satisfaction, and per- ceptions of health status[23]. Establishing two subdomains ‘psychosocial’ and ‘function’ might not be able to represent all other aspects of HRQoL. Although it indicated excellent reliability and validity in previous studies by using CTT methods, it was because careful clinical and psy- chometric criteria have been followed during the development of the AS-20. Besides, it might be improper for the AS-20 to include both physical and emotional functions together into the function subscale. For example, the item 17 (I feel stressed because of my eyes) and the item 18 (I worry about my eyes) were related to patients’ emotions while the item 14 (I have problems with depth perception) was related to symptoms. It was proved by the previous studies by using CTT and Rasch model that both methods indicated that the item 17 originally from the function subscale should be re-categoried into the psychosocial subscale. In terms of the psy- chosocial subscale, it also included items in different aspects (self-perception, social interaction, and psychological status) together. Therefore we assume that some new subdomains should be separated and established from the original two subscales and adding some items to cover 8 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 The category probability curves for (A) 5-response psychosocial subscale (representative item #1), (B) 5-response function subscale (representative item #11), (C) 4-response function subscale after the ‘rarely’ and ‘never’ response options were combined, (D) 4-response function subscale after the ‘rarely’ and ‘sometimes’ response options were combined. All curves at the extreme left represents ‘always’ and at the extreme right represents ‘never’. Response categories were properly oriented and distributed for all items in the psychosocial subscale (A). Using 5 response options for the function subscale, it was evident that the ‘rarely’ response was underutilized (B). When the ‘rarely’ option was combined with either the ‘never’ or the ‘sometimes’ option in the function subscale, response categories were properly oriented and distributed (C & D). Fig 3. The category probability curves for (A) 5-response psychosocial subscale (representative item #1), (B) 5-response function subscale (representative item #11), (C) 4-response function subscale after the ‘rarely’ and ‘never’ response options were combined, (D) 4-response function subscale after the ‘rarely’ and ‘sometimes’ response options were combined. All curves at the extreme left represents ‘always’ and at the extreme right represents ‘never’. Response categories were properly oriented and distributed for all items in the psychosocial subscale (A). Using 5 response options for the function subscale, it was evident that the ‘rarely’ response was underutilized (B). When the ‘rarely’ option was combined with either the ‘never’ or the ‘sometimes’ option in the function subscale, response categories were properly oriented and distributed (C & D). doi:10.1371/journal.pone.0142188.g003 more aspects of HRQoL might help improve the AS-20 performance. This assumption was supportive since that Yu et al. extracted six factors in PCA [10] and Leske et al.’s Rasch analysis indicated four new subscales [14]. The person separation index (PSI) and person separation reliability (PSR) refer to the num- ber of statistically distinct levels of person abilities identified by the instrument [24]. The Rasch-revised psychosocial subscale showed the PSI of 2.70 (reliability, 0.88), indicating satis- factory measurement precision to adequately discriminate different levels of quality of life [16]. In contrast, the PSI of the function subscale was suboptimal (1.88), with a reliability of 0.78, suggesting this subscale may not be sensitive and precise enough to distinguish the quality of life between high and low performers. Item separation index (ISI) means different from the PSI, which is used to confirm the item difficulty hierarchy (= construct validity). Quality of Life among Chinese Adult Patients with Strabismus 2. Person-item maps for (A) psychosocial subscale, (B) function subscale. The item discrimination matched well with the strabismus patients’ Q both subscales. M, Mean; S, 1 standard deviation; T, 2 standard deviations. Fig 2. Person-item maps for (A) psychosocial subscale, (B) function subscale. The item discrimination matched well with the strabismus patients’ QoL for both subscales. M, Mean; S, 1 standard deviation; T, 2 standard deviations. Fig 2. Person-item maps for (A) psychosocial subscale, (B) function subscale. The item discrimination matche for both subscales. M, Mean; S, 1 standard deviation; T, 2 standard deviations. doi:10.1371/journal.pone.0142188.g002 PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 9 / 13 Quality of Life among Chinese Adult Patients with Strabismus Fig 3. The category probability curves for (A) 5-response psychosocial subscale (representative item #1), (B) 5-response function subscale (representative item #11), (C) 4-response function subscale after the ‘rarely’ and ‘never’ response options were combined, (D) 4-response function subscale after the ‘rarely’ and ‘sometimes’ response options were combined. All curves at the extreme left represents ‘always’ and at the extreme right represents ‘never’. Response categories were properly oriented and distributed for all items in the psychosocial subscale (A). Using 5 response options for the function subscale, it was evident that the ‘rarely’ response was underutilized (B). When the ‘rarely’ option was combined with either the ‘never’ or the ‘sometimes’ option in the function subscale, response categories were properly oriented and distributed (C & D). doi:10.1371/journal.pone.0142188.g003 Fig 3. The category probability curves for (A) 5-response psychosocial subscale (representative item #1), (B) 5-response function subscale (representative item #11), (C) 4-response function subscale after the ‘rarely’ and ‘never’ response options were combined, (D) 4-response function subscale after the ‘rarely’ and ‘sometimes’ response options were combined. All curves at the extreme left represents ‘always’ and at the extreme right represents ‘never’. Response categories were properly oriented and distributed for all items in the psychosocial subscale (A). Using 5 response options for the function subscale, it was evident that the ‘rarely’ response was underutilized (B). When the ‘rarely’ option was combined with either the ‘never’ or the ‘sometimes’ option in the function subscale, response categories were properly oriented and distributed (C & D). Fig 3. PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 Quality of Life among Chinese Adult Patients with Strabismus The function subscale in its current form was not well matched to the Rasch model not only because of the unsatisfactory PSI and reliability, but also due to the need for revision of response options. Disordered response categories can occur when the neighboring categories may be indistinguishable to respondents and could be merged [21]. This was supported by Gothwal et al.’s findings that the response category did not function as expected for all items together in the AS-20. The reason they assumed was that the items of the AS-20 had too many response options for participants to distinguish between finer increments in response options. Therefore they reduced the response categories from the original 5-level to a new 3-level: ‘never’, ‘rarely/sometimes’ and ‘often/always’. They finally adopted the new 3-level rating scale since ideal PSI and targeting was achieved in the revised subscales. By comparison, in our study although the response categories in function subscale were properly oriented and distrib- uted after the combination of categories, the PSR and targeting did not improve. Since the higher PSR mainly depended on wider sample ability variance, longer test and more response categories, the decreased PSR of the function subscale seemed reasonable after the combination and the number of the response categories was reduced. However, given that the unsatisfactory PSR has already presented before the re-category, the insufficient item number may underlie the unsatisfactory PSR in the function subscale in Chinese population. The targeting provides an indication of how well targeted the items are for people in the sample by comparing of the mean score obtained for persons with that of the value of zero set for the items. The Rasch-revised psychosocial and function subscales demonstrated good tar- geting (i.e., the items are not too easy or too hard) of the items to the patients’ quality of life in our study cohort, because the mean location for strabismus patients were found around the value of zero (the mean value set for the items, 0.45 logits for the psychosocial subscale and 0.67 logits for the function one); in other words, the difference between the mean scores of the patients and the items were less than 1.0 logits. A positive mean value for the patients would indicate that the sample as a whole was located at a higher level of quality of life than the aver- age of the scale. Conclusions In conclusion, despite the need for re-categorization and unsatisfactory PSR in function sub- scale, the Chinese version of the AS-20 and its two subscales are generally reliable and valid tool among adult strabismus patients in China. Considering the lack of disease-specific QoL measurement for adult strabismus patients, undoubtedly this scale bridges the gap and pro- vides a useful tool to evaluate the HRQoL in this patient population. It is noted that culture plays an important to interpret HRQoL; therefore culturally-appropriate validation becomes necessary when the AS-20 applying in a new ethnic population with different culture. Further studies are needed to test out assumption that the performance of the Chinese version of the AS-20 would improve if several items are added and new subdomains are established. Both subscales of the Rasch-revised AS-20 showed high ISI and reliability, suggesting that the sample size was big enough to precisely locate the items difficulty hierarchy on the different levels of quality of life. 10 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 References 1. Gunton KB. Impact of strabismus surgery on health-related quality of life in adults. Curr Opin Ophthal- mol. 2014; 25(5):406–10. doi: 10.1097/ICU.0000000000000087 PMID: 25029092 2. Satterfield D, Keltner JL, Morrison TL. Psychosocial aspects of strabismus study. Arch Ophthalmol. 1993; 111(8):1100–5. PMID: 8166786 3. Menon V, Saha J, Tandon R, Mehta M, Khokhar S. Study of the psychosocial aspects of strabismus. J Pediatr Ophthalmol Strabismus. 2002; 39(4):203–8. PMID: 12148552 4. Hatt SR, Leske DA, Kirgis PA, Bradley EA, Holmes JM. The effects of strabismus on quality of life in adults. Am J Ophthalmol. 2007; 144(5):643–7. PMID: 17707329 5. Xu J, Yu X, Huang Y, Chen J, Yu H, Wang Y, et al. The psychosocial effects of strabismus before and after surgical correction in Chinese adolescents and adults. J Pediatr Ophthalmol Strabismus. 2012; 49 (3):170–5. doi: 10.3928/01913913-20110920-02 PMID: 22909123 6. Wang Z, Ren H, Frey R, Liu Y, Raphael D, Bian W, et al. Comparison of the Adult Strabismus Quality of Life Questionnaire (AS-20) with the Amblyopia and Strabismus Questionnaire (ASQE) among adults with strabismus who seek medical care in China. BMC Ophthalmol. 2014; 14:139. doi: 10.1186/1471- 2415-14-139 PMID: 25416453 7. Bian W, Li M, Wang Z, Wang X, Liu Y, Wu Y. Psychometric properties of the Chinese version of the Amblyopia and Strabismus Questionnaire (ASQE). Health Qual Life Outcomes. 2015; 13:81. doi: 10. 1186/s12955-015-0269-6 PMID: 26066333 8. Wang CW, Chan CL, Jin HY. Psychometric properties of the Chinese version of the 25-item National Eye Institute Visual Function Questionnaire. 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Health Qual Life Outcomes. 2013; 11:180. Supporting Information S1 Dataset. A summary of DIF results. This file includes all tables and figures for the DIF results of all the different selected variables. S2 Dataset. The original data of the 247 participants included in the study. This file includes the raw score of the AS-20 and participants’ demographic and clinical data. (XLS) 11 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 Quality of Life among Chinese Adult Patients with Strabismus Acknowledgments The authors sincerely appreciate the assistance from Ophthalmologists Dr. Dongmei Qi, Dr. d d l S h h l d C Zh The authors sincerely appreciate the assistance from Ophthalmologists Dr. Dongmei Qi, Dr. Jing Xie and Head nurse RN Xiaolei Wang in Southwest hospital and Dr. Cong Zhang in Xin- qiao Hospital. Author Contributions Conceived and designed the experiments: ZW JZ XW. Performed the experiments: ZW XL YX XS HY. Analyzed the data: ZW LC. Contributed reagents/materials/analysis tools: ZW LC. Conceived and designed the experiments: ZW JZ XW. Performed the experiments: ZW XL YX Conceived and designed the experiments: ZW JZ XW. Performed the experiments: ZW XL YX XS HY. Analyzed the data: ZW LC. Contributed reagents/materials/analysis tools: ZW LC. Wrote the paper: ZW JZ XW. Wrote the paper: ZW JZ XW. PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0142188 November 6, 2015 References doi: 10.1186/1477-7525-11-180 PMID: 24164742 13. Gothwal VK, Bharani S, Kekunnaya R, Chhablani P, Sachdeva V, Pehere NK, et al. Measuring Health- Related Quality of Life in Strabismus: A Modification of the Adult Strabismus-20 (AS-20) Questionnaire Using Rasch Analysis. PLoS One. 2015; 10(5):e0127064. doi: 10.1371/journal.pone.0127064 PMID: 26011430 14. Leske DA, Hatt SR, Liebermann L, Holmes JM. Evaluation of the Adult Strabismus-20 (AS-20) ques- tionnaire using Rasch analysis. 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PMID: 14707754 19. Khadka J, Pesudovs K, McAlinden C, Vogel M, Kernt M, Hirneiss C. Reengineering the glaucoma qual- ity of life-15 questionnaire with rasch analysis. Invest Ophthalmol Vis Sci. 2011; 52(9):6971–7. doi: 10. 1167/iovs.11-7423 PMID: 21810983 20. Hagquist C, Bruce M, Gustavsson JP. Using the Rasch model in nursing research: an introduction and illustrative example. Int J Nurs Stud. 2009; 46(3):380–93. doi: 10.1016/j.ijnurstu.2008.10.007 PMID: 19059593 21. Khadka J, Gothwal VK, McAlinden C, Lamoureux EL, Pesudovs K. The importance of rating scales in measuring patient-reported outcomes. Health Qual Life Outcomes. 2012; 10:80. doi: 10.1186/1477- 7525-10-80 PMID: 22794788 22. Zammit AR, Starr JM, Johnson W, Deary IJ. Profiles of physical, emotional and psychosocial wellbeing in the Lothian birth cohort 1936. BMC Geriatr. 2012; 12:64. doi: 10.1186/1471-2318-12-64 PMID: 23088370 23. Beaton DE, Schemitsch E. Measures of health-related quality of life and physical function. 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https://openalex.org/W2896012139	https://pure.eur.nl/files/48058501/Repub_111691_O-A.pdf	English	null	Association between Gestational Weight Gain, Gestational Diabetes Risk, and Obstetric Outcomes: A Randomized Controlled Trial Post Hoc Analysis	Nutrients	2,018	cc-by	8,497	Association between Gestational Weight Gain, Gestational Diabetes Risk, and Obstetric Outcomes: A Randomized Controlled Trial Post Hoc Analysis David Simmons 1,2,* , Roland Devlieger 3,4,5, Andre van Assche 3, Sander Galjaard 3,4,6 , Rosa Corcoy 7,8 , Juan M. Adelantado 8, Fidelma Dunne 9, Gernot Desoye 10 , Alexandra Kautzky-Willer 1, Peter Damm 11, Elisabeth R. Mathiesen 11, Dorte M. Jensen 12,13,14, Lise Lotte T. Andersen 14, Annunziata Lapolla 15, Maria G. Dalfra 15, Alessandra Bertolotto 16, Ewa Wender-Ozegowska 17, Agnieszka Zawiejska 17, David Hill 18, Frank J. Snoek 19 and Mireille N. M. nutrients nutrients nutrients nutrients Association between Gestational Weight Gain, Gestational Diabetes Risk, and Obstetric Outcomes: A Randomized Controlled Trial Post Hoc Analysis van Poppel 19,20 1 Institute of Metabolic Science, Addenbrooke’s Ho alexandra.kautzky-willer@meduniwien.ac.at 1 Institute of Metabolic Science, Addenbrooke’s Hospital, CB2 0QQ Cambridge, UK; alexandra.kautzky-willer@meduniwien.ac.at 1 Institute of Metabolic Science, Addenbrooke s Hospital, CB2 0QQ Cambridge, UK; alexandra.kautzky-willer@meduniwien.ac.at 2 alexandra.kautzky-willer@meduniwien.ac.at 2 Macarthur Clinical School, Western Sydney University, Locked Bag 1797, Campbelltown, Sydney, NSW 2760, Australia 3 Department of Development and Regeneration, KU Leuven, 3000 Leuven, Belgium; roland.devlieger@uz.kuleuven.ac.be (R.D.); andre.vanassche@kuleuven.be (A.v.A.); s.galjaard@erasmusmc.nl (S.G.) 4 Department of Obstetrics and Gynecology, University Hospitals Leuven, 3000 Leuven, Belgium 5 5 Department of Obstetrics, Gynecology and Fertility, GasthuisZusters Antwerpen Sint-Augustinus, 2610 Wilrijk, Belgium 6 Department of Obstetrics and Gynaecology, Division of Obstetrics and Prenatal Medicine, Erasmus MC, University Medical Centre, 3015 GD Rotterdam, The Netherlands 6 Department of Obstetrics and Gynaecology, Division of Obstetrics and Prenatal Medicine, Erasmus MC 7 CIBER Bioengineering, Biomaterials and Nanomedicine, Instituto de Salud Carlos III, Zaragoza, 50018 Spain; RCorcoy@santpau.cat 8 Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain; JAdelantado@santpau.cat 9 Galway Diabetes Research Centre (GDRC) and National University of Ireland, H91 CF50 Galway, Ireland; ﬁdelma.dunne@nuigalway.ie 10 Department of Obstetrics and Gynecology, Medizinische Universitaet Graz, A-8036 Graz, Austria; gernot.desoye@medunigraz.at 11 Center for Pregnant Women with Diabetes, Departments of Endocrinology and Obstetrics, Rigshospitalet and The Clinical Institute of Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, DK-1165 Copenhagen, Denmark; peter.damm@regionh.dk (P.D.); li b th thi @ h i h dk (E R M ) 11 Center for Pregnant Women with Diabetes, Departments of Endocrinology and Obstetrics, Rigshospitalet and The Clinical Institute of Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, DK-1165 Copenhagen, Denmark; peter.damm@regionh.dk (P.D.); elisabeth mathiesen@rh regionh dk (E R M ) 12 Department of Endocrinology, Odense University Hospital, DK-5000 Odense, Denmark; Dorte.Moeller.Jensen@ouh.regionsyddanmark.dk 12 Department of Endocrinology, Odense University Hospital, DK-5000 Odense, Denmark; Dorte.Moeller.Jensen@ouh.regionsyddanmark.dk 13 Department of Gynaecology and Obstetrics, Odense University Hospital, DK-5000 Odense, Denmark 14 Department of Clinical Research, Faculty of Health Sciences, University of Southern Denmark, DK-5230 Odense, Denmark; lise.lotte.andersen@rsyd.dk y 15 Department of Medical and Surgical Sciences. Università degli Studi di Padova, 35100 Padua, Italy; annunziata.lapolla@unipd.it (A.L.); mariagrazia.dalfra@sanita.padova.it (M.G.D.) 16 Department of Clinical and Experimental Medicine, University of Pisa, 56126 Pisa, Italy; alessandrabertolotto1959@yahoo.it (A.B.) 17 Department of Reproduction, Poznan University of Medical Sciences, 61-701 Poznan, Poland; ewaoz@post.pl (E.W.-O.); agazaw@post.pl (A.Z.) 18 Recherche en Santé Lawson SA, 9552 St. Association between Gestational Weight Gain, Gestational Diabetes Risk, and Obstetric Outcomes: A Randomized Controlled Trial Post Hoc Analysis Gallen, S 19 Department of Public and Occupational Health, Amsterdam Public Health Research Institute, Amsterdam UMC, Vrije Universiteit Amsterdam, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Netherlands ; fj snoek@vumc nl (F J S ); Mireille van-poppel@uni-graz at (M N M vP) 19 Department of Public and Occupational Health, Amsterdam Public Health Research Institute, Amsterdam UMC, Vrije Universiteit Amsterdam, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Netherlands.; fj.snoek@vumc.nl (F.J.S.); Mireille.van-poppel@uni-graz.at (M.N.M.v.P.) 20 Institute of Sports Sciences, University of Graz, A-8010 Graz, Austria 20 Institute of Sports Sciences, University of Graz, A-8010 Graz, Austria * Correspondence: da.simmons@westernsydney.edu.au; Tel.: +61-2-4620-3899; Fax: +61-2-4620-38 * Correspondence: da.simmons@westernsydney.edu.au; Tel.: +61-2-4620-3 Nutrients 2018, 10, 1568; doi:10.3390/nu10111568 www.mdpi.com/journal/nutrients Nutrients 2018, 10, 1568 2 of 13 Received: 27 September 2018; Accepted: 19 October 2018; Published: 23 October 2018 Received: 27 September 2018; Accepted: 19 October 2018; Published: 23 October 2018 Abstract: Excess gestational weight gain (GWG) is associated with the development of gestational diabetes mellitus (GDM). Lifestyle trials have not achieved much GWG limitation, and have largely failed to prevent GDM. We compared the effect of substantial GWG limitation on maternal GDM risk. Pregnant women with a body mass index (BMI) ≥29 kg/m2 <20 weeks gestation without GDM (n = 436) were randomized, in a multicenter trial, to usual care (UC), healthy eating (HE), physical activity (PA), or HE and PA lifestyle interventions. GWG over the median was associated with higher homeostasis model assessment insulin resistance (HOMA-IR) and insulin secretion (Stumvoll phases 1 and 2), a higher fasting plasma glucose (FPG) at 24–28 weeks (4.66 ± 0.43 vs. 4.61 ± 0.40 mmol/L, p < 0.01), and a higher rate of caesarean section (38% vs. 27% p < 0.05). The GWG over the median at 35–37 weeks was associated with a higher rate of macrosomia (25% vs. 16%, p < 0.05). A post hoc comparison among women from the ﬁve sites with a GWG difference >3 kg showed no signiﬁcance difference in glycaemia or insulin resistance between HE and PA, and UC. We conclude that preventing even substantial increases in GWG after the ﬁrst trimester has little effect on maternal glycaemia. We recommend randomized controlled trials of effective lifestyle interventions, starting in or before the ﬁrst trimester. Keywords: gestational diabetes mellitus; pregnancy; lifestyle intervention; randomised controlled trial; healthy eating; physical activity; overweight; motivational interviewing; prevention 2.1. Overall Study Design The methods used in this study have been described previously [8–10]. Brieﬂy, the DALI Lifestyle Study is an RCT Trial registration: ISRCTN70595832 that compared three different lifestyle approaches that could prevent GDM across the following ten European centers: Cambridge, (United Kingdon: coordinating center), Amsterdam (Netherlands), Leuven (Belgium), Barcelona (Spain), Galway (Ireland), Pisa/Padova (two sites; Italy), Vienna (Austria), Poznan (Poland), Copenhagen, and Odense (Denmark). The primary outcomes were changes in the GWG, fasting glucose, and HOMA-IR. The target GWG was 5 kg, which is at the lower end of the Institute of Medicine’s recommendations [11]. The women were randomized, and were stratiﬁed by site to usual care (UC), HE, PA, or HE and PA. The intervention has been described in detail previously [8,10]. After randomization, the women were assigned a lifestyle coach who provided ﬁve face-to-face, and up to four telephone coaching sessions, based on the principles of motivational interviewing. The coaches received standardized training and an intervention toolkit (including, e.g., a pedometer in the PA intervention) tailored to their culture/language. The coaching involved the discussion of seven HE and/or ﬁve PA “messages”, and a GWG <5 kg was targeted [10]. The HE intervention promoted a food-based, lower simple and complex carbohydrate, lower fat, higher ﬁber, and higher protein diet, including a focus on portion size, and therefore a more limited intake of total calories. The PA intervention promoted both aerobic and resistance physical activity. At least four face-to-face coaching sessions were expected to take place before the second measurement session (24–28 weeks), and the intervention was completed after 35 weeks of gestation. The HE intervention was associated with the self-reported reduced carbohydrate intake, reduced portion size, increased vegetable intake, and reduced sugary drinks intake, while the PA intervention was associated with a reduced sedentary time and increased moderate or vigorous activity [10]. The combined intervention was the only intervention associated with a signiﬁcant reduction in GWG. The participants were pregnant women aged ≥18 years, before 20 weeks gestation, with a pre-pregnancy body mass index (BMI) ≥29 kg/m2, and were recruited between January 2012 and February 2014. All of the women underwent an oral glucose tolerance test (OGTT), and those with GDM (International Association Diabetes Pregnancy Study Group (IADPSG)/World Health Organisation (WHO) 2013 criteria of fasting venous plasma glucose ≥5.1 mmol/L and/or one hour of glucose ≥10 mmol/L and/or two hours of glucose ≥8.5 mmol/L) [12] were excluded from the study. Nutrients 2018, 10, 1568 Nutrients 2018, 10, 1568 We hypothesized that the greater the GWG limitation, the greater the difference in GDM rates, fasting glucose, HOMA, and adverse obstetric outcomes between controls and intervention subjects. We tested this by initially comparing women above and below the median GWG, independent of intervention. As randomization in the DALI Lifestyle Study was stratiﬁed by site, we have gone on to treat each site within this study as a separate RCT, and tested whether the GDM risk was reduced in the ﬁve sites with the greatest GWG limitation, using the HE and PA intervention—the only intervention able to achieve GWG limitation. 1. Introduction The risk of gestational diabetes mellitus (GDM) increases with both obesity and gestational weight gain (GWG) [1] GDM, excess GWG, and overweight/obesity are all independently associated with an increased risk of macrosomia, operative delivery, and other adverse perinatal outcomes, including shoulder dystocia [1]. If increased GWG is causally related to an increase in GDM incidence, then limiting GWG should reduce the incidence of GDM. However, the results from randomized controlled trials (RCTs) for the prevention of GDM through lifestyle have been mixed [2]. The mean difference in GWG between the intervention and control groups has ranged between −9.07 kg and +3.5 kg in a meta-analysis in 2012, with a mean of −2.21 kg and no signiﬁcant reduction in GDM rates [3]. A recent meta-analysis of the individual participant data from randomized trials estimated a GWG reduction of 0.7 kg overall, with no overall reduction in GDM (unless data from studies without individual data were included) [4]. A GDM reduction was found with interventions involving physical activity alone (PA) [4]. The most recent Cochrane review (2017) showed no reduction in GDM or in adverse obstetric outcomes with lifestyle interventions [5]. However, another recent meta-analysis showed a reduction in GDM if intervention commenced in the ﬁrst, but not the second trimester [6]. Moreover, prevention occurred in some lifestyle intervention studies with minimal GWG difference (e.g., The Finnish Gestational Diabetes Prevention Study (RADIEL), where women with previous GDM and/or obesity were studied) [7]. More information is therefore needed on the relationship between GWG, the development of GDM, and obstetric outcomes. The Vitamin D and Lifestyle Intervention for GDM Prevention (DALI) [8–10] is a European multicenter RCT that tested different approaches for the reduction of GDM risk. The study was unique in that it had the following two limbs: (1) the DALI Lifestyle Study, which compared healthy eating (HE), PA, and mixed (HE and PA) with a control group, and (2) the DALI Vitamin D study that compared vitamin D supplementation with and without an HE and PA intervention. The main lifestyle RCT (the DALI Lifestyle Study) found the HE and PA intervention was associated with the least GWG, but there was no signiﬁcant reduction in the fasting glucose, GDM incidence, or insulin sensitivity (as measured with the homeostasis model assessment insulin resistance (HOMA-IR) [10]. 3 of 13 2.1. Overall Study Design Other exclusion criteria are reported elsewhere [8]. All of the women gave signed informed consent. The study was approved by the relevant ethical committees and was registered as an RCT (ISRCTN70595832). The assessments were made by the research midwife/nurse at four antenatal time points—before 20 weeks (baseline), between 24–28 weeks (visit 2), and between 35–37 weeks (visit 3) gestation. Maternal gestational weight gain (GWG) was deﬁned as the weight difference between the self-reported pre-pregnancy and the DALI antenatal measurement. Randomization was stratiﬁed by site, and the randomization method, and interventions have been described in detail previously [8]. Those involved in taking the measurements were kept blinded to the intervention. Nutrients 2018, 10, 1568 4 of 13 Information on the demographics, pre-pregnancy weight, smoking, alcohol consumption, past/current medical and obstetric history, and medication use was gathered using questionnaires. The data from the medical records were obtained regarding the co-morbidities, obstetric and perinatal outcomes, and birth weight. The women attended the three assessments while fasting, and undertook a standardized, sitting, 75 g OGTT, with blood samples taken at 0, 60, and 120 min after glucose ingestion. The women completed the questionnaire and anthropometric measurements between the blood tests. The local laboratories were used to rapidly obtain the OGTT results so as to assess their eligibility for the study, and to support referral for clinical care where needed. The blood samples were centrifuged and separated from the serum and plasma aliquots (1000 µL or 250 µL), placed in microrack tubes, and stored at −20 or −80 ◦C, until further analysis, in the central trial laboratory in Graz, Austria, which was certiﬁed according to ISO 9001 standards. y g The laboratory glucose and insulin analytical methods have been described previously [8–10]. The homeostatic model assessment-insulin resistance (HOMA-IR) was calculated as (glucose × insulin)/22.5 and the homeostatic model assessment-insulin secretion (HOMA-IS) (B) was calculated using the following formula of 20 × fasting insulin (µIU/mL)/fasting glucose (mmol/mL) −3.5 [13]. The Stumvoll ﬁrst and second phase indices are the surrogates of early and late insulin secretion, and were calculated as 1194 + 4.724 × Ins0 −117.0 × Gluc60 + 1.414 × Ins60 for the Stumvoll ﬁrst phase, and 295 + 0.349 × Ins60 −25.72 × Gluc60 + 1.107 × Ins0 for the Stumvoll second phase, as described earlier by Stumvoll et al. 2.1. Overall Study Design [14,15] The height was measured at baseline with a stadiometer (SECA 206, SECA, Birmingham, UK; Leicester Height Measure), and the average value of the two measurements was used. The women were weighed on calibrated electronic scales (SECA 888; SECA 877, SECA, Birmingham, UK) without shoes and wearing light clothes, to the nearest 0.1 kg; the average value of the two measurements was used. 2.2. Statistics The trial data were entered into a bespoke web-based electronic database using the Microsoft.Net development environment. The analyses were performed in SPSS22. The trial data were analyzed according to the intention-to-treat principle. Two-sided p < 0.05 was taken as signiﬁcant. The discrete variables were described as crude %, with comparisons adjusted for sites using logistic regression, and were reported with 95% conﬁdence intervals. The continuous variables were compared using generalized linear modelling, again adjusting for sites, with the mean ± SEM shown. The comparisons were also adjusted for the other covariates, as described. The following two analyses were undertaken in this post hoc study: The ﬁrst was to compare the characteristics of the women above and below the median GWG at each gestation. This comparison was undertaken rather than above and below the lower limit of the Institute of Medicine (IOM) [11] targets so as to maximize the numbers in both groups, and only a minority of women achieved the 5 kg limitation. The second analysis used the RCT framework to compare the impact of a substantial GWG limitation on metabolic the and obstetric outcomes. A substantial GWG limitation was deﬁned as >50% higher than the mean DALI Lifestyle Trial GWG difference between the UC and intervention groups (i.e., >3 kg difference). This was only achieved with the HE and PA intervention in ﬁve sites, and hence the post hoc comparison was only undertaken between UC, and HE and PA in these ﬁve sites. 3. Results The gestational age on entry ranged from 8 to 19+6 weeks. The median GWG between pre-pregnancy and 24–28 weeks was 5.5 (IQR 2.3–9.1) kg, and at 35–37 weeks it was 9.5 (IQR 5.3–14.3) kg. Table 1 shows the baseline data for those above and below the median GWG at baseline, 24–28 weeks overall, and at 35–37 weeks, excluding those developing GDM by 24–28 weeks gestation. The women with the greatest GWG commenced at a lower weight and body mass index (BMI), and were more likely to be smokers and nulliparous (35–37 weeks group only). Those with the greatest GWG at 24–28 5 of 13 Nutrients 2018, 10, 1568 weeks had a lower fasting glucose, while those with the greatest GWG at 35–37 weeks (excluding those with GDM by 24–28 weeks) had lower fasting, and 1 h and 2 h glucose concentrations at baseline. Table 2 shows that the HOMA-IR, Stumvoll phase 1, and Stumvoll phase 2 were signiﬁcantly higher among those with the greatest GWG by 24–28 weeks and 35–37 weeks. HOMA-IS was also statistically higher in those with the greatest GWG at 24–28 weeks. The fasting glucose was higher in those with the greatest GWG at 24–28 weeks and 35–37 weeks. The women with the greatest GWG by 35–37 weeks (but not by 24–28 weeks) had signiﬁcantly larger babies. There were no other signiﬁcant obstetric differences. There were no differences in the gender of the babies between groups by GWG. Nutrients 2018, 10, x FOR PEER REVIEW 5 of 12 those with the greatest GWG at 24–28 weeks and 35–37 weeks. The women with the greatest GWG by 35–37 weeks (but not by 24–28 weeks) had significantly larger babies. There were no other significant obstetric differences. There were no differences in the gender of the babies between groups by GWG. Figure 1 shows the ﬂow of the participants in the ﬁve sites with a high GWG difference throughout the post hoc analysis. The numbers that were randomized were comparable. Table 3 compares the maternal characteristics and the pregnancy outcome measures between usual care (UC), and HE and PA. The women in the HE and PA group had 2.6 kg less GWG at 24–28 weeks and 4.3 kg at 35–37 weeks compared with the UC group. The glucose levels, measures of insulin resistance and secretion, and GDM rates were not signiﬁcantly different. 3. Results The combined lifestyle intervention was associated with a signiﬁcantly lower large for gestational age (LGA) rate. Figure 1 shows the flow of the participants in the five sites with a high GWG difference throughout the post hoc analysis. The numbers that were randomized were comparable. Table 3 compares the maternal characteristics and the pregnancy outcome measures between usual care (UC), and HE and PA. The women in the HE and PA group had 2.6 kg less GWG at 24–28 weeks and 4.3 kg at 35–37 weeks compared with the UC group. The glucose levels, measures of insulin resistance and secretion, and GDM rates were not significantly different. The combined lifestyle intervention was associated with a significantly lower large for gestational age (LGA) rate. Figure 1. Recruitment flowchart for five sites achieving the greatest gestational weight gain limitation. Figure 1. Recruitment ﬂowchart for ﬁve sites achieving the greatest gestational weight gain limitation. Figure 1. Recruitment flowchart for five sites achieving the greatest gestational weight gain limitation. Figure 1. Recruitment ﬂowchart for ﬁve sites achieving the greatest gestational weight gain limitation. 6 of 13 6 of 13 Nutrients 2018, 10, 1568 Table 1. Maternal baseline characteristics in the Vitamin D and Lifestyle Intervention for Gestational Diabetes Mellitus (GDM) Prevention (DALI) participants, above and below median gestational weight gain from pre-pregnancy to baseline, to 24–28 weeks, and to 35–37 weeks gestation. 3. Results The latter excludes women with GDM at 24 28 weeks Gestational Weight Gain Group <1.80 kg at Baseline ≥1.80 kg at Baseline <5.65 kg to 24–28 Weeks ≥5.65 kg to 24–28 Weeks <9.5 kg to 35–37 Weeks † ≥9.5 kg to 35–37 Weeks † n = 215 n = 216 n = 203 n = 201 n = 158 n = 159 Age (years) 31.5 ± 5.3 32.4 ± 5.4 32.0 ± 5.2 32.1 ± 5.4 32.7 ± 5.1 31.7±5.4 Pre-pregnancy weight (kg) 94.1 ± 14.0 91.4 ± 12.0 * 94.9 ± 14.0 90.5 ± 11.9 * 94.7 ± 13.9 90.8 ± 11.4 Pre-pregnancy BMI (kg/m2) 34.2 ± 4.2 33.3 ± 3.6 34.6 ± 4.2 33.0 ± 3.5 * 34.4 ± 4.1 33.0 ± 3.5 ** European descent 184/215 (86%) 189/216 (88%) 177/203 (87%) 176/201 (88%) 137/158 (87%) 142/159 (89%) Nullipara 109/215 (51%) 106/216 (49%) 100/203 (49%) 102/201 (51%) 69/158 (44%) 84/159 (53%) * Smokers 26/215 (12%) 41/216 (19%) 19/203 (9%) 42/201 (21%) ** 14/158 (9%) 29/159 (18%) * First degree relative with diabetes 52/215 (24%) 48/216 (22%) 48/2103 (24%) 41/201 (20%) 33/158 (21%) 38/159 (24%) Previous GDM 7/136 (5%) 10/137 (7%) 8/133 (6%) 5/120 (4%) 4/112 (4%) 6/91(7%) Fasting glucose (mmol/L) 4.6 ± 0.4 4.6 ± 0.4 4.7 ± 0.4 4.6 ± 0.4 4.7 ± 0.3 4.6 ± 0.4 1-h glucose (mmol/L) 6.9 ± 1.4 6.7 ± 1.3 6.9 ± 1.4 6.7 ± 1.4 6.9 ± 1.3 6.5 ± 1.3 2-h glucose (mmol/L) 5.9 ± 1.2 5.8 ± 1.0 5.9 ± 1.1 5.8 ± 1.1 5.9 ± 1.0 5.7 ± 1.1 * HOMA-IR (IQR) 2.5 (2.0, 3.4) 2.8 (2.0, 3.7) 2.7 (2.0, 3.6) 2.6 (2.0, 2.4) 2.7 (2.1, 3.4) 2.5 (1.9, 3.4) HOMA insulin secretion (IQR) 217 (170, 312) 256 (180, 363) 220 (166, 324) 245 (181, 346) 215 (158, 295) 251 (180, 358) Stumvoll phase 1 (IQR) 1590 (1221, 2067) 1521 (1255, 2045) 1560 (1219, 2083) 1502 (1258, 2016) 1595 (1224, 2099) 1507 (1261, 2042) Stumvoll phase 2 (IQR) 409 (318, 532) 388 (327, 520) 408 (319, 533) 386 (326, 519) 410 (319, 534) 388 (327, 519) For continuous outcomes, the differences between the groups were tested using multilevel regression models (country and individual as levels), and were adjusted for gestational age at the outcome measurement. 3. Results For the dichotomous outcomes, logistic regression models were performed, and were adjusted for country and gestational age at the outcome measurement. * p < 0.05; p < 0.01; * p < 0.001. † Excluding women with GDM at 24–28 weeks, based on local glucose values. BMI—body mass index; HOMA-IR—homeostasis model assessment insulin resistance. Numbers in bold highlight statistically signiﬁcant comparisons. For continuous outcomes, the differences between the groups were tested using multilevel regression models (country and individual as levels), and were adjusted for gestational age at the outcome measurement. For the dichotomous outcomes, logistic regression models were performed, and were adjusted for country and gestational age at the outcome measurement. * p < 0.05; p < 0.01; * p < 0.001. † Excluding women with GDM at 24–28 weeks, based on local glucose values. BMI—body mass index; HOMA-IR—homeostasis model assessment insulin resistance. Numbers in bold highlight statistically signiﬁcant comparisons. 7 of 13 Nutrients 2018, 10, 1568 Table 2. Metabolic status at 24–28 weeks, 35–37 weeks, and birth outcomes in DALI participants, according to gestational weight gain from pre-pregnancy. 3. Results Gestational Weight Gain Group <1.80 kg at Baseline ≥1.80 kg at Baseline <5.65 kg to 24–28 Weeks ≥5.65 kg to 24–28 Weeks <9.5 kg to 35–37 Weeks † ≥9.5 kg to 35–37 Weeks † n = 215 n = 216 n = 203 n = 201 n = 156 n = 158 24–28 weeks Weight (kg) 96.5 ± 12.4 100.7 ± 12.8 ** 96.7±12.7 100.4±12.7* 96.7 ± 12.7 100.2 ± 12.6 * Gestational weight gain (kg) # 2.3 ± 3.9 9.3 ± 4.3 * 1.7±3.3 9.9±3.8* 2.0 ± 3.5 9.4 ± 4.3 *** Fasting blood glucose (BG) (mmol/l) # 4.62 ± 0.40 4.66 ± 0.42 * 4.61±0.40 4.66±0.43** 4.58 ± 0.36 4.57 ± 0.37 * 1-h glucose (mmol/l) # 7.73 ± 1.58 7.81 ± 1.66 7.74±1.59 7.83±1.68 7.52 ± 1.29 7.39 ± 1.49 2-h glucose (mmol/l) # 6.34 ± 1.23 6.21 ± 1.24 6.33±1.26 6.22±1.19 6.18 ± 1.02 5.98 ± 1.13 HOMA-IR # 2.88 (2.17, 3.83) 3.13 (2.39, 4.45) * 2.80 (2.07, 3.76) 3.15 (2.46, 4.47) * 2.84 (2.17, 3.69) 3.03 (2.26, 4.29) * HOMA insulin secretion # 256 (190, 366) 285 (200, 393) 264 (189, 345) 283 (204, 407) * 269 (193, 343) 300 (208, 420) Stumvoll phase 1 # 1757 (1292, 2308) 1888 (1404, 2347) * 1675 (1276, 2256) 1919 (1403, 2378) * 1612 (1287, 2255) 1929 (1494, 2366) * Stumvoll phase 2 # 454 (337, 588) 484 (365, 598) * 433 (335, 576) 496 (368, 607) * 418 (336, 576) 495 (380, 604) * 35–37 weeks Weight (kg)† 100.3 ± 12.3 104.9 ± 13.0 100.1 ± 12.6 105.2 ± 12.8 99.4 ± 12.8 105.7 ± 12.3 * Gestational weight gain (kg) #† 6.2 ± 5.3 13.4 ± 5.7 * 5.4 ± 4.5 14.5 ± 4.9 * 4.7 ± 3.7 14.9 ± 4.4 * Fasting BG (mmol/l) #$ 4.57 ± 0.45 4.60 ± 0.51 4.55 ± 0.45 4.61 ± 0.51 * 4.49 ± 0.43 4.53 ± 0.43 ** 1-h glucose (mmol/l) #$ 8.20 ± 1.61 8.46 ± 1.57 8.21 ± 1.54 8.45 ± 1.67 * 8.01 ± 1.37 8.17 ± 1.47 * 2-h glucose (mmol/l) #$ 6.74 ± 1.28 6.58 ± 1.21 6.82 ± 1.31 6.51 ± 1.14 * 6.63 ± 1.21 6.43 ± 1.09 HOMA-IR #$ 3.11 (2.35, 4.46) 3.38 (2.55, 4.60) 2.96 (2,25, 4.32) 3.45 (2.62, 4.76) 2.66 (2.21, 4.04) 3.47 (2.70, 4.74) * HOMA insulin secretion #$ 345 (234, 565) 354 (250, 483) 346 (227, 478) 351 (252, 531) 324 (217, 445) 367 (256, 531) Stumvoll phase 1 #$ 2469 (1722, 3174) 2518 (1898, 3117) 2403 (1723, 3124) 2561 (1917, 3200) 2383 (1732, 3026) 2684 (2162, 3354) * Stumvoll phase 2 #$ 629 (441, 801) 639 (497, 786) 618 (450, 790) 652 (497, 810) 608 (452, 766) 684 (554, 841) * Birth N = 198 N = 195 N = 194 N = 194 N = 154 N = 158 Gestation at birth (weeks) 39.5 ± 2.6 39.6 ± 1.7 39.8 ± 1.6 39.5 ± 1.7 39.8 ± 1.4 39.8 ± 1.3 Gender (male) 102/198 (52%) 94/195 (48%) 101/194 (52%) 94/194 (49%) 84/154 (55%) 74/158 (47%) Birthweight 3490 ± 538 3479 ± 557 3457 ± 541 3505 ± 551 3477 ± 503 3602 ± 515 Birthweight ≥4 (kg) 37/195 (19%) 34/195 (17%) 33/192 (17%) 35/193 (18%) 25/153 (16%) 39/157 (25%) * Birthweight <2.5 (kg) 8/195 (4%) 8/195 (4%) 8/192 (4%) 8/193 (4%) 5/153 (3%) 2/157 (1%) Large for Gestational Age 26/187 (14%) 25/186 (13%) 23/186 (12%) 26/182 (14%) 13/150 (9%) 31/150 (21%) ** Small for Gestational Age 12/186 (7%) 16/186 (9%) 15/186 (8%) 13/182 (7%) 10/150 (7%) 9/150 (6%) Preterm birth 8/194 (4%) 14/195 (7%) 8/192 (4%) 14/192 (7%) 2/153 (1%) 4/157 (3%) Induction of labor or planned caesarean section 70/188 (37%) 82/187 (44%) 76/186 (41%) 75/184 (41%) 61/151 40% 60/148 41% Caesarean section 51/190 (27%) 73/190 (38%) * 58/188 (31%) 65/187 (35%) 45/153 (29%) 53/151 (35%) Pre-eclampsia 4/181 (2%) 10/187 (5%) 7/181 (4%) 8/183 (4%) 4/147 (3%) 5/148 (3%) Neonatal Intensive Care Unit admission 19/170 (11%) 18/185 (10%) 19/173 (11%) 19/179 (11%) 14/145 (10%) 14/144 (10%) GDM total 71/183 (39%) 61/175 (35%) 68/185 (37%) 63/175 (36%) 40/153 (26%) 45/155 (29%) For the continuous outcomes, the differences between the groups were tested with multilevel regression models (country and individual as levels), and adjusted for the gestational age at the outcome measurement. 3. Results For the dichotomous outcomes, logistic regression models were performed, and were adjusted for country and gestational age at outcome measurement. The data are adjusted for the pre-pregnancy BMI and fasting glucose at baseline. * p < 0.05; p < 0.01; * p < 0.001. #—regression models were additionally adjusted either for value at baseline or for pre-pregnancy BMI when GWG was the outcome. $—value of 24–28 weeks carried forward to 35–37 weeks when GDM was diagnosed at 24–28 weeks. †—excluding women with GDM at 24–28 weeks, based on local glucose values. Numbers in bold highlight statistically signiﬁcant comparisons. eeks, and birth outcomes in DALI participants, according to gestational weight gain from pre-pregnancy. For the continuous outcomes, the differences between the groups were tested with multilevel regression models (country and individual as levels), and adjusted for the gestational age at the outcome measurement. For the dichotomous outcomes, logistic regression models were performed, and were adjusted for country and gestational age at outcome measurement. The data are adjusted for the pre-pregnancy BMI and fasting glucose at baseline. * p < 0.05; p < 0.01; * p < 0.001. #—regression models were additionally adjusted either for value at baseline or for pre-pregnancy BMI when GWG was the outcome. $—value of 24–28 weeks carried forward to 35–37 weeks when GDM was diagnosed at 24–28 weeks. †—excluding women with GDM at 24–28 weeks, based on local glucose values. Numbers in bold highlight statistically signiﬁcant comparisons. 8 of 13 Nutrients 2018, 10, 1568 Table 3. Maternal and neonatal characteristics of women at the ﬁve DALI sites with maternal gestational weight gain >3 kg difference at 35–37 weeks between healthy eating (HE), and physical activity intervention (HE and PA) and usual care (UC). 3. Results Baseline UC HE + PA n = 50 n = 53 Age (years) 32.1 ± 6.0 31.9 ± 5.0 Pre-pregnancy weight (kg) 94.7 ± 11.4 94.7 ± 13.6 Pre- pregnancy BMI (kg/m2) 33.6 ± 3.3 34.8 ± 4.1 Fasting (F) BG (mmol/L) 4.69 ± 0.33 4.64 ± 0.36 1-h BG (mmol/L) 6.80 ± 1.36 6.84 ± 1.23 2-h BG (mmol/L) 5.69 ± 1.25 5.72 ± 0.99 HOMA-IR 2.62 (1.94, 4.30) 2.49 (2.19, 2.82) HOMA insulin secretion 229 (142, 365) 215 (168, 313) Stumvoll phase 1 1498 (1211, 2144) 1489 (1236, 1948) Stumvoll phase 2 379 (313, 554) 382 (322, 501) European descent 45/50 90.0% 47/53 88.7% Nullipara 27/50 46.2% 27/53 49.1% Smokers 6/50 12.0% 5/53 9.4% First degree relative with diabetes 13/50 26.0% 6/53 11.3% 24–28 weeks n = 50 n = 53 Weight gain from pre-pregnancy 6.7 ± 5.9 (n = 49) 4.1 ± 5.0 (n = 46) * Fasting Blood Glucose (BG) 4.59 ± 0.40 4.57 ± 0.43 1-h BG 7.86 ± 1.64 7.93 ± 1.60 2-h BG 6.23 ± 1.31 6.09 ± 1.20 HOMA-Insulin Resistance 2.86 (2.18, 3.82) 2.54 (2.16, 3.02) HOMA-Insulin secretion 284 (203, 394) 249 (198, 352) Stumvoll phase 1 1762 (1386, 2371) 1943 (1311, 2327) Stumvoll phase 2 455 (359, 606) 501 (346, 594) GDM at 24–28 weeks 9/49 (18.4%) 9/45 (20.0%) 35–37 weeks N = 46 N = 42 Weight gain from pre-pregnancy † 11.3 ± 6.7 (n = 40) 7.0 ± 6.0 (n = 36) * FBG & 4.53 ± 0.46 4.49 ± 0.51 1-h BG & 8.57 ± 1.31 8.32 ± 1.44 2-h BG & 6.70 ± 1.23 6.59 ± 1.01 HOMA-Insulin Resistance & 2.89 (2.07, 4.44) 2.56 (2.28, 3.84) HOMA insulin secretion & 309 (233, 504) 337 (242, 411) Stumvoll phase 1 & 2644 (1793, 3179) 2577 (2001, 3189) Stumvoll phase 2 & 674 (457, 802) 654 (512, 811) GDM at 35–37 weeks 9/39(23.1%) 5/36 (13.9%) Birth outcomes N = 45 N = 47 Gestational age at birth 39.8 ± 1.5 39.8 ±1.2 LGA 7/42 16.7% 2/44 4.5% * SGA 1/42 2.4% 4/44 9.1% Birthweight 3588 ± 524 3455 ± 463 Preterm birth 2/44 4.5% 0/45 0% Caesarean section 14/43 30.2% 14/47 29.8% Pre-eclampsia 4/42 9.5% 2/43 4.7% Birthweight ≥4 kg 9/45 20.0% 7/45 15.6% Birthweight <2.5 kg 1/45 2.2% 1/45 2.2% NICU admission 7/41 17.1% 6/44 13.6% GDM total 15/46 32.6% 13/44 29.5% in bold highlight statistically signiﬁcant comparisons. 4. Discussion We have shown that GWG above the median is indeed associated with increased fasting glucose, insulin resistance, and insulin secretion. However, there was no increased risk of GDM. In a post hoc analysis in the ﬁve sites with an average 4.3 kg GWG reduction in the HE and PA intervention by 35–37 weeks, there were similar rates of GDM and no difference in the glycaemia, HOMA-IR, or measures of insulin secretion. A GWG above the median in the ﬁrst and third trimesters was also associated with more caesarean sections and greater macrosomia/LGA, respectively. The HE and PA intervention with its associated GWG limitation showed a signiﬁcant reduction in LGA—something not shown in previous meta-analyses [4,5] with their lower GWG limitation. No other large study has achieved this degree of GWG limitation [3]. We believe that this post hoc study clearly demonstrates that GDM cannot be prevented in overweight/obese women with lifestyle intervention initiated in the second trimester, even with substantial GWG limitation. Previous studies have failed to conclusively demonstrate this lack of effect, because of the limited effect of their lifestyle interventions on GWG [4]. There are two key questions that arise, as follows: ﬁrstly, why is it that there was no effect on the risk of GDM, and, secondly, what other strategies might be effective? y g g There are several theories behind why lifestyle change, even when resulting in a signiﬁcant GWG limitation, is insufﬁcient to prevent GDM in overweight/obese women. The ﬁrst is that there is simply insufﬁcient time between commencing the intervention and the OGTT at 24 weeks of gestation for the intervention to be effective. Certainly, some subjects in the DALI study only experienced 4–8 weeks of the intervention. A further possibility is that the reduction in insulin resistance achievable with lifestyle is insufﬁcient. While the mean insulin resistance was reduced by 10–15%, this was not statistically signiﬁcant and was unlikely to be physiologically meaningful, given the fact that the overall increase in insulin resistance during pregnancy is two- to three-fold [16]. A parallel situation occurs with the use of metformin for the prevention of GDM. 3. Results The HE and PA comparison alone is reporte ion group used for selecting the sites, and the intervention with a signiﬁcant effect on GWG lim s only one woman with previous GDM in the HE and PA group. All: mean ± SD or n (%). The dif he groups at baseline were tested using T-test or Mann–Whitney U-test, as appropriate. For the con , the differences between groups at 24–28 and 35–37 weeks were tested in mixed models, adju alues, and for the outcome weight gain adjusted for pre-pregnancy BMI. For the dichotomous ou regression was performed, and was adjusted for country. * p < 0.05 ** p < 0.01 vs. UC. &—values 3) carried forward to 35–37 weeks (T4) when GDM was present at T3. &–excluding women wit Numbers in bold highlight statistically signiﬁcant comparisons. The HE and PA comparison alone is reporte i i d f l i h i d h i i i h i iﬁ ff GWG li Numbers in bold highlight statistically signiﬁcant comparisons. The HE and PA comparison alone is reported, as the intervention group used for selecting the sites, and the intervention with a signiﬁcant effect on GWG limitation. There was only one woman with previous GDM in the HE and PA group. All: mean ± SD or n (%). The differences between the groups at baseline were tested using T-test or Mann–Whitney U-test, as appropriate. For the continuous outcomes, the differences between groups at 24–28 and 35–37 weeks were tested in mixed models, adjusted for baseline values, and for the outcome weight gain adjusted for pre-pregnancy BMI. For the dichotomous outcomes, a logistic regression was performed, and was adjusted for country. * p < 0.05 ** p < 0.01 vs. UC. &—values of 24–28 weeks (T3) carried forward to 35–37 weeks (T4) when GDM was present at T3. &–excluding women with GDM at T3. Nutrients 2018, 10, 1568 9 of 13 4. Discussion However, the mechanism for such a trajectory setting is unclear. increased risk of GDM, but a similar relationship between GWG and GDM risk was not found in the second trimester. However, the mechanism for such a trajectory setting is unclear. If the trajectory is set in the ﬁrst trimester, then there are two possible approaches to prevent GDM through standard (i.e., through some non-tested strategy) lifestyle changes among obese women. The ﬁrst is for interventions to commence early in the ﬁrst trimester, as suggested by Song [6]. However, for many women, the ﬁrst contact with the health service is near the end of (or after) the ﬁrst trimester, and an intervention may be challenging to commence. Nevertheless, this is certainly an aspect that warrants an RCT commencing between, for example, 4–8 weeks’ gestation. There is no evidence that such an RCT would increase spontaneous miscarriage. This may either involve a community wide intervention or personalized strategies, and may be before the ﬁrst pregnancy or between pregnancies. One possible recommendation would be for obese women to plan their pregnancies in a comparable manner to women with pre-existing diabetes. We have also shown that a substantial GWG limitation (mean 4.3 kg vs. usual care) is associated with reduced rates of LGA. This was shown in both the comparison of GWG above and below the median at 35–37 weeks, and with the RCT of usual care vs. the combined HE and PA intervention. Few studies have shown such an effect in pregnancy. The LIMIT RCT found a reduction after a post hoc analysis [27], but overall no beneﬁcial effect has been shown [5]. The post hoc analysis undertaken here suggests that lifestyle interventions can reduce LGA rates, but that the degree of GWG reduction needs to be sufﬁcient. There was a non-signiﬁcantly higher rate of small for gestational age (SGA), and larger RCTs are needed to assess whether this degree of GWG limitation can restrict growth excessively. This study has a number of strengths. It is one of the larger RCTs on lifestyle to prevent GDM, and it compared three different interventions. DALI was across nine European countries, with the participants encompassing a range of lifestyles and cultures, making it more widely representative than studies within a single site or country. The intervention was clearly effective, and being within site randomization allowed for the post hoc analysis. 4. Discussion While metformin prevents type 2 diabetes over time [17], no reduction in the development of GDM was seen in either the Effect of Metformin on Maternal and Fetal Outcomes in Obese Pregnant Women (EMPOWaR) or in the Metformin in Obese Non-Diabetic Pregnant Women (MOP) RCTs of metformin for the prevention of GDM [18,19], although MOP did show a reduction in insulin resistance [20]. However, one would expect that the intervention would have been effective in reducing GDM in a proportion of women, but this was not seen. The relationship between insulin resistance and insulin secretion follows a clear hyperbola [21], and as long as the insulin secretory capacity is sufﬁcient, the glucose homeostasis would be predicted to remain steady, and prevent the development of GDM. An increase in insulin resistance was shown in our comparison of women with GWG above and below the median, and insulin secretion was also greater, suggesting a compensatory increase. In women with previous GDM, the development of type 2 diabetes outside of pregnancy was prevented with troglitazone, a thiazolidinedione that also reduces insulin resistance [22]. The proposed mechanism was that the reduced insulin resistance reduced the insulin secretion, thereby preventing/delaying the onset of beta cell exhaustion, and the drop off from the set insulin resistance–insulin secretion hyperbola. While an estimated increase in insulin sensitivity of 88% was seen within three months [23], prevention occurred over a median of 30 months, much longer than the time available during pregnancy, and without the need to adapt to rapidly changing insulin requirements. One possibility is that the trajectory of insulin secretion by the beta cell is already established by the second trimester (i.e., some form of “programming” has occurred in the ﬁrst trimester) [24]. The possibility of maternal metabolic trajectory setting in the ﬁrst trimester as an explanation for the failure to prevent GDM in DALI is supported by the recent meta-analysis by Song [6], and another recent study introducing physical activity in the ﬁrst trimester, which resulted in a substantial reduction in GDM [25]. The possibility of trajectory setting is also supported by the study by Hedderson et al. [26], where women with GWG in the highest tertile in the ﬁrst trimester had an Nutrients 2018, 10, 1568 10 of 13 increased risk of GDM, but a similar relationship between GWG and GDM risk was not found in the second trimester. 4. Discussion On the other hand, there are some shortcomings. In retrospect, the number of patients successfully recruited was sub-optimal to answer deﬁnitively important questions regarding a reduction in fasting glucose with lifestyle change and its relationship to the development of GDM. Nevertheless, the sample size provided 82% power to detect a 0.2 mmol/L difference in the fasting glucose, representing a clinically meaningful difference in the context of pregnancy. This is a post hoc analysis, with all of the statistical limitations that result from such an analysis. On the other hand, achieving GWG reduction goals is remarkably difﬁcult, and to only select sites with an achieved GWG above the median is a novel way to test this within an RCT framework. We have not compared above and below the IOM lower target (5 kg), as only a limited number of women achieved this goal. Women coming into an RCT are more motivated than most women, and some women in the control arm appeared to be motivated to improve their lifestyle as a result of participation, thus reducing the differences between the intervention and control group in some sites. A further issue is the exclusion of women with GDM at baseline. DALI is one of the few to include this design feature, and while this allows a test of the impact of GWG limitation on the incidence of GDM, those with hyperglycemia at baseline may have had greater beneﬁt from the intervention [28,29]. References 1. Simmons, D. Diabetes and obesity in pregnancy. Best Pract. Res. Clin. Obstet. Gynaecol. 2011, 25, 25–36. [CrossRef] [PubMed] 1. Simmons, D. Diabetes and obesity in pregnancy. Best Pract. Res. Clin. Obstet. Gynaecol. 2011, 25, 25–36. [CrossRef] [PubMed] 2. Simmons, D. Prevention of gestational diabetes mellitus: Where are we now? Diabetes Obes. Metab. 2015, 17, 824–834. [CrossRef] [PubMed] 2. Simmons, D. Prevention of gestational diabetes mellitus: Where are we now? Diabetes Obes. Metab. 2015, 17, 824–834. [CrossRef] [PubMed] 3. Oteng-Ntim, E.; Varma, R.; Croker, H.; Poston, L.; Doyle, P. Lifestyle interventions for overweight and obese pregnant women to improve pregnancy outcome: Systematic review and meta-analysis. BMC Med. 2012, 10. [CrossRef] [PubMed] 3. Oteng-Ntim, E.; Varma, R.; Croker, H.; Poston, L.; Doyle, P. Lifestyle interventions for overweight and obese pregnant women to improve pregnancy outcome: Systematic review and meta-analysis. BMC Med. 2012, 10. [CrossRef] [PubMed] 4. The International Weight Management in Pregnancy (i-WIP) Collaborative Group. Effect of diet and physical activity based interventions in pregnancy on gestational weight gain and pregnancy outcomes: Meta-analysis of individual participant data from randomised trials. BMJ 2017, 358. [CrossRef] 4. The International Weight Management in Pregnancy (i-WIP) Collaborative Group. Effect of diet and physical activity based interventions in pregnancy on gestational weight gain and pregnancy outcomes: Meta-analysis of individual participant data from randomised trials. BMJ 2017, 358. [CrossRef] 5. Shepherd, E.; Gomersall, J.C.; Tieu, J.; Han, S.; Crowther, C.A.; Middleton, P. Combined diet and exercise interventions for preventing gestational diabetes mellitus. Cochrane Database Syst. Rev. 2017, 11. [CrossRef] [PubMed] 5. Shepherd, E.; Gomersall, J.C.; Tieu, J.; Han, S.; Crowther, C.A.; Middleton, P. Combined diet and exercise interventions for preventing gestational diabetes mellitus. Cochrane Database Syst. Rev. 2017, 11. [CrossRef] [PubMed] Song, C.; Li, J.; Leng, J.; Ma, R.C.; Yang, X. Lifestyle intervention can reduce the risk of gestational diabetes A meta-analysis of randomized controlled trials. Obes. Rev. 2016, 17, 960–969. [CrossRef] [PubMed] 7. Koivusalo, S.B.; Rönö, K.; Klemetti, M.M.; Roine, R.P.; Lindström, J.; Erkkola, M.; Kaaja, R.J.; Pöyhönen-Alho, M.; Tiitinen, A.; Huvinen, E.; et al. Gestational diabetes mellitus can be prevented by lifestyle intervention: The Finnish Gestational Diabetes Prevention Study (RADIEL): A randomized controlled trial. Diabetes Care 2016, 39, 24–30. [CrossRef] [PubMed] 8. Jelsma, J.G.; van Poppel, M.N.; Galjaard, S.; Desoye, G.; Corcoy, R.; Devlieger, R.; van Assche, A.; Timmerman, D.; Jans, G.; Harreiter, J.; et al. 5. Conclusions In conclusion, we have shown in a lifestyle intervention study RCT cohort that GWG is associated with an increase in insulin resistance and glycaemia and some worse obstetric outcomes, however, a post hoc analysis of RCT sites with the greatest GWG limitation showed no reduction in GDM or its risk, but a reduction in LGA. We conclude that lifestyle intervention in the second trimester is too late for GDM prevention, and more RCTs are needed in the ﬁrst trimester, which includes sub-studies, so as to understand the mechanisms behind a putative “locking in” of the metabolic trajectory. 11 of 13 Nutrients 2018, 10, 1568 11 of 13 Nutrients 2018, 10, 1568 Author Contributions: All of the authors contributed to the conception and/or design of the trial, read and corrected draft versions of the manuscript, and approved the ﬁnal manuscript. D.S. wrote the ﬁrst draft of the paper and M.N.M. undertook statistical analyses. D.S. is Guarantor. Funding: The project described has received funding from the European Community’s 7th Framework Programme (FP7/2007–2013) under grant agreement no. 242187. In the Netherlands, additional funding was provided by the Netherlands Organization for Health Research and Development (ZonMw) (grant no. 200310013). In Poland, additional funding was obtained from Polish Ministry of Science (grant no. 2203/7. PR/2011/2). In Denmark, additional funding was provided by the Odense University Free Research Fund. In the United Kingdom, the DALI team acknowledge the support received from the NIHR Clinical Research Network: Eastern, especially the local diabetes clinical and research teams based in Cambridge. In Spain, additional funding was provided by CAIBER 1527-B-226. The funders had no role in any aspect of the study beyond funding. Acknowledgments: We thank the participants, coaches, research midwives/nurses, and health professio ollaborating in the recruitment. Conﬂicts of Interest: The authors declare no conﬂicts of interest. All of the authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare no support from any organization for the submitted work beyond that below; no ﬁnancial relationships with any organizations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have inﬂuenced the submitted work. References DALI: Vitamin D and lifestyle intervention for gestational diabetes mellitus (GDM) prevention: An European multicentre, randomised trial–study protocol. BMC Pregnancy Childbirth 2013, 13. [CrossRef] [PubMed] 9. Simmons, D.; Jelsma, J.G.; Galjaard, S.; Devlieger, R.; van Assche, A.; Jans, G.; Corcoy, R.; Adelantado, J.M.; Dunne, F.; Desoye, G.; et al. Results from a european multicenter randomized trial of physical activity and/or healthy eating to reduce the risk of gestational diabetes mellitus: The DALI Lifestyle Pilot. Diabetes Care 2015, 38, 1650–1656. [CrossRef] [PubMed] 10. Simmons, D.; Devlieger, R.; van Assche, A.; Jans, G.; Galjaard, S.; Corcoy, R.; Adelantado, J.M.; Dunne, F.; Desoye, G.; Harreiter, J.; et al. Effect of physical activity and/or healthy eating on GDM risk: The DALI Lifestyle Study. J. Clin. Endocrinol. Metab. 2017, 102, 903–913. [CrossRef] [PubMed] 11. Institute of Medicine. Weight gain during pregnancy: Reexamining the guidelines. In Institute of Medicine (US) and National Research Council (US) and Committee to Reexamine IOM Pregnancy Weight Guidelines; National Academies Press (US): Washington, DC, USA, 2009. 12 of 13 Nutrients 2018, 10, 1568 12. World Health Organization. Diagnostic Criteria and Classiﬁcation of Hyperglycaemia First Detected in Pregnancy; WHO/NMH/MND/13.2; World Health Organization: Geneva, Switzerland, 2013. 13. Matthews, D.R.; Hosker, J.P.; Rudenski, A.S.; Naylor, B.A.; Treacher, D.F.; Turner, R.C. Homeostasis model assessment: Insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985, 28, 412–419. [CrossRef] [PubMed] 14. Stumvoll, M.; Mitrakou, A.; Pimenta, W.; Jenssen, T.; Yki-Jarvinen, H.; Van Haeften, T.; Renn, W.; Gerich, J. Use of oral glucose tolerance test to assess insulin release and insulin sensitivity. Diabetes Care 2000, 23, 295–301. [CrossRef] [PubMed] 15. Stumvoll, M.; Van Haeften, T.; Fritsche, A.; Gerich, J. Oral glucose tolerance test indexes for insulin sensitivity and secretion based on various availabilities of sampling times. Diabetes Care 2001, 24, 796–797. [CrossRef] [PubMed] 16. Catalano, P.M.; Tyzbir, E.D.; Roman, N.M.; Amini, S.B.; Sims, E.A.H. Longitudinal changes in insulin release and insulin resistance in nonobese pregnant women. Am. J. Obstet. Gynecol. 1991, 165, 1667–1672. [CrossRef] 17. Ratner, R.E.; Christophi, C.A.; Metzger, B.E.; Dabelea, D.; Bennett, P.H.; Pi-Sunyer, X.; Fowler, S.; Kahn, S.E. The Diabetes Prevention Program Research Groupa. Prevention of diabetes in women with a history of gestational diabetes: Effects of metformin and lifestyle interventions. J. Clin. Endocrinol. Metab. 2008, 93, 4774–4779. [CrossRef] [PubMed] 18. References Chiswick, C.; Reynolds, R.M.; Denison, F.; Drake, A.J.; Forbes, S.; Newby, D.E.; Walker, B.R.; Quenby, S.; Wray, S.; Weeks, A.; et al. Effect of metformin on maternal and fetal outcomes in obese pregnant women (EMPOWaR): A randomised, double-blind, placebo-controlled trial. Lancet Diabetes Endocrinol. 2015, 3, 778–786. [CrossRef] 19. Syngelaki, A.; Nicolaides, K.H.; Balani, J. Metformin versus placebo in obese pregnant women without diabetes mellitus. N. Engl. J. Med. 2016, 374, 434–443. [CrossRef] [PubMed] 20. Balani, J.; Hyer, S.; Syngelaki, A.; Akolekar, R.; Nicolaides, K.H.; Johnson, A.; Shehata, H. Association between insulin resistance and preeclampsia in obese non-diabetic women receiving metformin. Obstet. Med. 2017, 10, 170–173. [CrossRef] [PubMed] 21. Kahn, S.E.; Prigeon, R.L.; McCulloch, D.K.; Boyko, E.J.; Bergman, R.N.; Schwartz, M.W.; Neiﬁng, J.L.; Ward, W.K.; Beard, J.C.; Palmer, J.P.; et al. Quantiﬁcation of the relationship between insulin sensitivity and -cell function in human subjects: Evidence for a hyperbolic function. Diabetes 1993, 42, 1663–1672. [CrossRef] [PubMed] 22. Buchanan, T.A.; Xiang, A.H.; Peters, R.K.; Kjos, S.L.; Marroquin, A.; Goico, J.; Ochoa, C.; Tan, S.; Berkowitz, K.; Hodis, H.N.; et al. Preservation of pancreatic beta-cell function and prevention of type 2 diabetes by pharmacological treatment of insulin resistance in high-risk hispanic women. Diabetes 2002, 51, 2796–2803. [CrossRef] [PubMed] 23. Berkowitz, K.; Peters, R.; Kjos, S.L.; Goico, J.; Marroquin, A.; Dunn, M.E.; Xiang, A.; Azen, S.; Buchanan, T.A. Effect of troglitazone on insulin sensitivity and pancreatic -cell function in women at high risk for non-insulin-dependent diabetes mellitus. Diabetes 1996, 45, 1572–1579. [CrossRef] [PubMed] 24. Banerjee, R.R. Piecing together the puzzle of pancreatic islet adaptation in pregnancy. Ann. N. Y. Acad. Sci. 2018, 1411, 120–139. [CrossRef] [PubMed] 25. Wang, C.; Wei, Y.; Zhang, X.; Zhang, Y.; Xu, Q.; Sun, Y.; Su, S.; Zhang, L.; Liu, C.; Feng, Y.; et al. A randomized clinical trial of exercise during pregnancy to prevent gestational diabetes mellitus and improve pregnancy outcome in overweight and obese pregnant women. Am. J. Obstet. Gynecol. 2017, 216, 340–351. [CrossRef] [PubMed] 26. Hedderson, M.M.; Gunderson, E.P.; Ferrara, A. Gestational weight gain and risk of gestational diabetes mellitus. Obstet. Gynecol. 2010, 115, 597–604. [CrossRef] [PubMed] 27. Dodd, J.M.; Turnbull, D.; McPhee, A.J.; Deussen, A.R.; Grivell, R.M.; Yelland, L.N.; Crowther, C.A.; Wittert, G.; Owens, J.A.; Robinson, J.S. Antenatal lifestyle advice for women who are overweight or obese: LIMIT randomised trial. BMJ 2014, 348, g1285. [CrossRef] [PubMed] 28. 29. Landon, M.B.; Spong, C.Y.; Thom, E.; Carpenter, M.W.; Ramin, S.M.; Casey, B.; Wapner, R.J.; Varner, M.W.; Rouse, D.J.; Thorp, J.M., Jr.; et al. A multicenter, randomized trial of treatment for mild gestational diabetes. N. Engl. J. Med. 2009, 361, 1339–1348. [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/). References Crowther, C.A.; Hiller, J.E.; Moss, J.R.; McPhee, A.J.; Jeffries, W.S.; Robinson, J.S. Effect of treatment of gestational diabetes mellitus on pregnancy outcomes. N. Engl. J. Med. 2005, 352, 2477–2486. [CrossRef] [PubMed] 13 of 13 Nutrients 2018, 10, 1568 © 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/).
https://openalex.org/W2954154485	http://www.scielo.br/pdf/mr/v22n4/1516-1439-mr-22-04-e20180917.pdf	English	null	Biomimetic Coating of Mechanochemically Synthesized Zirconium Titanate	Materials research	2,019	cc-by	4,865	1. Introduction As mentioned previously, zirconium titanate is a material that has great qualities such as, high performance, good stability at high temperature and high resistance to heat and corrosive environment; and the compound may crystallize in the orthorhombic system, which is related to its electrical properties. The zirconium and titanium ions are distributed randomly occupying octahedral sites 5. The traditional scope of zirconium titanate materials is that of electroceramics, as dielectric resonators due to their high dielectric constant (κ ≈ 35-46), high quality factor (Q ≈ 2000-8000) measured between 1 and 10 GHz and their small variation of the resonance frequency with temperature (τf ≈ 0 ppm ºC-1) 6-15. These properties make the zirconium titanate materials useful in the field of telecommunications as tuners, filters and voltage oscillators tuneable by voltage 6,11. In addition, the dielectric properties of zirconium titanate have been determined in thin films prepared by laser ablation 11. On the other hand, zirconium titanate materials are used in catalytic applications. Zirconium titanate has catalytic activity in some reactions, such as the non-oxidative dehydrogenation of ethylbenzene, which is important in the production of styrene. Likewise, it has been used in a large number of catalytic reactions as catalyst support12-15. In addition, zirconium titanate has photocatalytic activity. Applications of zirconium titanate as a pigment 16 and as a hydrocarbon (methane, propane, toluene and hexane) 13,17 and humidity sensor have also been described 18,19. The biomaterials science has gained great importance because the human being wanted to improve their quality of life. The sophistication of implants and prostheses in recent years has led to the development of new materials with the necessary requirements to biomedical applications. And to gather the required properties in a single material, which have become the greatest challenge of our times 1. And one of these possible materials can be composed of ZrO2 and TiO2, such as zirconium titanate, which we will now talk about its characteristics both electric, and biocompatibility. Zirconium titanate is a material that has the potential for such application, due to its high performance, good stability at high temperature and high resistance to heat and corrosive environment; it is widely used in electronic components, that is, for telecommunication purposes such as resonators, capacitors, etc. 2. Biomimetic Coating of Mechanochemically Synthesized Zirconium Titanate D id R í Z ó * J Al Dí G illé D Ali i C é H á d b S i A biomimetic method was used to promote bioactivity of ZrTiO4. Zirconium titanate was obtained by using a combination of techniques such as mechanical milling and heat treatment of zirconia and titania powders. The effect of milling time on the evolution of the ZrTiO4 phase formation was studied. Powders were uniaxially pressed followed by heat treatment for 6 h at different temperatures within the range between 700 and 1500 ºC. Single phase ZrTiO4 was obtained after ball-milling for 30 h followed heat treating in air at 1500 ºC for 6 h. For the biomimetic coatings were obtained by immersion of the samples in a simulated body fluid (SBF) for 7, 14 and 21 days on a bed of wollastonite powder, and were characterized by SEM, EDS, XRD, FT-IR and TEM. For comparison purposes, experiments were also performed without using the bioactive powder bed. A bone-like apatite layer was formed on zirconium titanate after 21 days of immersion in SBF using a bed of wollastonite powder. Keywords: zirconium titanate, biomaterials, biomimetic method, HA coating, mechanical milling e-mail: drenteria@itsaltillo.edu.mx Materials Research. 2019; 22(4): e20180917 DOI: http://dx.doi.org/10.1590/1980-5373-MR-2018-0917 ; ( ) Biomimetic Coating of Mechanochemically Synthesized Zirconium Titanate David Rentería-Zamarróna , Jose Alonso Díaz-Guilléna, Dora Alicia Cortés-Hernándezb, Sagrario Martinez-Montemayorc, Claudia Magdalena Lopez-Badillod, Antonio Fernandez-Fuentesb Received: December 20, 2018; Revised: April 08, 2019; Accepted: May 19, 2019 aDivisión de Estudios de Posgrado e Investigación, Tecnológico Nacional de México, Instituto Tecnológico de Saltillo, Saltillo, Coahuila, México bDepartamento de Ingeniería Cerámica, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV), Saltillo, Coahuila, México cDepartamento de Materiales Avanzados, Centro de Investigación en Química Aplicada (CIQA), Saltillo, Coahuila, México dFacultad de Ciencias Químicas, Universidad Autónoma de Coahuila - UAdeC, Saltillo, Coahuila, México 2.2 Biomimetic method A Simulated Body Fluid (SBF) with ionic concentration nearly equal to that of human blood plasma (SBF) was used. The solution was prepared according to Kokubo´s recipe 24, dissolving reagent grade sodium chloride (NaCl), sodium hydrogen carbonate (NaHCO3), potassium chloride (KCl), dipotassium hydrogen phosphate (K2HPO4·3H2O), magnesium chloride hexahydrate (MgCl2·6H2O), calcium chloride dihydrate (CaCl2·2H2O) and sodium sulfate (Na2SO4) in deionized water and buffered with tri(hydroxymethyl)-aminomethane and 1N HCl at 36.5 ºC. The substrates were washed with acetone and deionized water, and then immersed in 150 mL of SBF at 36.5 ºC in an incubator, and under static conditions. The biomimetic coatings were obtained by the immersion of samples in SBF for 7, 14 and 21 days with or without the presence of a bed of wollastonite powder, as a calcium ions supplier, these ions are released into the SBF increasing the ionic activity, which generates the formation of apatite on the surface of the materials that contain it. The pills of the material were placed on a wollastonite bed in a container, one face of the pill was on the wollastonite bed, and the other face was exposed to the contact with the fluid. After immersion periods, substrates were removed from the bottles, gently washed with deionized water and dried at room temperature 22, 24. Biomimetic processes have been widely studied for growing a bonelike apatite layer on different substrates by immersion in simulated body fluids. In the case of the wollastonite, calcium ions released into the solution increase the ionic activity product of the apatite in the fluid and the Si-OH groups formed on the surface provide sites for apatite nucleation 22. After the apatite nuclei are formed, they grow spontaneously since the solution is already saturated with respect to apatite. In this case, calcium and silicate ions dissolved from the bioactive material induce apatite nucleation on the materials placed nearby 20,22. The aim of this study is to obtain the ZrTiO4 ceramic material by mechanical milling followed by heat treatment, with the possibility of being used as a biomaterial. And so to face the problem of corrosion in physiological fluids that have certain metals and alloys, when used as implants in the body. The effects of milling time and heat treatment temperature on the evolution of the ZrTiO4 formation are presented. 2.1 Zirconium titanate preparation Single phase ZrTiO4 materials were prepared by mechanical milling. This powder processing method has already been successfully used to prepare zirconate and titanate materials at room temperature 4,23. Stoichiometric mixtures of high- purity (≥ 99%, Sigma Aldrich) TiO2 and ZrO2 reagent grade chemicals were placed into zirconia containers along with 20 mm diameter zirconia balls as grinding media (balls to powder mass ratio = 10:1). Dry mechanical milling was carried out in air in a planetary ball mill using a rotating disc speed of 400 rpm for different milling times (1, 3, 6, 20 and 30 h). Phase evolution on milling was periodically analyzed by X-Ray Diffraction (XRD) in a Philips X'pert diffractometer (0.01º/s) using Ni-filtered CuKα radiation (λ=1.5418 Å) and reactions were considered complete when no traces of the starting reagents were evident by this technique. Pills (10 mm diameter and ~1 mm thickness) were obtained by uniaxial pressing (38 MPa for 20 sec) of the fine powders prepared by mechanical milling. To increase their mechanical strength and obtain dense samples, pills were heat treated at different temperatures 700, 1000 and 1500 ºC for 6 h (heating and cooling rates 2 ºC/min). 1. Introduction Different methods have been employed to prepare zirconium titanate ceramic materials such as solid state reaction method, high energy ball milling, DC magnetron sputtering, co-precipitation, sol-gel and mechanochemical processing 3. Mechanochemical processing has become very popular since it is a simple method to employ, in which there is no need to use solvents and with which sufficient volume of the material to be treated can be obtained in an economically viable manner 4. Rentería-Zamarrón et al. 2 Materials Research On the other hand, zirconia has been used in combination with other oxides such as SiO2 and Al2O3; studies on biological behaviour have shown that zirconia and silica create favourable non-toxic layers with good biocompatibility 20. In addition, ZrO2 exhibits high mechanical strength, high fracture resistance and corrosion, making it a suitable material for implants 21. Zirconium dioxide (ZrO2) is a bioinert ceramic material generally known for its excellent mechanical strength and hardness. Materials based on both ZrO2 and Al2O3 are considered bioinert under physiological conditions and, therefore, in surface modification treatments 21. Due to the excellent biocompatibility of titanium dioxide (TiO2), recent studies have shown the manufacture of a highly porous ceramic material (scaffolding) from TiO2 foams for bone growth and the ability to promote adhesion and proliferation of osteoblasts and mesenchymal cells (hMSC) on the entire surface of the scaffold in in vitro testing 20,21. On the other hand, crystalline TiO2 has shown to have bioactive properties since its surface can support chemical bonds with bone, through the formation of an apatite layer similar to that of bone; however it lacks mechanical strength and hardness of ZrO2. Therefore, combining the favourable osteogenic properties of TiO2 with the excellent mechanical properties of ZrO2 may allow the manufacture of osteoconductive materials with sufficient mechanical strength to withstand the stress to which they will be subjected, while maintaining biocompatibility 20,21. 2.2 Biomimetic method In addition, selected samples were biomimetically treated on views to the potential use of this materials in biomedical applications, such as orthopaedic and dental implants. Biomimetic Coating of Mechanochemically Synthesized Zirconium Titanate Biomimetic Coating of Mechanochemically Synthesized Zirconium Titanate 3 3 The surface of the biomimetically treated substrates was analyzed by X-Ray Diffraction (XRD) (Philips, X’Pert), then substrates were Au-Pd coated and analyzed by Scanning Electron Microscopy (SEM) (Philips, XL-30 ESEM) and Energy Dispersive Spectroscopy (EDS) (Philips, XL-30 ESEM). Microstructural studies were also performed using High-Resolution Transmission Electron Microscopy (HRTEM) (FEI TITAN HRTEM), while Infrared spectroscopy (IR, NICOLET 500) was employed to assess the growth of the phosphate groups (PO4)3-, which are characteristics groups of apatite. The width and low intensity of these reflections is related with the high structural disorder in these materials generated by mechanical activation. This pattern also reveals a slight increase of the reflections intensity with respect to the sample milled for 20 hours, which is practically amorphous. Either the crystallites growth of the formed phase or the increase in their number with milling time may be the cause for this effect. The presence of remnant reflections of starting reagents after 30 hours of milling made necessary a thermal treatment to complete the reaction of this mechanically activated composition.f Figure 2 shows the effect of heat treatment temperature on the structural characteristics of the Ti-Zr materials. Figure 2a shows XRD patterns of ball-milled samples for 30 h followed by heat treatment at different temperatures (700-1000 ºC) for 6 h. As observed, as the heat treatment temperature is increased, an increase in the reflections intensity and a decrease in their width are observed, leading to a material with high crystallinity and crystallite size. It is also clear that patterns of fired samples correspond to the ZrTiO4 phase. Rentería-Zamarrón et al. Materials Research treatments, such as calcium phosphate coatings, are necessary to produce a bioactive material surface which may allow direct contact between the material surface and bone tissue without fibrous encapsulation 20,22,24,25. However, some authors suggest that ZrO2 is bioactive, because the ZrO2 with tetragonal structure has presented apatite formation on its surface, it is also mentioned that in the presence of nanometer-sized particles of ZrO2 it is also possible to make bioactive material; it is believed to be one of the reasons why the ZrO2 is bioactive in comparison with ZrO2 ceramics, which is considered bioinert 26. Figure 3 shows SEM images of the studied composition at different magnifications after 30 h of milling and sintered at 1500 °C for 6 h. It is a well densified microstructure with dispersed grain sizes. The corresponding EDS spectrum confirms the presence of Pd, because the sample was coated with this to be analyzed. Figure 4 shows SEM images of sintered (1500 °C) ZrTiO4 after 7, 14 and 21 days of immersion in SBF without using a wollastonite powder bed. As observed, no microstructural changes are observed, that is, there is no indication of the formation of a Ca, P-rich layer on the surface; this is probably due to the fact that the material is constituted by ZrO2, which is already known as a bioinert material 22,24. The EDS spectra confirm the presence of mainly Zr and Ti. During the immersion of bioinert samples in SBF, with no wollastonite powder bed, the nuclei formed are not able to grow since an excess of Ca and P ions is necessary. Thus, the nuclei formed tend to dissolve again 22,24. Zirconium dioxide (ZrO2) is a biocompatible ceramic material generally known for its excellent mechanical strength and toughness. However, this material is considered bioinert under physiological conditions, and therefore surface modification According to the results presented above, the sample selected for biomimetic treatment was that ball-milled for 30 h and heat treated at 1500 ºC. Figure 5 shows SEM images of ZrTiO4 after 7, 14 and 21 days of immersion in SBF in the presence of a wollastonite powder bed. The EDS spectrum of the sample corresponding to 7 days of immersion (Fig. 5a) demonstrates that the compound formed on the surface contains calcium and silicon due to the presence of the wollastonite powder bed used as a supplier of calcium ions. 3. Results and Discussion The evolution of the ZrTiO4 phase as a function of milling time can be observed in Figure 1, which shows the XRD patterns of samples after different milling times (1, 3, 6, 20 and 30 h). For comparison purposes, the starting mixture is also included. The XRD pattern reported in the International Centre for Diffraction Data, ICDD (PDF 49- 1642) for the fluorite cubic structure of ZrO2, was used as reference. The characteristic reflections of the anatase-TiO2 (PDF 00-078-2486) are also shown. Figure 2. XRD patterns of balled-milled samples for 30 h treated at different temperatures for 6 h, (a) 700-1000 ºC, and (b) 1500 ºC. (PDF 00-078-2486) are also shown. Figure 1. XRD patterns of samples after different milling times. Figure 1. XRD patterns of samples after different milling times. Fi 1 XRD f l f diff illi i Figure 1. XRD patterns of samples after different milling times. An important decreasing in intensity and broadening of the characteristic reflections of starting oxides are observed after the first 3 h of milling as a consequence of a considerable decrease in particle size and the introduction of a large number of structural defects due to the impact of balls to the powders. After 6 h of milling, XRD patterns show no appreciable changes. After 30 h, the powder mixture shows a pattern where the main reflection of the ZrTiO4 (PDF 00-01-080- 6058) is evident. Reflections of remnant TiO2 and ZrO2 are also present. Figure 2. XRD patterns of balled-milled samples for 30 h treated at different temperatures for 6 h, (a) 700-1000 ºC, and (b) 1500 ºC. Figure 2b shows the XRD pattern of the ball-milled sample for 30 h after heat treatment at 1500 ºC for 6 h. A single and highly crystalline phase, identified as ZrTiO4 (PDF 00-01-080- 6058), was observed. Rentería-Zamarrón et al. 4 Rentería-Zamarrón et al. This wollastonite powder may play an important role in the nucleation and growth of bioactive crystals in the next stages of immersion. Figure 3. SEM images of sintered (1500 °C) ZrTiO4 samples: Microstructures (a and b) and EDS spectrum (c). Figure 3. SEM images of sintered (1500 °C) ZrTiO4 samples: Microstructures (a and b) and EDS spectrum (c). Biomimetic Coating of Mechanochemically Synthesized Zirconium Titanate 5 Figure 4. SEM images and EDS spectrum of sintered (1500 °C) ZrTiO4 after 7 (a), 14 (b) and 21 days (c) of immersion in SBF without using a wollastonite powder bed. Figure 4. SEM images and EDS spectrum of sintered (1500 °C) ZrTiO4 after 7 (a), 14 (b) and 21 days (c) of immersion in SBF without using a wollastonite powder bed. On the other hand, if a wollastonite bed is not used during the immersion of samples in SBF for 21 days, no changes were observed on the material surface 24. After 14 days of immersion in SBF (Fig. 5b), a new compound, with a microstructure consisting of agglomerates of a smaller size than that of the ZrTiO4 grains, has been formed. According to the corresponding EDS spectrum, even though the Zr peak overlaps with that of P, the elements detected, apart from those of the substrate, are Ca, P and Si. Figure 6 shows the FT-IR spectra of the untreated sample (before immersion in SBF), the sample after 21 d of immersion in SBF and the biomimetically-treated sample after 21 d of immersion in SBF on a bed of wollastonite powder. Regarding to the untreated ZrTiO4, the absorption band at 466 cm-1 corresponds to the vibration of the Zr-O bond and the absorption bands at 650 and 520 cm-1 correspond to Ti-O and Zr-O-Ti groups, respectively 25. The spectrum of the sample immersed in SBF with no wollastonite powder bed is similar to that of the untreated ZrTiO4 sample. In the FT-IR spectrum of the biomimetically treated sample, the absorption band at 574 cm-1 correspond to (PO4)3- groups, several authors report the most intense signal of hydroxyapatite, the antisymmetric vibration of calcium phosphates υ3as(PO4)3- at 1040 and 1091 cm-1. 26-28 and the stretching bands at 3645 and 2930 cm-1 correspond to O-H. After 21 days of immersion in SBF, a homogeneous Ca, P-rich layer has been formed on the surface, this according to the corresponding EDS spectrum (Fig. 5c). Rentería-Zamarrón et al. Results showed that 30 hours of milling followed by heat treatment at 1500 ºC for 6 h are necessary to consider the reactions completed. No bioactive compound was formed on the samples immersed for 21 days in SBF. On the other hand, when a wollastonite d b d d d i h i i f l i Figure 6. FT-IR spectra of sintered (1500 °C) ZrTiO4: untreated sample, sample after 21 d of immersion in SBF and biomimetically treated sample for 21 d in SBF using a wollastonite powder bed. Figure 7. TEM images and EDS spectrum of sintered (1500 ºC) ZrTiO4 after biomimetic treatment for 21 days. 5. Acknowledgements This work was financially supported by CONACYT ( CB 2011 01 166 995 PEI 250174 d CB2013 Figure 7. TEM images and EDS spectrum of sintered (1500 ºC) ZrTiO4 after biomimetic treatment for 21 days. 5. Acknowledgements This work was financially supported by CONACYT (grants CB-2011-01-166 995, PEI 250174 and CB2013- 01-221701) and Tecnológico Nacional de México (grant These results are in agreement with those obtained by SEM and EDS and the presence of (PO4)3- and O-H groups may indicate the formation of a bioactive compound on ZrTiO4 after 21 days of immersion in SBF on a bed of wollastonite powder. Figure 6. FT-IR spectra of sintered (1500 °C) ZrTiO4: untreated sample, sample after 21 d of immersion in SBF and biomimetically treated sample for 21 d in SBF using a wollastonite powder bed. Figure 6. FT-IR spectra of sintered (1500 °C) ZrTiO4: untreated sample, sample after 21 d of immersion in SBF and biomimetically treated sample for 21 d in SBF using a wollastonite powder bed. Figure 6. FT-IR spectra of sintered (1500 °C) ZrTiO4: untreated sample, sample after 21 d of immersion in SBF and biomimetically treated sample for 21 d in SBF using a wollastonite powder bed. Figure 7. TEM images and EDS spectrum of sintered (1500 ºC) ZrTiO4 after biomimetic treatment for 21 days. In the aim to identify the bioactive compound formed on the surface of biomimetically treated ZrTiO4, TEM and HRTEM analyses were performed (Figure 7). The TEM image (Figure 7) shows the morphology of the material after the biomimetic process. The HRTEM image shows the lattice fringes of (211) and (112) planes of a hydroxyapatite crystal with a lattice spacing of 0.292 and 0.289 nm, respectively. 4. Conclusions The viability of using a combination between the mechano- synthesis method and the application of a heat treatment to obtain ZrTiO4 was demonstrated. Results showed that 30 hours of milling followed by heat treatment at 1500 ºC for 6 h are necessary to consider the reactions completed. No bioactive compound was formed on the samples immersed for 21 days in SBF. On the other hand, when a wollastonite powder bed was used during the immersion of samples in SBF, apatite was formed on ZrTiO4 samples. These results indicate that biomimetically treated zirconium titanate may be a potential material for biomedical applications. Figure 7. TEM images and EDS spectrum of sintered (1500 ºC) ZrTiO4 after biomimetic treatment for 21 days. Rentería-Zamarrón et al. This indicates that the growing layer on ZrTiO4 may correspond to hydroxyapatite, HA 27-30. In order to verify this information, the chemical composition of the bioactive compound was evaluated by EDS. The resulting analysis revealed a Ca/P ratio = 1.54, which is lower than that of HA (1.67) and very similar to the apatite formed on the existing bioactive systems 27-32. Thus, the biomimetically treated ZrTiO4 obtained in this work may exhibit a bone-bonding ability through the apatite layer. Rentería-Zamarrón et al. Silicon is also detected in a lower quantity than that corresponding to the sample immersed for 14 days (Fig. 5b). This may indicate that the wollastonite particles that were observed on the sample after 7 and 14 days of immersion have been dissolved into the SBF and an ionic exchange between H+, from the SBF, and Ca2+, from the wollastonite powder, takes place. This eventually increases pH and the ionic activity product of Ca and P ions in solution, leading to the nucleation and growth of the Ca, P-rich compound. Rentería-Zamarrón et al. Materials Research 6 Figure 5. SEM images and EDS spectra of sintered (1500 °C) ZrTiO4 after 7 days (a), 14 days (b) and 21 days (c), of immersion in SBF on a wollastonite powder bed. Figure 5. SEM images and EDS spectra of sintered (1500 °C) ZrTiO4 after 7 days (a), 14 days (b) and 21 days (c), of immersion in SBF on a wollastonite powder bed. 7 Biomimetic Coating of Mechanochemically Synthesized Zirconium Titanate Biomimetic Coating of Mechanochemically Synthesized Zirconium Titanate These results are in agreement with those obtained by SEM and EDS and the presence of (PO4)3- and O-H groups may indicate the formation of a bioactive compound on ZrTiO4 after 21 days of immersion in SBF on a bed of wollastonite powder. In the aim to identify the bioactive compound formed on the surface of biomimetically treated ZrTiO4, TEM and HRTEM analyses were performed (Figure 7). The TEM image (Figure 7) shows the morphology of the material after the biomimetic process. The HRTEM image shows the lattice fringes of (211) and (112) planes of a hydroxyapatite crystal with a lattice spacing of 0.292 and 0.289 nm, respectively. This indicates that the growing layer on ZrTiO4 may correspond to hydroxyapatite, HA 27-30. In order to verify this information, the chemical composition of the bioactive compound was evaluated by EDS. The resulting analysis revealed a Ca/P ratio = 1.54, which is lower than that of HA (1.67) and very similar to the apatite formed on the existing bioactive systems 27-32. Thus, the biomimetically treated ZrTiO4 obtained in this work may exhibit a bone-bonding ability through the apatite layer. 4. Conclusions The viability of using a combination between the mechano- synthesis method and the application of a heat treatment to obtain ZrTiO4 was demonstrated. 6. References 1. Salahinejad E, Hadianfard MJ, Macdonald DD, Sharifi Asl S, Mozafari M, Walker KJ, et al. Surface modification of stainless steel orthopedic implants by Sol-Gel ZrTiO4 and ZrTiO4- PMMA coatings. Journal of Biomedical Nanotechnology. 2013;9(8):1327-1335. 18. Gajović A, Šantić A, Djerdj I, Tomašić N, Moguš-Milanković A, Su DS. Structure and electrical conductivity of porous zirconium titanate ceramics produced by mechanochemical treatment and sintering. Journal of Alloys and Compounds. 2009;479(1-2):525-531. 2. Akhtar N, Rafique HM, Atiq S, Aslam S, Razaq A, Saleem M. Dielectric based energy storage capacity of sol-gel synthesized Sr-doped ZrTiO4 nanocrystallites. Ceramics International. 2018;44(6):6705-6712. 19. Williams MC, Strakey JP, Surdoval WA. The U. S. Department of Energy, Office of Fossil Energy Stationary Fuel Cell Program. Journal of Power Sources. 2005;143(1-2):191-196. 3. Gajović A, Furić M, Musić S, Djerdj I, Tonejc A, Tonejc AM, et al. Mechanism of ZrTiO4 Synthesis by Mechanochemical Processing of TiO2 and ZrO2. Journal of the American Ceramic Society. 2006;89(7):2196-2205. 20. Śmieszek A, Donesz-Sikorska A, Gresiak J, Krzak J, Marycz K. Biological effects of sol-gel derived ZrO2 and SiO/ZrO2 coatings on stainless steel surface--In vitro model using mesenchymal stem cells. Journal of Biomaterials Applications. 2014;29(5):699-714. 4. Fuentes AF, Takacs L. Preparation of multicomponent oxides by mechanochemical methods. Journal of Materials Science. 2013;48(2):598-611. 21. Tianinen H, Eder G, Nilsen O, Haugen HJ. Effect of ZrO2 addition on the mechanical properties of porous TiO2 bone scaffolds. Materials Science and Engineering: C. 2012;32(6):1386-1393. 5. George A, Solomon S, Thomas JK, John A. Characterizations and electrical properties of ZrTiO4 ceramic. Materials Research Bulletin. 2012;47(11):3141-3147. 22. Nogiwa-Valdez AA, Cortés-Hernández DA, Almanza-Robles JM, Chávez-Valdez A. Bioactive Zirconia Composites. Materials Science Forum. 2006;509:193-198. 6. Park Y, Dim Y. Influence on cooling rate on the physical properties of tin modified zirconium titanate. 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Acknowledgements This work was financially supported by CONACYT (grants CB-2011-01-166 995, PEI 250174 and CB2013- 01-221701) and Tecnológico Nacional de México (grant 6798.18-P). D. Rentería -Zamarrón thanks to CONACYT for the posdoctoral scholarship. Rentería-Zamarrón et al. 8 Materials Research 17. Ferrari V, Marioli D, Taroni A, Ranucci E. Multisensor array of mass microbalances for chemical detection based on resonant piezo-layers of screen-printed PZT. Sensors and Actuators B: Chemical. 2000;68(1-3):81-87. 32. Reséndiz-Hernández PJ, Cortés-Hernández DA, Saldívar-Ramírez MMG, Acuña-Gutiérrez IO, Flores-Valdés A, Torres-Rincón S, et al. Novel bioactive materials: silica aerogel and hybrid silica aerogel/pseudo wollastonite. Boletin de la Sociedad Espanola de Ceramica y Vidrio. 2014;53(5):235-239. 31. Huang D, Yin M, Lin Q, Qin Y, Wei Y, Hu Y, et al. Aligned hydroxyapatite nano-crystal formation on a polyamide surface. RSC Advances. 2017;7(68):43040-43046. Biomimetic Coating of Mechanochemically Synthesized Zirconium Titanate 6. References Chemical Processing and Microwave Characteristics of (Zr,Sn)TiO4 Microwave Dielectrics. Journal of the American Ceramic Society. 1991;74(6):1320-1324. 25. Ortega-Chavarría S, Cortés-Hernández DA, Nogiwa-Valdez AA. Biomimetic Coating on Zirconia Composites Induced by Chemical Treatment. Materials Science Forum. 2007;560:127- 132. 10. Christoffersen R, Davies PK, Wei X, Negas T. Effect of Sn Substitution on Cation Ordering in (Zr1-xSnx)TiO4 Microwave Dielectric Ceramics. Journal of the American Ceramic Society. 1994;77(6):1441-1450. 26. Liu X, Huang A, Ding C, Chu PK. Bioactivity and cytocompatibility of zirconia (ZrO2) films fabricated by cathodic arc deposition. Biomaterials. 2006;27(21):3904-3911. 27. Dos Santos V, Bergmann CP. Synthesis and Characterization of Crystalline Zirconium Titanate Obtained by Sol-Gel. In Mastai Y, ed. Advances in Crystallization Processes. London: IntechOpen; 2012. Available from: http://www.intechopen. com/books/advances-in-crystallizationprocesses/synthesis-and- characterization-of-crystalline-zirconium-titanate-obtained-by- sol-gel 11. Victor P, Bhattacharyya S, Krupanidhi SB. Dielectric relaxation in laser ablated polycrystalline ZrTiO4 thin films. Journal of Applied Physics. 2003;94(8):5135-5142. 12. Liu SW, Song CF, Lü MK, Wang SF, Sun DL, Qi YX, et al. A novel TiO2/ZrxTi1-xO2 composite photocatalytic films. Catalysis Communications. 2003;4(7):343-346. 13. Reddy BM, Khan A. Recent Advances on TiO2-ZrO2 Mixed Oxides as Catalysts and Catalyst Supports. Catalysis Reviews - Science and Engineering. 2005;47(2):257-296. 28. Fathi MH, Hanifi A, Mortazavi V. Preparation and bioactivity evaluation of bone-like hydroxyapatite nanopowder. Journal of Materials Processing Technology. 2008;202(1-3):536-542. 14. Mazurkevich YS, Kobasa IM. ZrO2-TiO2 Materials. Inorganic Materials. 2001;37(12):1285-1288. 29. Deng Y, Sun Y, Chen X, Zhu P, Wei S. Biomimetic synthesis and biocompatibility evaluation of carbonated apatites template- mediated by heparin. Materials Science and Engineering: C. 2013;33(5):2905-2913. 15. Sohn JR, Lee SH. Effect of TiO2-ZrO2 composition on catalytic activity of supported NiSO4 for ethylene dimerization. Applied Catalysis A: General. 2007;321(1):27-34. 30. Xiao J, Zhu Y, Ruan Q, Liu Y, Zeng Y, Xu F, et al. Biomacromolecule and Surfactant Complex Matrix for Oriented Stack of 2-Dimensional Carbonated Hydroxyapatite Nanosheets as Alignment in Calcified Tissues. Crystal Growth and Design. 2010;10(4):1492-1499. 16. Costa CEF, Crispim SCL, Lima SJG, Paskocimas CA, Longo E, Fernandes VJ Jr., et al. Synthesis and thermal characterization of zirconium titanate pigments. Journal of Thermal Analysis and Calorimetry. 2004;75(2):467-473. 9
https://openalex.org/W4304204407	https://hal.science/hal-04269018/document	English	null	Value components in equestrian self-organization	HAL (Le Centre pour la Communication Scientifique Directe)	2,022	cc-by	11,364	Value Components in Equestrian Self-Organization Camille Eslan, Sandrine Costa, Céline Vial To cite this version: Camille Eslan, Sandrine Costa, Céline Vial. Value Components in Equestrian Self-Organization. Management & Organisations du Sport, In press. hal-04269018v2 Value Components in Equestrian Self-Organization Camille Eslan, Sandrine Costa, Céline Vial To cite this version: Camille Eslan, Sandrine Costa, Céline Vial. Value Components in Equestrian Self-Organization. Management & Organisations du Sport, In press. hal-04269018v2 Camille Eslan, Sandrine Costa, Céline Vial. Value Components in Equestrian Self-Organization. Management & Organisations du Sport, In press. hal-04269018v2 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-04269018 https://hal.science/hal-04269018v2 Submitted on 17 May 2024 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 Les composantes de la valeur dans l'auto-organisation équestre Camille ESLAN1,2,3; Sandrine COSTA2; Céline VIAL2,3 Affiliation: 1 UniLaSalle, InTerACT, Rouen, France ; 2 MoISA, univ Montpellier, CIRAD, CIHEAM- IAMM, INRAE, Institut Agro, IRD, Montpellier, France ; 3 IFCE, department development innovation and research, 61310 Exmes, France Résumé : Objectifs : L'auto-organisation dans les activités sportives et de loisirs a récemment augmenté mais reste peu connue. Cette tendance est la même dans les activités équestres, générant des enjeux économiques et sociétaux pour les institutions et les professionnels du secteur. L'auto- organisation consiste en l'organisation quotidienne, en autonomie, d'une ou plusieurs tâches qui pourraient être sous-traitées. Elle est la conséquence, pour certains pratiquants, d'une inadéquation entre leur demande et l'offre de services disponible. Afin de mieux comprendre les déterminants de l’auto-organisation équestre, cet article étudie la valeur de consommation associée à ce choix organisationnel. Problématique : Le service sportif, dissocié du bien matériel, est un contrat d'échange immatériel qui requiert la participation du consommateur. Dans ce contexte, cet article analyse les composantes de la valeur de l'auto-organisation sportive. En outre, cette recherche exploratoire vise à comprendre quelles composantes de la valeur influencent la valeur globale de l'auto-organisation, dans le cas des activités équestres, et si cette valeur globale est aussi influencée par l'attachement à l’animal et l'auto-efficacité. Méthodologie : La méthodologie repose sur une enquête quantitative réalisée en ligne via les réseaux sociaux (Facebook, X - Twitter et Instagram) pendant six mois en 2021, en France auprès de 615 répondants. L’analyse effectuée mobilise des modèles d’équations structurelles. Résultats : Les résultats montrent que l’attachement à l’animal, l’auto-efficacité, la valeur économique, la valeur hédonique, la valeur éthique liée au bien-être sont des composantes clés de la valeur de l’auto-organisation équestre. De plus, une nouvelle composante est mise en évidence par la valeur de transmission. Cette étude explore aussi l’effet des risques d’erreur perçus dans l’auto-organisation équestre. Originalité : Ces résultats ouvrent des perspectives pour de nouvelles stratégies managériales à destination des usagers auto-organisés, dans les activités équestres et plus généralement dans le domaine du sport, impliquant notamment une prise en compte des souhaits pour une 1 1 pratique permettant une transmission à ses proches et un alignement avec ses préoccupations éthiques. Mots clés : auto-organisation, activités équestres ; valeur de transmission ; valeur éthique ; attachement ; risques perçus ; modèle d’équation structurelle. Financement : Ce travail a été soutenu par le Conseil scientifique de la filière équine française, la Fédération française d'équitation (FFE) et l'Agence nationale de la recherche et de la technologie (ANRT) [contrat n°2018/1491]. Abstract: Aim: Self-organization in sport and leisure activities has increased recently but remains little known. This trend is the same in equestrian activities, generating economic and societal issues for institutions and professionals of the sector. Self-organization is the daily autonomous organization of one or more tasks that could otherwise be subcontracted. For some practitioners, it is the result of a mismatch between their demand and the supply of available services. To better understand the determinants of equestrian self-organisation, this article examines the consumer value associated with this organizational choice. Question: The sports service, dissociated from the material good, implies an immaterial exchange contract requiring consumer participation. In this context, this article analyses the value components of sports self-organization, in the case of equestrian activities. In addition, this exploratory research aims to understand which value components influence the overall value of self-organization and whether this overall value is also influenced by attachment to the animal and self-efficacy. Methodology: The methodology is based on a quantitative survey conducted online via social networks (Facebook, X - Twitter and Instagram) over six months in 2021, in France with 615 respondents. Structural equation models are used in the analysis. Results: The results reveal that attachment to the animal, self-efficacy, economic value, hedonic value, ethical value associated with well-being are pivotal elements in the value of equestrian self-organization. Additionally, a novel component, transmission value, has been identified. This study also explore the influence of the perceived risk of error in equestrian self- organization. 2 Originality: These findings open-up perspectives for new managerial strategies towards self- organized users, in equestrian activities, and more generally in the field of sport. In particular, this means considering the consumers’ expectations of a practice that can be shared with loved ones and that is consistent with everyone's ethical concerns. Keywords: self-organization, equestrian activities; transmission value; ethical value; attachment; perceived risks; PLS-SEM. 3 3 Introduction What is the value attributed to an activity? The consumption value is the value of an object or a service according to the consumer's perception. Detailed value components qualify the relationship of consumers with the object or service in question using typologies (Aurier et al., 2004; Holbrook, 1994). The value co-creation framework of Leclercq et al. (2016) analyzes the process of consumption value formation. Is the value different when the consumer participates in the service's production, and if so, what are the value components in a self-organized setting? Self-organization consists of the daily organization, in autonomy, for one or several tasks that could be subcontracted. It can concern various practices or activities. In the field of sport and leisure, self-organized practices are little known. They are the result, for some practitioners, of a mismatch between their demand and the available service supply, with clubs that do not fulfil practitioners' needs for autonomy, or freedom. This autonomization process concerns all sport and leisure activities, such as running, cycling, or horse riding, and it generates economic and societal issues for institutions and professionals. We focus specifically on equestrianism, which has the specificity to involve an animal (a horse) in the activity, which questions the role of attachment to the animal. Detached from the material good, a sports service is an intangible exchange contract requiring consumer participation. Therefore, we believe that understanding the value of performing the activity can lead to a better understanding of the desired service and, consequently, to better adapting the offer to the demand. In this context, this paper analyzes the value components of self-organization in equestrian sports. Moreover, this research 4 analyzes the influence of attachment and self-efficacy on the overall value of self- organization, and whether there are some mediating effects of the value components. The conceptual framework reviews the literature on self-organization and the elements that can influence value creation in equestrian activities. Then, based on an exploratory qualitative survey, we propose a hypothetical theoretical model and test it through a quantitative study. The results are then presented and discussed. Value Creation through Self-Organization In this study, the principle of self-organization is defined as taking care of activities that could be delegated to a professional, but that can also be self-organized, while using professional services or not. In this perspective, consumer behavior literature observes consumer participation in service activities. The particularity of services is the added value to the offer, created through customer participation (Gabriel et al., 2014). Thus, the experience of service use or purchase creates value. The level of consumer involvement in value creation can vary. Various literature shows the different roles or behaviors of the consumer assuming tasks previously realized by the provider, such as designing, coproducing, or providing information, according to their resources (Dong et Sivakumar, 2017). In the case of sports services, the consumer must necessarily participate in the activity to benefit from the service. However, self-organization and, more generally, the participation of consumers in a production process depends on their experience and the value they perceive to be created and gained. The value created by the consumer experience differs according to the situation and includes several components (Aurier et al., 2004). Table 1 proposes an integrative approach of value components in equestrian self-organization adapted from the literature. We hypothesize 5 that these value components are essential in this specific consumption experience, based on scientific review and field experience. Table 1 Hypothetical integrative approach of value components in equestrian self-organization, adapted from Holbrook (2002, 2006), Aurier et al. (2004), Richins (1994) and Medberg and Heinonen (2014). Value Creation through Self-Organization Extrinsic (means-end relation in which consumption is appreciated for its functional/banal instrumentality to achieve a goal – e.g., a hammer) Intrinsic (consumption experience is appreciated as an end in itself, for its own sake, as self- justified, playful, or self-sustaining - e.g., the pleasure of a day at the beach) Self-Oriented (an aspect of selfish consumption for my own good or for the effect it has on me - e.g., a sweater is valuable because it keeps me warm) Instrumental value Utilitarian (product meets practical needs - the concept of efficiency) Economic value Knowledge (permanent info-seeking behavior, different from pre-purchase info-seeking) Hedonic value Playful (ability to have fun, the difference between work and play) Experiential stimulation (the ability of the experience to stimulate the senses of the individual) Others orientated (Consumption experience is evaluated for the effect it has on others- e.g., buying a car to impress neighbours) Social value Self-expression/status (the ability of the product or service to project self- expression) Social link (product as an aid to social interaction, to exchange in the form of conversations. Two-way communication: other ↔ self) Heritage (customer's and family's experience within the company and their consumption experience) Altruistic value Spirituality (Experience as a means to a later end but rather as an end valued for its own sake) Shared moral value (adequacy between the ethical standards of the clients and the standards of the other stakeholders) Table 1 Hypothetical integrative approach of value components in equestrian self-organization, adapted from (2002, 2006), Aurier et al. (2004), Richins (1994) and Medberg and Heinonen (2014). Others orientated (Consumption experience is evaluated for the effect it has on others- e.g., buying a car to impress neighbours) Others orientated (Consumption experience is evaluated for the effect it has on others- e.g., buying a car to impress neighbours) 6 In their netnography, Medberg et Heinonen (2014) introduce invisible values, as the non-visible result of benefits or sacrifices regarding a customer's experience and relationship with the environment. Following a customer-dominant logic, the invisible values appear from the customer's point of view rather than that of business. Even if Medberg et Heinonen (2014) identified four invisible value factors in bank-customer relationships, one (in red in Table 1) seem relevant in this case study. First, "Heritage value concerns the history […] initiated by parents or relatives of the customer" (Medberg et Heinonen, 2014, p. 599). Value Creation through Self-Organization The more self-organization meets the self- organized users' horse needs or responds to a specific ethical value, the more they think self- organization has a high overall value. We pose that the different value components can positively influence the perceived overall value of self-organization. Thus, the more users think that a specific value dimension is important, the more important is the overall value (H1). Value Creation through Self-Organization In equestrian self- organization, the heritage value may gain importance as management often occurs within the family. The second invisible value relevant in our study case, is the shared moral value. This value "refers to the compatibility between a customer's own moral standards and the 6 perceived moral standards of the bank" (Medberg et Heinonen, 2014, p. 599). Shared moral value is positioned in the altruistic component, as it refers to the adequacy between the client's ethical standards and the other stakeholders' standards. In this case study, considering the ethical value component related to the animal's welfare seems important as animal's welfare depends on how equines are housed. The altruistic value component (Holbrook, 2006), based on how the consumer experience is an ethical way of consuming, can be transposed to how people consume ethically for themselves and manage their equine for their welfare in equestrian activities. Creativity can lead to autonomous practices in sport as professional structures have too much of a constraining frame to offer sufficiently diversified experiences (Galewicz, 2017; Riffaud, 2018). Consequently, the hedonic value component includes experiential stimulation (Aurier et al., 2004) and the pleasure of practicing the desired activities (Hirschman et Holbrook, 1982). Self-organized equestrian users may also be motivated by the social value component, including self-expression, status, and social link (Aurier et al., 2004). Self-organized equestrians are passionate about their activity and wish to exchange with others who share the same goals and values, or to have a more comprehensive social network, as observed for runners (Xie et al., 2020). The status dimension related to the prestige of the self-organization enables members to achieve their goals, enhancing the need for identification and self-realization concerning the image projected to others (Bhattacharya et al., 1995). Despite the many similarities between the different recreational practices, such as representing a lifestyle, self-organization within equestrian activities has the specificities to be expensive, either for equestrian activities or caring for the horse. Thus, the influence of economic value could be essential. This dimension is part of the instrumental value component. This component also includes a knowledge dimension related to a permanent 7 7 search for information, stimulating the individual (Aurier et al., 2004). Moreover Eslan et al. (2023) emphasized the importance of animal welfare for self-organization. Consequently, the shared moral value (Medberg et Heinonen, 2014) initially considered finally seems to relate to the ethical value linked to equine welfare. Attachment to the Animal Attachments and interactions between humans and animals can be of different natures. Equines may be used as "equipment" for many practical and recreational activities or provide aesthetic value and pleasure to consumers. Attachment to an animal can also represent a felt and expressed emotional bond between the animal and its owner (St. George et al., 1998). This concept has already been introduced in the literature by Bowlby (2002) under the principle of attachment theory, firstly applied to the relationship between the child and the mother. The fundamental principle of this theory, in addition to its applicability to all mammal species, is that a child, to develop both emotionally and socially, must create an attachment relationship with a reference person, the caregiver (Rockett et Carr, 2014). In equestrian activities, Le Clinche et al. (2017) observe the type of attachment in the sporting equestrian event and see the equine as an extension of the family. This link also reflects a real passion for the equine, as observed in Eslan et al. (2023). It can lead to behaviors that are sometimes addictive but mostly linked to the playful and experiential pleasure that the relationship with the animal provides. This attachment relationship can develop into a passion with harmonious or obsessive behavior (Rousseau et al., 2002). In a self-organization context, equine owners can live close to their horse and take care of it how they wish, being able to give free rein to their attachment to their equine. Consequently, we suppose that the more users are attached to their animal, the more self-organization has a high overall value (H4), and that the attachment with the animal will positively influence different value components (H5). Self-Efficacy Self-efficacy concept, introduced by the psychologist Bandura (1997), refers to an individual's beliefs about his ability to complete a task, learn, challenge, or change. In a self- organization context, self-efficacy is the perceived ability to participate successfully and achieve desired outcomes in the production process, because consumers believe they can produce by themselves better and more responsive products (Xie et al., 2008) in challenging tasks. Self-efficacy underlies the motivation to engage in action and perseverance in achieving the goal. People with a keen sense of efficacy tend to be more proactive in attaining goals, anticipating, and preventing stressors. Self-efficacy measures perceive competence (Vaughan‐ Johnston et Jacobson, 2020) and includes awareness of the possible risk of error (Park et John, 2014). We pose that the more people are confident in their skills, the more they think self-organization has a high overall value (H2). We also suppose that self-efficacy positively influences different value components (H3). 8 8 General Methodology Based on the literature review and a qualitative survey (Eslan et al., 2023), an online quantitative survey via social networks (Facebook, X-Twitter, and Instagram) was carried out during six months in 2021, in France among 615 respondents. Because there is no primary sampling database, this dissemination method makes it easier to reach an unknown population with a convenient sample. The quantitative step seeks to show the influence of self-efficacy and attachment to the animal on overall self-organization value and the effect of the different value components listed in Table 1. Development of a Conceptual Analysis Framework Thus, we pose that the risk of error can mediate the relationship between the overall value of self-organization (H6) and both its antecedents, self-efficacy (H7a) and animal attachment (H7b). his own actions and interacting effectively with the environment (Gillet et al., 2008). This situation creates hazards that can cause mistakes, which can be source of concern for the users. Moreover, according to the environment and people, worries can greatly fluctuate. Therefore, this study proposes to measure the perceived risks of errors in the context of equestrian self-organization introducing the risks of errors as a mediator. Thus, we pose that the risk of error can mediate the relationship between the overall value of self-organization (H6) and both its antecedents, self-efficacy (H7a) and animal attachment (H7b). Development of a Conceptual Analysis Framework We consider different value components in the initial framework according to the literature (figure 1). 9 Figure 1 Hypothetical model of the influence of self-efficacy and attachment on different value components and the overall value of self-organization Figure 1 Hypothetical model of the influence of self-efficacy and attachment on different value components and the overall value of self-organization g yp f f f f ff y ff p overall value of self-organization Eslan et al. (2023) highlight the fear of being unable to meet equines' needs or of doing mistakes in horses’ care, in the context of self-organization. Self-organization also implies many sacrifices since it costs time and money. Eslan et al. (2023) highlight the fear of being unable to meet equines' needs or of doing mistakes in horses’ care, in the context of self-organization. Self-organization also implies many sacrifices since it costs time and money. Eslan et al. (2023) highlight the fear of being unable to meet equines' needs or of doing mistakes in horses’ care, in the context of self-organization. Self-organization also implies many sacrifices since it costs time and money. The notion of sacrifice shown by Aurier et al. (2004) appears, but the concept of risk is mainly related to strategic management (Mitchell, 1999), or environmental influence in decision-making (Paulraj et Chen, 2007). In tourism, risk assessments are categorized as uncontrollable or self-induced (Wang et al., 2010). Sjöberg et Engelberg (2005) consider risk perception in relation to lifestyles without conclusive results on consumer behavior. In sport literature, risks are more observed according to environmental difficulties rather than organizational ones (Siebert et Kolleck, 2013). 10 The exploratory qualitative survey (Eslan et al., 2023) emphasizes the need for autonomy and the search for improvement in the process. These findings corroborate Gillet and al.’s work who show that autonomy and competency perceptions influence the motivation to participate in a sport. The need for autonomy and competence implies being at the origin of 10 his own actions and interacting effectively with the environment (Gillet et al., 2008). This situation creates hazards that can cause mistakes, which can be source of concern for the users. Moreover, according to the environment and people, worries can greatly fluctuate. Therefore, this study proposes to measure the perceived risks of errors in the context of equestrian self-organization introducing the risks of errors as a mediator. Identifying the Constructs In the quantitative survey, 53 items were initially used to measure 12 constructs. To this aim, items were selected from the literature review and completed following the approach proposed by Rossiter (2002). Relying on the results of the qualitative study, items were created to be integrated into the quantitative survey. Therefore, we introduced items related to animal welfare, risk of error, and transmission value. All the items used, and their source are presented in appendix. 11 Exploratory analysis was performed with SMART-PLS©4.0 (Ringle et al., 2022) to verify the scales' structure. Some constructs were not validated and thus discarded, and others were redesigned. Sample Description 671 respondents answered the quantitative survey, they all own at least one equine at home. 56 respondents with more than 11 missing answers were excluded. Finally, the 615 respondents are between 15 and 78 years old and have various socioeconomic statuses. The average duration of self-organization is 10.7 years (Table 2). Table 2 Sample general description Age (number in the sample) 15-20 yrs. (17), 21-35 yrs. (294), 36-49 yrs. (173), 50 yrs and over (100) Gender Men 7%, Women 93% Job category Farmers 6%, Intermediary profession 6%, Unemployed 6%, Students 7%, Craftsman and Business owners 13%, Executives and Senior intellectuals 27%, Employees/Workers 35% Geographical area Urban area = 18%, Suburban area = 46%, Rural area = 36% Number of equines owned Average of three equines [1;45] Average time spent per week with equine(s) Practicing activities (riding, grooming) = 6.66 hours Husbandry (feeding, pasture care) = 8.79 hours Average previous duration in a professional structure 9.9 yrs. Average duration in self- organization 10.7 yrs. Table 2 Sample general description Age (number in the sample) 15-20 yrs. (17), 21-35 yrs. (294), 36-49 yrs. (173), 50 yrs and over (100) Gender Men 7%, Women 93% Job category Farmers 6%, Intermediary profession 6%, Unemployed 6%, Students 7%, Craftsman and Business owners 13%, Executives and Senior intellectuals 27%, Employees/Workers 35% Geographical area Urban area = 18%, Suburban area = 46%, Rural area = 36% Number of equines owned Average of three equines [1;45] Average time spent per week with equine(s) Practicing activities (riding, grooming) = 6.66 hours Husbandry (feeding, pasture care) = 8.79 hours Average previous duration in a professional structure 9.9 yrs. Average duration in self- organization 10.7 yrs. Table 2 Sample general description Exploratory Factor Analysis (EFA) Bartlett’s sphericity test shows a significant difference, thus rejecting the correlation hypothesis and verifying the adequacy of the common variance of matrix. The validity of the sample measure quality KMO is good (MSA= 0.8). The EFA carried out on the data collected online shows a correlation between some of the measurement scales from the literature. The 12 measurement scale is purified with cutoff at 0.32 and removing constructs with only one item. The final scale includes 26 items for 8 constructs with a discriminant validity Heterotrait- monotrait ratio (HTMT) < O.9, no items have a higher cross-loading with another construct and VIF < 0.2 for all items. Table 3 present the eight constructs finally selected. Table 3 AFE results after cleaning the measuring scales (loading cutoff = 0.32) and construct reliability and convergent validity Construct Code Self- efficacy Attach- ment Risk of error Ethical value Transmis- sion value Econom ic value Hedonic Value Overall value Selfeff1 0.757 Selfeff2 0.753 Selfeff3 0.795 Selfeff4 0.701 Attach2 0.634 Passion1 0.684 Passion3 0.732 Passion4 0.689 Riskerr1 0.832 Riskerr2 0.831 Riskerr3 0.764 Riskerr4 0.588 Ethic1 0.823 Ethic2 0.844 Ethic3 0.685 Trans1 0.800 Trans2 0.839 Trans3 0.462 Eco1 0.848 Eco2 0.848 Hedo1 0.753 Hedo2 0.786 Hedo3 0.685 Val1 0.814 Val2 0.783 Val3 0.672 Cronbach’s Alpha 0.743 0.623 0.750 0.618 0.514 0.610 0.592 0.628 Rho C 0.838 0.780 0.841 0.798 0.750 0.793 0.785 0.802 AVE 0.563 0.470 0.577 0.573 0.518 0.669 0.550 0.576 le 3 AFE results after cleaning the measuring scales (loading cutoff = 0.32) and construct reliability and convergent validity 13 The risk of error construct is formed from four items, two from the qualitative survey, one from the autonomy measuring scale, and the last one from the knowledge devaluation scale. They are associated with each other during the EFA with satisfactory nomological validity, as their meanings are in line with each other and the construct theme (Rossiter, 2002). The same phenomenon appears in the attachment, as this construct combines one item from the attachment scale and three items from the passion scale. The coefficients α and ω of the attachment, ethical value, economic value, and overall value constructs are above the 0.6 thresholds in exploratory analysis (Hair et al., 2020), as their AVE is close to or above 0.5 (Table 3), so we kept them for the next phase. Exploratory Factor Analysis (EFA) We assumed the inheritance of heritage value; however, during the EFA, the items correlated with heritage value are all oriented toward a nomological validity of transmission to heirs such as children or friends. Therefore, we retained the construct of transmission value, even if the indices are a little under the threshold, as it seems interesting to investigate this value component in the case of a leisure. It posits that the more self-organized users are attached to their equine, the more they think that being autonomous allows them to transmit and share their passion with their loved ones. In addition, the more self-organized users think that being autonomous enables them to transmit and share their passion with their relatives, the more important overall self-organization value is for them (H1b). Similar to transmission value, hedonic value also has a low alpha coefficient that is close to 0.6, which is the threshold value acceptable during exploration studies. However, the concept of status value does not meet the necessary thresholds, so we did not retain this construct for the modeling phase. Table 4 presents the measuring scales of the 8 final constructs with the retained items used for the SEM model presented hereafter. Table 4 Measuring scales of the retained items for the quantitative survey with AFE results. Origin Original item in the literature Original item in English Code Concept Aurier et al. (2004) Globalement, je considère qu’aller au cinéma, ça vaut bien l’énergie que j’y consacre Overall, being autonomous in the management and maintenance of my equine(s) is well worth the energy and Val1 Overall Value Table 4 Measuring scales of the retained items for the quantitative survey with AFE results. Table 4 Measuring scales of the retained items for the quantitative survey with AFE results. Origin Original item in the literature Original item in English Code Concept Aurier et al. Exploratory Factor Analysis (EFA) (2004) Globalement, je considère qu’aller au cinéma, ça vaut bien l’énergie que j’y consacre Overall, being autonomous in the management and maintenance of my equine(s) is well worth the energy and Val1 Overall Value 14 time I put into it Globalement, le cinéma ça vaut bien les sacrifices que je consens Overall, being autonomous in the management of my equine(s) is well worth the sacrifices I make Val2 Qualitative survey Having my horse(s) at home is irreplaceable Val3 Scholz et al., (2002) I am confident that I could deal efficiently with unexpected events I am confident that I could deal efficiently with unexpected events Selfeff1 Self-efficacy I can remain calm when facing difficulties because I can rely on my coping abilities I can remain calm when facing difficulties because I can rely on my coping abilities Selfeff2 I can handle whatever comes my way I can handle whatever comes my way Selfeff3 When faced with a problem, I can find several solutions When faced with a problem, I can find several solutions Selfeff4 Mathwick et al. (2001) XYZ are good economic value This mode of organization is a good economic value Eco1 Economic value Overall, I'm happy with XYZ’s prices Overall, I'm happy with the costs associated with this type of organization Eco2 Qualitative survey Being autonomous in the husbandry of my equine(s) allows me to meet the needs of my equine(s) Ethic1 Ethical Value Being autonomous in the husbandry of my equine(s) respects their wellbeing Ethic2 Innocent et François- Lecompte (2020) 1- Faire des économies d’électricité, je le fais pour la planète 2- Pour moi, faire attention à mes consommations d’électricité, c’est aussi pour le bien de la collectivité I find it ethical to be autonomous in the husbandery of my horse(s). Ethic3 Mathwick et al. Exploratory Factor Analysis (EFA) (2001) I think XYZ’s internet site is very entertaining Being autonomous in the husbandry of my equine(s) is entertaining Hedo1 Hedonic value I shop from the XYZ’s internet site for the pure enjoyment of it Being autonomous in the husbandry of my equine(s) gives me a sense of happiness Hedo2 Innocent et François- Lecompte (2020) Maîtriser sa consommation électrique, pour moi, c’est contraignant Being autonomous in the husbandry of my equine(s) is restrictive Hedo3 J’aime partager mon expérience au sujet de la consommation d’électricité avec ma famille et mes relations I like to share my experience about the husbandry of my equine(s) with my family and my relatives Trans1 Transmission value Qualitative survey Being autonomous in the husbandry of my equine(s) allows me to transmit my knowledge to my friends and family Trans2 Being autonomous in the management and care of my horse(s) allows me to continue my family's habits Trans3 Qualitative survey I am afraid of making mistakes or errors in the husbandry of my equine(s) Risqerr1 Risk of error Innocent et François- Lecompte (2020) Dans le domaine de la consommation d’électricité je suis gêné par le fait de ne pas toujours savoir comment faire In the field of husbandry of my equine(s), I am bothered by the fact that I do not always know how to do things Risqerr2 Gillet et al. (2008) Dans mon sport, souvent je ne me sens pas très compétent In my organization outside the structure, I often don't feel very competent Risqerr3 Qualitative survey I'm worried or stressed about what might happen to my horse(s) Risqerr3 Bures et al. (2019) When you feel bad, do you seek your pet for comfort? When I feel bad, I seek comfort from my equine(s) Attach2 Attachment Rousseau et al. (2002) I cannot live without this gambling game I can't live without my equine(s) Passion1 The new things that I discover with this gambling game allow me to appreciate it even more The new things I discover with my equine(s) game allow me to appreciate it even more Passion3 This gambling game allows me to live a variety of experiences Being with my equine(s) allows me to live a variety of experiences Passion4 15 Structural Equation Models Structural Equation Models Following this exploratory analysis, we tested different models to observe the distinct influence of value components. In the rest of this paper, we present the structural equation model based on the hypotheses proposed on Figure 2, which is considered as the best model. The fit indices are acceptable: SRMR = 0.074, and d-ULS = 2.167; d-G =0.469 and Chi- square= 1661.971. The discriminant validity between all constructs is good (<0.9). This model explains 44.3% of the variance of the overall value of self-organization. Considering the first hypothetical model proposed (Figure 1), we derived each hypothesis from both antecedents on the different value components: influence of attachment on value components: H5(a to d); influence of self-efficacy on value components: H3(a to d). Similarly, the influence of each value component on overall value constitutes hypothesis H1(a to d) (Figure 2). Figure 2 Model presenting the overall value of the self-organized equestrians under the effect of two antecedents: attachment and self-efficacy and different value components (significancy * = 0 to 1%; = 1 to 5%; * = 5 to 10%) Figure 2 Model presenting the overall value of the self-organized equestrians under the effect of two antecedents: attachment and self-efficacy and different value components (significancy * = 0 to 1%; = 1 to 5%; * = 5 to 10%) 16 The findings show a significant positive link between self-efficacy and attachment on the overall value of self-organization (Table 5). The direct influence of both antecedents demonstrates their importance as the more self-organized users are attached to their horse or feel confident about their skills, the greater the value of self-organization, confirming H2 and H4. Moreover, the ethical value (H1a), transmission value (H1b) and hedonic value (H1c) components have a strong positive significance on overall value. This confirms not only the importance given to equine welfare by self-organized users, but also the pleasure while caring for their animals. The negative influence of transmission value on overall value of self- organization (H1b) suggests that the more self-organized users appreciate being able to share with their loved ones, the less they value self-organization. Our initial hypothesis concerning the link between these two constructs is confirmed, but the influence is not the one expected. This might be explained by the fact that when you are self-organized you have time constraints linked with everyday tasks (e.g. cleaning pastures and stock management). Table 5 presents the results of this model. Regressions of the first model as presented in Figure 1 with the direct effect of self-efficacy and attachment on the all value of self-organization (H2 and H4) (significant p-value in bold: *≤ 0.01; 0.1<≤ 0.05; 0.05<<0.1) Table 5 Regressions of the first model as presented in Figure 1 with the direct effect of self-efficacy and attachment on the overall value of self-organization (H2 and H4) (significant p-value in bold: ≤ 0.01; 0.1<≤ 0.05; 0.05<<0.1) Regressions Estimate Std err p-value Ethical value ~ Attachment 0.315 0.038 0.000 ~ Self-efficacy 0.277 0.040 0.000 * Transmission value ~ Attachment 0.409 0.043 0.000 * ~ Self-efficacy 0.152 0.049 0.002 * Risk of error ~ Attachment 0.163 0.034 0.000 * ~ Self-efficacy -0.582 0.028 0.000 * Hedonic value ~ Attachment 0.532 0.037 0.000 * ~ Self-efficacy 0.167 0.043 0.000 * Economic value ~ Attachment 0.080 0.043 0.061 * ~ Self-efficacy 0.199 0.046 0.000 * Overall value ~ Attachment 0.260 0.058 0.000 * ~ Self-efficacy 0.143 0.040 0.000 * ~ Ethical value 0.200 0.043 0.000 * ~ Transmission value -0.093 0.036 0.002 * ~ Risk of error -0.031 0.038 0.414 (ns) ~ Hedonic value 0.316 0.045 0.000 * ~ Economic value 0.062 0.035 0.074 * Structural Equation Models These constraints reduce the available time that people can have with their loved ones to share their passion for horses. Economic value has a low significant coefficient proving that even if it is an important motivation to choose self-organization (Eslan et al., 2023), economical elements do not give high value to self-organization (H1d). Risk of error does not influence overall value, invalidating H6. Self-efficacy negatively influences risk of error, confirming H7a. This result shows that the more you feel confident in your skills, the less you fear making mistakes in equestrian self-organization. This result confirms that self-efficacy related to self-organization influences the need to consider the risks inherent in autonomous practice, as shown by the qualitative interviews, and is often underestimated. Attachment to the equine positively influences all value components (H5a, b, c, d), and risk of errors (H7b). These findings show that the more people are attached to their equine, the more willing they are to transmit their passion to their 17 entourage, the more they want to offer good welfare conditions to their equines, and the more they enjoy being self-organized. In addition, they give more importance to the risk of making mistakes. Table 5 presents the results of this model. Mediation Effects This model also demonstrates the mediating effect of i) ethical value, transmission value, and hedonic value between attachment and overall value and ii) ethical value, transmission value, and hedonic value between self-efficacy and overall value. Table 6 presents the type of mediation effect of the different value components between self-efficacy and overall value, and Table 7 presents the mediation effect concerning attachment, according to the interpretation of Zhao et al. (2011). 18 The indirect effect of self-efficacy on the overall value is mediated by ethical value, transmission value, and hedonic value. The complementary mediation of ethical value and hedonic value is considered as a medium size effect (est <0.3). This indirect effect of self- efficacy on overall value is significatively stronger than the direct effect of self-efficacy on ethical value. We observe a similar mediation concerning the effect of ethical value and hedonic value between attachment and overall value. A non-mediation effect occurs for risk of error and economic values. These findings tend to prove that ethical value and hedonic value mediates the attachment to the equine, self-efficacy, and overall value of self- organization. This implies that overall value will be lower if ethical value and hedonic value are not respected and present during self-organization. Concerning the mediation effect of transmission value, the results show a competitive mediation of transmission value between self-efficacy and attachment with overall value. The indirect effect of the mediation is indeed negative (Table 7). This effect can be interpreted as a sign of an omitted indirect path in the model (Zhao et al., 2011). Mediation Effects Table 6 Mediation effects of the different value components between self-efficacy and overall value in the model (Figure 2) (significant p-value in bold: *≤ 0.01; 0.1<≤ 0.05; 0.05<<0.1) Self-efficacy Standardized estimate (std_all) Std err p-value Type of mediation Direct effect self-efficacy => Overall value 0.125 0.029 0.000 Complementary mediation of ethical and hedonic value Competitive mediation of transmission value Indirect effect ethical value 0.055 0.015 0.000 * Indirect effect hedonic value 0.053 0.016 0.001 * Indirect effect transmission value -0.038 0.015 0.010 Indirect effect risk of error 0.018 0.023 0.419 (ns) Non-mediation of risk of error and economic value of the construct of self-efficacy on Indirect effect economic 0.012 0.008 0.106 (ns) Table 6 Mediation effects of the different value components between self-efficacy and overall value in the model (Figure 2) (significant p-value in bold: *≤ 0.01; 0.1<≤ 0.05; 0.05<<0.1) 19 value overall value Table 7 Mediation effects of the different value components between attachment and overall value in the model (Figure 2) (significant p-value in bold: ≤ 0.01; 0.1<≤ 0.05; 0.05<<0.1) Attachment Standardized estimate (std_all) Std err p-value Type of mediation Direct effect attachment => Overall value 0.193 0.035 0.000 Complementary mediation of ethical and hedonic value Competitive mediation of transmission value Indirect effect ethical value 0.063 0.015 0.000 * Indirect effect hedonic value 0.168 0.028 0.000 * Indirect effect transmission value -0.038 0.015 0.010 Indirect effect risk of error -0.005 0.006 0.422 (ns) Non-mediation of risk of error and economic value of the construct of attachment on overall value Indirect effect economic value -0.000 0.006 0.237 (ns) Discussion Triggers for Self-Organization Table 7 Mediation effects of the different value components between attachment and overall value in the model (Figure 2) (significant p-value in bold: *≤ 0.01; 0.1<≤ 0.05; 0.05<*<0.1) Discussion Triggers for Self-Organization Triggers for Self-Organization In the equestrian context, this research highlights the importance of specific antecedents and value components in the overall value attributed to self-organization. Our study shows that, contrary to received ideas about elitism and social representation associated with horse riding (Bourdieu, 1979; Le Mancq, 2007), the status value does not seem to influence equestrian self-organization. On the contrary, other dimensions, such as transmission value, ethical value, and hedonic value, are important. Ethical value, in our case, is strongly associated with equine welfare, and not with the ethical preservation of resources or the impression of participating in something bigger as shown in electricity consumption (Innocent et François-Lecompte, 2020). This highlights the 20 importance of the ethical component of value but also shows the necessity to adapt it to each study case, as different kinds of ethical arguments can be considered according to the context. In our case, equine welfare appears essential for self-organized equestrian people. These results confirm the finding about the importance of equine housing for horse-owners, shown by Hemsworth (2017) and Ruet et al. (2019). This research brings an original point of view on transmission value. The literature showed the influence of a heritage value inherited from the entourage. This notion, shown by Medberg et Heinonen (2014) in the context of banking, influences providers' choice according to their family's previous experiences. However, in equestrian self-organization, the results reflect the idea of transmission to relatives. The transmission value consists in social sharing with its entourage. This transmission value deserves to be developed and investigated in new research in other sports. Concerning autonomy, this research is promising. Specific factors like the different value components influence the importance of overall self-organization value, hence the choice to be self-organized. Unfortunately, the constructs of autonomy was not confirmed during the EFA, so not included in the model, but the strong influence of self-efficacy on the overall value of self-organization, hedonic value, economic value, ethical value, and risk of error proves the importance of trusting one’s own ability to really value self-organization. The non-significant effect of status value underlines an ambivalence about horse riding, which is often considered as elitist and as the self-expression of high social status. In equestrian self-organization, the status does not seem important in the overall value. The practice of equestrian activities is often perceived as being reserved for the higher socioeconomic classes (Bourdieu, 1979) with a prestigious status. Triggers for Self-Organization The image of horses in magnificent stud farms with white fences is often representative of the privileged status of the owner. However, self-organized practitioners are more likely to come from diverse 21 backgrounds (Chantelat et al., 1998; Galewicz, 2017). People become self-organized either because of the passion for the animal or the passion for the activity, rather than for the status they acquire (Eslan et al., 2023). The status value of having a self-organizing equine is thus inconclusive. The economic value is seen by Aurier et al. (2004) as part of the utilitarian value. The results are in line with these conclusions, as the economic value influences the overall value of self-organization. However, even if economic arguments are considered in the choice to self-organize (Eslan et al., 2023), this value is not as important as we expected. The non-mediation effect result of risk of error suggests further investigation. Mitchell (1999) specifically calls for perceived risk research in leisure activities as empirical studies tend to focus on a medical approach of injuries risks, which are not the exact scope of this paper. Nevertheless, in these last ones, the notion of risk corresponds to the risks incurred in terms of health. The risk of error is thus composed by the perception of the risks incurred and the ability to manage these risks (Meyer et Verlhiac, 2004). In our case study, we can assume that people are worried about major risks for their horses, or worried about endangering their horses. This creates possible mental load and stress for self-organized users. Furthermore, this variable could be linked to people's skills, beliefs, and constraints. Adding constructs related to mental load and sacrifices could enrich the theoretical model to better understand the effect of the risk of error. These elements are in line with the results on the transmission value, which underline an omitted element that could be time constraints. Self-organized users sacrifice their available time to transmit their passion and organization. Finally, a question arises about the effect of sacrifices, constraints, and risks on overall value of self- organization, which opens the way to research avenues. Moreover, according to Zhao et al. (2011)’s interpretation, considering the risk of error as a moderator rather than a mediator should also be investigated in future research to enrich and improve the theoretical model. 22 The object of attachment, here the horse, is the self-organization keystone. Triggers for Self-Organization The direct influence on overall value as well as its indirect effects via ethical value and hedonic value are of high importance in self-organization’s overall value. These results are in line with the work of Le Clinche et al. (2017) who show that attachment to the horse is also the motivation to participate in equestrian shows. However, the link between attachment and transmission is new. Indeed, Medberg et Heinonen (2014) only showed that a form of relational inheritance was involved in the decision to choose a bank. This study, in self-organization context, shows that the attachment to the horse makes one want to transmit to relatives. Measurement Scales Evaluation The measurement of overall value in our study follows the work of Aurier et al. (2004). This measuring scale compares benefits and sacrifices of self-organization. This scale raises the question of how to measure the overall value in general. It would also be interesting to explore how constraints influence the model and the overall value, in line with Bornemann (2020) findings. Indeed, she shows that depending on how a person is engaged with his/her horse, the way time is consumed fluctuates, as the constraints differ according to individuals and the environment (Dashper et Brymer, 2019). Even if this model seems promising, some limitations persist in this study, such as the low reliability of some constructs (i.e. attachment, transmission value), although sufficient for this type of modelling. Overall, the measurement scales need reworking to confirm the theoretical model, especially for the risk of error, transmission value, hedonic value, attachment, and overall value. This would enable to extend the use of this model to other self- organized activities. The number and variability of individuals in the sample could be increased. Thus, reworking the scales and the sample are prospective avenues. 23 Managerial Implications Managerial Implications With a focus on the consumer and the value components, this study allows a better understanding of the needs and expectations of self-organized users. Therefore, businesses' tasks are to adapt and innovate to meet individual consumers' expectations better. These results show the importance of adapting the services offer to the self-organized equestrian user’s needs. This work raises the question of organization in terms of learning and the role of companies in training self-organized users. A learning program through apprenticeship should reinforce the search for autonomy instead of dependence for self-organized users. The self- organization phenomenon implies a strong contribution from the consumer-producer and a minor contribution from the providers. The provider role is seen more as a support than a creator of the final service (Rayna et al., 2015). Breaking away from a federal sport model, self-organized users are nevertheless sometimes forced to use commercial or private service providers for support (Gaubert, 2016). Thus, as observed in innovation on online platforms, equestrian companies can co-create new services according to the identified needs of their clients, to support them in their project. 24 This first approach of analyzing value components in equestrian self-organization shows the importance of specific values. Considering these results, a user typology approach would highlight the importance of specific values according to the different profiles of self- organized users and according to the organization types. Professional structures need to address this issue, to at least keep their customers or develop co-creation activities with these users. The notion of unperceived sacrifices, constraints, and risks remains to be considered. According to the self-organized users’ expectations, several types of services are relevant. The attachment to their equine, their desire to respect its welfare, and the wish to transmit their way of living with their horse must be considered by professionals. A comparative analysis between self-organized users and users of professional structures would be 24 interesting as their values may differ and therefore influence the overall value of organization differently. Conclusion To conclude, this paper analyzes the effects of self-efficacy, attachment, and value components on overall value of self-organization. Economic value is not the main and only value to consider. Hedonic, ethic, and transmission values appear as major components and mediators of the influence of attachment and self-efficacy on the overall value. Nevertheless, these mediators’ effects of value components can vary greatly according to the environment and the skills of the individuals, which limits the globalization of the results. Among all the effects on overall value of self-organization, we can notice the high importance of ethical value and the attachment to the animal. This research adds the concept of transmission value to the literature on autonomy in sport. At the same time, our results raise questions about the effects of risks, constraints, and sacrifices that are not completely encompassed in this study and open the way to further research on self-organization. This study provides valuable results for the equine industry, suggesting the importance of adapting professionals' offer to self-organized equestrians to value components that are essential for the organization of these users. Equestrian services offered in the professional structures would then serve as a support by offering a network and adapted advice for self- organized people. Moreover, the case of self-organized equestrian users could be transferable, on the one hand, to other activities with animals, such as canicross or dog mushing, but also to activities without animals, as the roles of attachment and ethical value could be found in natural settings with hikers or the mountain with climbers, as suggested by Crockett et al. (2022). 25 This research warns of the need to keep an open mind about new self-organized practices and consumers’ expectations that they reflect. Moreover, in the animal field research, animal welfare and its perceptions have to be deeply studied. Acknowledgements: This research was funded by the French Equestrian Federation (FFE), the Scientific Council of the French equine industry and the ANRT grant n°2018/1491. 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Reconsidérer Baron et kenny : mythes et vérités à propos de l'analyse de médiation [Reconsidering Baron and Kenny: Myths and Truths about Mediation Analysis]. Recherche Et Applications En Marketing (RAM), 26(1), 81–96. Appendix : Table of initial items for quantitative survey constructs (in French) Authors Initial items Variable name Presence in final scale Gillet et al. (2008) 6 items AUTONOMY Dans mon organisation Hors Structure, souvent je ne me sens pas très compétent Risqerr3 X Dans mon organisation Hors Structure, j'ai le sentiment de bien réussir Aut2 Dans mon organisation Hors Structure, je peux prendre des décisions pour la gestion de mon/mes équidé(s) Aut3 Dans mon organisation Hors Structure, je me sens libre d'exprimer mes idées et mes opinions Aut4 Mon mode d'organisation Hors Structure me permet d'être en contact avec des personnes pour lesquelles j'ai beaucoup de sympathie Aut5 Dans mon organisation Hors structure, je me sens à l'aise avec les autres Aut6 Bures et al. REFERENCES (2019) 3 items ANIMAL ATTACHMENT Je passe du temps chaque jour à jouer ou faire de l’exercice avec mon/mes équidé(s) Attach1 Je considère mon/mes équidé(s) comme un/des membre(s) de ma famille Attach2 Quand je me sens mal, je cherche du réconfort auprès de mon/mes Attach3 X Appendix : Table of initial items for quantitative survey constructs (in French) Appendix : Table of initial items for quantitative survey constructs (in French) 30 31 équidé(s) Rousseau et al. (2002) 4 items PASSION Je ne peux pas me passer de mon/mes équidé(s) Passion1 X Les choses nouvelles que je découvre avec mon/mes équidé(s), me permettent de l'apprécier davantage Passion2 X J'ai un sentiment qui est presque obsessif pour mon/mes équidé(s) Passion3 Être avec mon équidé me permet de vivre des expériences variées. Passion4 X Scholz et al. (2002) 5 items SELF-EFFICACY J’ai confiance dans le fait que je pourrais faire face efficacement à des évènements inattendus Selfeff1 X Je peux rester calme lorsque je suis confronté(e) à des difficultés car je peux me fier à mes habiletés pour faire face aux problèmes Selfeff2 X Peu importe ce qui arrive, je suis généralement capable d’y faire face. Selfeff3 X Je suis certain de pouvoir accomplir mes objectifs Selfeff4 X Lorsque je suis confronté(e) à un problème, je suis capable de trouver plusieurs solutions Selfeff5 Medberg et Heinonen (2014) 3 items HERITAGE VALUE -> TRANSMISSION VALUE Être autonome pour la gestion et l’entretien de mon/mes équidé(s) me permet de continuer des habitudes familiales Her1 X Ma famille m'a transmis ses connaissances dans la gestion et l'entretien de mon/mes équidé(s) Her2 Être autonome dans la gestion et l'entretien de mon/mes équidé(s) me permet de transmettre mes connaissances à mes proches (amis ou famille) Trans2 X Matthwick et al. REFERENCES (2001) 2 items ECONOMIC VALUE Ce mode d’organisation est avantageux d’un point de vue économique Eco1 X Dans l'ensemble, je suis content(e) des coûts liés à ce mode d'organisation Eco2 X From qualitative verbatim 6 items RISK OF ERROR / SACRIFICES Être autonome pour la gestion et l’entretien de mon/mes équidé(s) est lourd à gérer au quotidien Risqauto Il est plus risqué pour mes équidés d’être Hors structure que d’être dans une structure professionnelle Risqequi Je suis inquiet(e) ou stressé(e) de ce qui pourrait arriver à mon/mes équidé(s) Risqerr4 X Je ne trouve pas le temps de faire autre chose que de m’occuper de mes équidés Risqtps Mon/mes équidé(s) m'oblige(nt) à être présent et m'empêche(nt) de partir en vacances Risqvac J’ai peur de me tromper ou de faire des erreurs dans la gestion et l’entretien de mon/mes équidé(s) Risqerr1 X Innocent et François- Lecomte (2020) 3 items HEDONIC VALUE Être autonome dans la gestion et l’entretien de mon/mes équidé(s), c’est contraignant Hedo3 X Pour moi, être autonome dans la gestion et l’entretien de mon/mes équidé(s), c’est comme un jeu Hedo4 Quand je suis autonome dans la gestion et l’entretien de mon/mes équidé(s) et que mon/mes équidé(s) est/sont bien, c’est une récompense à mes efforts Hedo5 Matthwick et al. (2001) 2 items Être autonome dans la gestion et l’entretien de mon/mes équidé(s) est distrayant Hedo1 X Être autonome dans la gestion et l’entretien de mes équidés me procure un sentiment de bonheur Hedo2 X Innocent et François- Lecomte (2020) 4 items SOCIAL VALUE J’aime partager mon expérience au sujet de la gestion et de l’entretien de mon/mes équidé(s) avec ma famille et mes relations Trans1 X Ce n’est pas toujours bien vu d’être autonome dans la gestion et l’entretien de ses équidés Soc2 Être attentif à la gestion et l’entretien de mon/mes équidé(s) entraîne parfois des tensions au sein de mon foyer Soc3 Je me sens un peu isolé(e) dans ma démarche d’être autonome dans la Soc4 équidé(s) Rousseau et al. (2002) 4 items PASSION Je ne peux pas me passer de mon/mes équidé(s) Passion1 X Les choses nouvelles que je découvre avec mon/mes équidé(s), me permettent de l'apprécier davantage Passion2 X J'ai un sentiment qui est presque obsessif pour mon/mes équidé(s) Passion3 Être avec mon équidé me permet de vivre des expériences variées. Passion4 X Scholz et al. REFERENCES (2002) 5 items SELF-EFFICACY J’ai confiance dans le fait que je pourrais faire face efficacement à des évènements inattendus Selfeff1 X Je peux rester calme lorsque je suis confronté(e) à des difficultés car je peux me fier à mes habiletés pour faire face aux problèmes Selfeff2 X Peu importe ce qui arrive, je suis généralement capable d’y faire face. Selfeff3 X Je suis certain de pouvoir accomplir mes objectifs Selfeff4 X Lorsque je suis confronté(e) à un problème, je suis capable de trouver plusieurs solutions Selfeff5 Medberg et Heinonen (2014) 3 items HERITAGE VALUE -> TRANSMISSION VALUE Être autonome pour la gestion et l’entretien de mon/mes équidé(s) me permet de continuer des habitudes familiales Her1 X Ma famille m'a transmis ses connaissances dans la gestion et l'entretien de mon/mes équidé(s) Her2 Être autonome dans la gestion et l'entretien de mon/mes équidé(s) me permet de transmettre mes connaissances à mes proches (amis ou famille) Trans2 X Matthwick et al. (2001) 2 items ECONOMIC VALUE Ce mode d’organisation est avantageux d’un point de vue économique Eco1 X Dans l'ensemble, je suis content(e) des coûts liés à ce mode d'organisation Eco2 X 31 gestion et l’entretien de mon/mes équidé(s) Bhattacharya et al. (1995) 2 items Dans mon groupe social, c'est considéré comme prestigieux de gérer ses équidés en autonomie Statut1 Les personnes qui gèrent de façon autonome leurs équidés n'ont pas une très bonne réputation dans mon groupe social. REFERENCES Statut2 ALTRUISTIC VALUE / ETHICAL VALUE From qualitative verbatim 2 items Être autonome dans la gestion et l’entretien de mon/mes équidé(s) permet de répondre aux besoins de mon/mes équidé(s) Ethic1 X Être autonome dans la gestion et l’entretien de mon/mes équidé(s) respecte leur bien-être Ethic2 X Innocent et François (2020) 2 items Je trouve éthique d’être autonome dans la gestion et l’entretien de mon/mes équidé(s) Ethic3 X Être autonome dans la gestion et l’entretien de mon/mes équidé(s) donne l’impression de participer, à son niveau, à quelque chose de plus grand Ethic4 Innocent et François- Lecomte (2020) 4 items INSTRUMENTAL VALUE : KNOWLEDGE J’aime en savoir plus sur la gestion et entretien des équidés Conn1 J’aime observer mon/mes équidé(s) Conn2 Cela m’est difficile d’obtenir les informations nécessaires pour être autonome dans la gestion et l’entretien de ses équidés Conn3 Dans le domaine de la gestion et l’entretien de mon/mes équidé(s), je suis gêné par le fait de ne pas toujours savoir comment faire Risqerr2 X OVERALL VALUE Aurier et al. REFERENCES (2004) 3 items Globalement, être autonome dans la gestion et l’entretien de mon/mes équidé(s) vaut bien l'énergie et le temps que j'y consacre Val1 X Globalement, être autonome dans la gestion de mon/mes équidé(s) vaut bien les sacrifices auxquels je consens Val2 X Globalement, être autonome dans la gestion de mon/mes équidé(s) vaut bien les risques que je dois prendre pour le faire Valglo3 From qualitative verbatim 2 items Être autonome dans la gestion de mon/mes équidé(s), c’est au-dessus de tout, ce n’est pas mesurable Novaleur1 Le fait d’avoir mon/mes équidé(s) chez moi, c’est irremplaçable Val 3/ Novaleur2 X Appendix 2 Cross loadings matrix Appendix 2 Cross loadings matrix Animal attachment Economic value Risk of error Ethical value Hedonic value Overall value Self- efficacy Transmission value attach2 0,554 0,026 0,093 0,209 0,279 0,265 0,011 0,148 passion1 0,613 -0,019 0,069 0,197 0,309 0,348 0,037 0,177 passion3 0,777 0,152 0,047 0,309 0,437 0,364 0,138 0,381 passion4 0,760 0,114 -0,023 0,270 0,472 0,392 0,254 0,407 eco1 0,043 0,617 -0,088 0,002 0,040 0,036 0,082 0,088 eco2 0,123 0,978 -0,171 0,164 0,231 0,231 0,223 0,152 risqerr1 0,106 -0,153 0,831 -0,117 0,029 -0,078 -0,447 0,016 risqerr2 -0,035 -0,138 0,852 -0,190 -0,082 -0,189 -0,519 -0,086 risqerr3 0,000 -0,069 0,771 -0,181 -0,041 -0,137 -0,419 -0,031 risqerr4 0,155 -0,190 0,544 -0,022 0,053 -0,004 -0,231 0,086 ethic1 0,301 0,112 -0,229 0,830 0,357 0,385 0,336 0,245 ethic2 0,282 0,072 -0,142 0,817 0,294 0,349 0,236 0,211 ethic3 0,246 0,151 -0,009 0,603 0,276 0,307 0,168 0,215 hedo1 0,368 0,186 -0,016 0,284 0,711 0,348 0,196 0,312 hedo2 0,446 0,139 -0,020 0,358 0,807 0,505 0,200 0,357 hedo3 0,436 0,154 -0,020 0,265 0,703 0,366 0,198 0,335 val1 0,373 0,169 -0,188 0,379 0,443 0,808 0,302 0,208 32 val2 0,373 0,156 -0,097 0,332 0,438 0,774 0,284 0,191 val3 0,404 0,154 -0,047 0,338 0,381 0,689 0,212 0,207 selfeff1 0,096 0,117 -0,401 0,231 0,112 0,204 0,724 0,133 selfeff2 0,101 0,168 -0,463 0,208 0,203 0,256 0,756 0,132 selfeff3 0,119 0,139 -0,402 0,267 0,181 0,242 0,772 0,139 selfeff4 0,225 0,206 -0,391 0,292 0,278 0,337 0,750 0,261 trans1 0,386 0,084 0,013 0,249 0,385 0,251 0,175 0,836 trans2 0,363 0,111 -0,034 0,239 0,355 0,179 0,168 0,823 trans3 0,130 0,195 -0,047 0,128 0,207 0,126 0,170 0,421 33
https://openalex.org/W4311852519	https://chemrxiv.org/engage/api-gateway/chemrxiv/assets/orp/resource/item/638d884744ccbcb23f0e63fe/original/central-role-of-entropy-in-thermodynamics.pdf	English	null	Central role of entropy in thermodynamics	null	2,022	cc-by	8,516	ABSTRACT Providing a simple description of entropy as the foundation of thermodynamics is necessary for researchers in explaining physical phenomena in the universe. Here we dive deep into the meaning of entropy starting from the basics and ending with Newton’s second law of motion which may be rooted in a hidden dimension as the foundation of entropy. Toward this end, after coming up with a general relation for the net entropic energy of a process regardless of its irreversibility, we recover the entropy production relation. Using this relation, we show how the inequality in macroscopic states between work and Helmholtz free energy during the non-equilibrium process converts to equality between reversible microscopic works of microscopic degrees of freedom and Helmholtz free energy change of maximum useful work. In the end, we provide a mathematical solution to this equality that may tell us about the foundation of entropy from underlying degrees of freedom that currently remain unknown to us. Keywords Thermodynamics · Entropy · Information · Force · Free Energy 1 Introduction Entropy etymologically is a derivative of a combination of two Greek words, Entropy etymologically is a derivative of a combination of two Greek words, (1) Entropy = ϵν + τρoπ´η (1) Entropy = ϵν + τρoπ´η where ϵν means inside and τρπ´η means the built-in energy of a system. The Greek root of the entropy term is an interpretative description of this phenomenal concept. More accessible states results in higher multiplicity (w) and entropy (S) within a system, S = k ln w (2) (2) S = k ln w A PREPRINT Mohsen Farshad Department of Chemistry Temple University Philadelphia, PA 19122 mohsen.farshad@temple.edu Mohsen Farshad Department of Chemistry Temple University Philadelphia, PA 19122 mohsen.farshad@temple.edu December 7, 2022 S = k ln w Toward this end, we begin by elaborating on why this concept was founded by comparing thermodynamics with Newtonian mechanics. Then we discuss the reversibility of a process and how an irreversible process from a macroscopic perspective is encompassed by reversible microscopic motions of particles within the system. In the end, we drive Newton’s second law of motion from the work and energy equality relation of non-equilibrium statistical mechanics. extra energy to the environment through seemingly endless morphological changes in the self-assembled topological landscape with the infinitely jagged and wiggly fractal curve at the system’s boundaries of the infinite perimeter.(4) These changes can manifest through shoreline evolution, the shape and height of the sand bars in beach breaker regions, and beach dunes. These collective changes result in a temporarily new quasistatic equilibrium state— time-dependent dynamic equilibrium— with relative stability in the properties of water and land at the interface and beyond. The water and land ultimately reach an equilibrium state where their properties only are subjected to slight fluctuations. However, the scale of these fluctuations, for instance, the change in the shoreline position, is extremely different from the fluctuations that are commonly used in statistical mechanics for microscopic degrees of freedom when the system is in equilibrium with its surounding. The question that is risen in mind is: Can a large macroscopic system or universe be considered in an equilibrium state but still with ongoing random oscillations on a proportional scale resulting from the collective microscopic fluctuations of constituting particles? While in this study, we implicitly provide insight to answer this question, our main focus remains to clarify the meaning of entropy in a fluent language. Toward this end, we begin by elaborating on why this concept was founded by comparing thermodynamics with Newtonian mechanics. Then we discuss the reversibility of a process and how an irreversible process from a macroscopic perspective is encompassed by reversible microscopic motions of particles within the system. In the end, we drive Newton’s second law of motion from the work and energy equality relation of non-equilibrium statistical mechanics. S = k ln w where k is Boltzmann’s proportionality factor. Reversely, more integrity in the stiffness of a system that results in the decrease of the system’s entropy is caused by entropic forces on the constituents of the system. Before we get into elegant mathematical expressions developed on basis of the entropy concept and their physical implications, we first use the beauty of nature (1; 2) to ease its explanation in a plain context. We picture in mind a mechanistic view through which an out-of-equilibrium system reaches an equilibrium state through the dissipation of entropic energy in and out of its body. Inspired by ecology and landscape, we choose our system of interest in nature to be a beach where the water and land interface with each other separated by a shoreline.(3) Imagine our hypothetically closed system undergoes an applied external energy driving it away from equilibrium to a non-equilibrium state of energy. The external field can lead to an increase in the water level or the height of the waves. In this scenario, the system strives to dissipate its unbearable Central role of entropy in thermodynamics A PREPRINT extra energy to the environment through seemingly endless morphological changes in the self-assembled topological landscape with the infinitely jagged and wiggly fractal curve at the system’s boundaries of the infinite perimeter.(4) These changes can manifest through shoreline evolution, the shape and height of the sand bars in beach breaker regions, and beach dunes. These collective changes result in a temporarily new quasistatic equilibrium state— time-dependent dynamic equilibrium— with relative stability in the properties of water and land at the interface and beyond. The water and land ultimately reach an equilibrium state where their properties only are subjected to slight fluctuations. However, the scale of these fluctuations, for instance, the change in the shoreline position, is extremely different from the fluctuations that are commonly used in statistical mechanics for microscopic degrees of freedom when the system is in equilibrium with its surounding. The question that is risen in mind is: Can a large macroscopic system or universe be considered in an equilibrium state but still with ongoing random oscillations on a proportional scale resulting from the collective microscopic fluctuations of constituting particles? While in this study, we implicitly provide insight to answer this question, our main focus remains to clarify the meaning of entropy in a fluent language. 2 Results and Discussion The total energy of a system in classical mechanics is described by the sum of the potential and kinetic energies: gy of a system in classical mechanics is described by the sum of the potential and kinetic energies: E = Ek + Ep (3) (3) E = Ek + Ep As it is shown in Eq. 3 the entropy effect is not local to the total energy, and therefore the explicit implementation of the entropy in the classical mechanics of macroscopic bodies required further modification of the concept of conservation of energy. On the other hand, the total energy in thermodynamics is founded on the concept of entropy which results from random fluctuations of microscopic degrees of freedom within the bodies. In principle, part of the total energy of the system is expended to organize and force these Brownian degrees of freedom to move in a specific direction to perform useful work. To intuitively understand the connection between Newtonian mechanics and thermodynamics, we create an arbitrary example where the change of the total energy is concerned rather than its absolute value. Because physically compre- hending the concept of entropy and its relation to absolute potential energy for useful work is extremely counterintuitive. 2 Results and Discussion Our descriptive example is as follows: If we apply external energy (Eex) to the system based on the conservation of energy If we apply external energy (Eex) to the system based on the conservation of energy (4) Etot,i + Eex = Ek,i + Ep,i + Eex (4) a portion of the external energy initially in the form of potential energy converts to the movement of the body as the kinetic energy and the other part will be stored in the system as shown by the following equation: a portion of the external energy initially in the form of potential energy converts to the movement of the body as the kinetic energy and the other part will be stored in the system as shown by the following equation: Etot,f = Ek,f + Ep,f (5) (5) then, the change of energy will be: then, the change of energy will be: ∆E = ∆Ek + ∆Ep (6) ∆E = ∆Ek + ∆Ep (6) ∆E = ∆Ek + ∆Ep (6) This equation reminds us of the first law of thermodynamics where the total energy of a system is: This equation reminds us of the first law of thermodynamics where the total energy of a system is: (7) ∆E = ∆Ek,mirco + ∆Ep,micro (7) ∆E = ∆Ek,mirco + ∆Ep,micro the measurement of the collective kinetic and potential energies composed of all microscopic degrees of freedom in a chaotic system is abstract. However, the total energy change is just the difference between the heat that the system absorbs from its surrounding and the work that the system performs on the surrounding: ∆E = Q −W (8) ∆E = Q −W (8) 2 Central role of entropy in thermodynamics A PREPRINT Where Q is the applied heat from surrounding to the system and W is part of the energy that system expend to perform work on the surrounding. While Q is reminiscent of the external energy, we show that W in the form of expansion work in absence of additional works (Wnon−pv) is equal to the potential energy change in classical mechanics theory. Below we elaborate on this similarity with an illustrative explanation of each work with the aid of a schematic representation for these two theories in Figure 1. 𝐴! = 𝐸! −𝑇𝑆! 𝑄 Δ𝐸!"!,$%%&' = 𝐹Δ𝑥 𝐸𝑒𝑥 𝐸" = 𝑚𝑔ℎ" 𝐴" = 𝐸" −𝑇𝑆" Δ𝐸!"!,$%%&' = 𝑄+ 𝐹Δ𝑥 𝐸! = 𝑚𝑔ℎ! 2 Results and Discussion Figure 1: A simple depiction of the change of energy in Newtonian mechanics and thermodynamics due to an external force. Figure 1: A simple depiction of the change of energy in Newtonian mechanics and thermodynamics due to an external force. le depiction of the change of energy in Newtonian mechanics and thermodynamics due to an externa Imagine we apply external energy to a piston that has separated two boxes initially at equilibrium with each other (Ek,i = 0). According to Newtonian mechanics, the piston moves in the direction of the external energy in the absence of other forces 1. After the piston reaches a new equilibrium state (Ek,f = 0), the stored energy in the upper box will be: ∆E = F∆h (9) (9) F is the force that has displaced the box of mass m. The average force applied on the upper box is: F = mg (10) (10) F = mg therefore: (11) ∆E = mg∆h (11) On the other hand, instead of mechanical applied energy, imagine you transfer heat to a closed system with a piston that is initially at equilibrium. Part of this energy converts to expansion work in absence of other forces except for entropic force. Under these circumstances, the change in total energy of the system will be: On the other hand, instead of mechanical applied energy, imagine you transfer heat to a closed system with a piston that is initially at equilibrium. Part of this energy converts to expansion work in absence of other forces except for entropic force. Under these circumstances, the change in total energy of the system will be: (12) ∆Esys,lower = Q −P∆Vsys,lower (12) ∆Esys,lower = Q −P∆Vsys,lower Q is a measure of the applied entropic energy that is transferred to the system from surrounding. In Eq. 12 when one side of the system expands the other side compresses (P∆Vsys,upper = −P∆Vsys,lower). We assume the cross-section area of the system stays constant (A). In this regard, the total thermodynamic energy for the upper system will be (13) ∆Esys,upper = Q + F∆hsys,upper (13) ∆Esys,upper = Q + F∆hsys,upper Comparing Eq. 13 and Eq. 9, we understand that the difference between the total energy change in Newtonian mechanics and thermodynamics theories is the presence of heat in thermodynamics. Central role of entropy in thermodynamics 14: Now, we substitute the total energy of the system (∆Esys) into Eq. 14: Now, we substitute the total energy of the system (∆Esys) into Eq. 14: ∆Asys = Qsys −{T∆S}sys −Wsys (15) ∆Asys = Qsys −{T∆S}sys −Wsys (15) (15) the amount of heat transfer to the system as a result of the entropy change of the surrounding from the system’s perspective is: the amount of heat transfer to the system as a result of the entropy change of the surrounding from the system’s perspective is: ∆Ssur = Z f i {Q T }sur (16) (16) where i and f denote the final and initial equilibrium states of the surrounding. Because we assume the universe is infinitely large, therefore in a coupled system and surrounding entity with a limited size of the system, the surrounding is infinitely large. Consequently, its temperature remains constant during heat transfer and we can write: ∆Ssur = {Q T }sur (17) (17) with substitution of Eq. 17 into Eq. 15, we derive following: ∆Asys = {T∆S}sur −{T∆S}sys −Wsys (18) (18) In this relation—if we put the work that is done on the surrounding aside—the net entropic energy that is absorbed by the system and released from it would be equal to the Gibbs free energy change (∆G). if we put the work that is done on the surrounding aside—the net entropic energy that is absorbed by leased from it would be equal to the Gibbs free energy change (∆G). ∆Gsys = T∆Ssys,net = T∆Ssur −T∆Ssys (19) (19) ∆Gsys = T∆Ssys,net = T∆Ssur −T∆Ssys G is a measure of the non-expansion work that a system can perform on the surrounding in a given process, and ∆G is the maximum non-expansion work that can be extracted from the system. Furthermore, ∆G can determine the spontaneity of a process. Principally, a process that releases more energy than it receives is spontaneous, and vice versa. Therefore, the absolute value of T∆Ssys should be larger than T∆Ssur as we discuss in the following. 2 Results and Discussion We know that the transferred heat that performs expansion work and therefore converts to the potential energy of the upper system is originally rooted in the entropic energy of the surrounding. This heat transfer to the system causes an increase in the system’s entropy which in turn leads to the return of the heat to the surrounding as the system expands and loses its energy. In fact, Comparing Eq. 13 and Eq. 9, we understand that the difference between the total energy change in Newtonian mechanics and thermodynamics theories is the presence of heat in thermodynamics. We know that the transferred heat that performs expansion work and therefore converts to the potential energy of the upper system is originally rooted in the entropic energy of the surrounding. This heat transfer to the system causes an increase in the system’s entropy which in turn leads to the return of the heat to the surrounding as the system expands and loses its energy. In fact, 3 Central role of entropy in thermodynamics Central role of entropy in thermodynamics A PREPRINT it is the increase in the stochastic fluctuations of microscopic degrees of freedom that lead to an increase in the heat and when these motions reach the system’s boundaries, the heat transmits to the surrounding. Also, for the system to perform useful work, some of its energy is lost as dissipated work owing to the very random Brownian motions of particles. On the basis of this information, we comprehend that to extract equivalent work in thermodynamics to Newtonian mechanics in a real system, the required amount of heat should be larger than the corresponding applied external energy in Newtonian mechanics. To understand this further we need to discuss Helmholtz free energy. If we subtract the reciprocal heat from total energy, the Helmholtz free energy change of the system will return as follows: it is the increase in the stochastic fluctuations of microscopic degrees of freedom that lead to an increase in the heat and when these motions reach the system’s boundaries, the heat transmits to the surrounding. Also, for the system to perform useful work, some of its energy is lost as dissipated work owing to the very random Brownian motions of particles. On the basis of this information, we comprehend that to extract equivalent work in thermodynamics to Newtonian mechanics in a real system, the required amount of heat should be larger than the corresponding applied external energy in Newtonian mechanics. To understand this further we need to discuss Helmholtz free energy. If we subtract the reciprocal heat from total energy, the Helmholtz free energy change of the system will return as follows: ∆Asys = ∆Esys −{T∆S}sys (14) (14) ∆Asys = ∆Esys −{T∆S}sys A is a measure of the useful work that a system can perform on the surrounding. ∆A determines the maximum amount of energy within the system that can convert to useful work (W = WpV + Wnon−pV ) during a thermodynamic process. The Helmholtz free energy change accounts for both expansion (WpV ) and non-expansion Wnon−pV works. A is a measure of the useful work that a system can perform on the surrounding. ∆A determines the maximum amount of energy within the system that can convert to useful work (W = WpV + Wnon−pV ) during a thermodynamic process. The Helmholtz free energy change accounts for both expansion (WpV ) and non-expansion Wnon−pV works. te the total energy of the system (∆Esys) into Eq. Central role of entropy in thermodynamics Central role of entropy in thermodynamics A PREPRINT Under these circumstances, we comprehend how in a spontaneous process within a system ∆Gsys < 0 or ∆Asys + p∆Vsys < 0. Conversely, when ∆G > 0 or ∆Asys + p∆Vsys > 0 the process is not spontaneous but still feasible when the applied heat from the surrounding is more than the net heat from the system to the surrounding. This does not violate the second law of thermodynamics which states a real physical process is always accompanied by an increase in the total entropy of the universe. Certainly, in an expanding universe, the total free energy of the universe always decreases as in a real process (∆Guni < 0). The heat transfer from the surrounding to the system causes an increase in the entropy of the system, and from the system to the surrounding leads to a decrease in the entropy of the system. Be aware that in either system and surrounding free energy change formulas, we calculate the difference in the transferred heat and the change in the entropy which accounts for the heat transfer from the surrounding to the system and from the system to the surrounding—from the system’s and surrounding’s perspectives. As the surrounding loses heat during the heat transfer to the system and Q is negative, the (Eq. 17) from surrounding’s perspective is: ∆Ssur = −{Q T }sur (22) (22) and (Eq. 20) accordingly from surrounding’s perspective is: ∆Ssys = − Z f i {δQ T }sys (23) (23) consequently, for the surrounding, the following is the relation of the Helmholtz free energy: consequently, for the surrounding, the following is the relation of the Helmholtz free energy: ∆Asur = {T∆S}sys −{T∆S}sur −Wsur (24) (24) and accordingly, Gibbs’s free energy relation is: ∆Gsur = {T∆S}sur,net = {T∆S}sys −{T∆S}sur (25) (25) We can say that while a process accompanies the decrease of free energy change within the system, it increases in surrounding for the same process, and the sum of the free energies of the system and surrounding, based on the conservation of energy must be zero. This is similar to Newton’s third law of motion where two bodies that are in contact apply forces on each other, these forces that are in opposite directions would have the same magnitude. This means, we just look at the same process from two perspectives of the system and surrounding. Central role of entropy in thermodynamics Due to the increase of the system’s temperature during the heat transfer, its entropy change relationship with infinitesimal changes of heat (δQ) is: ∆Ssys = Z f i {δQ T }sys (20) (20) The nonlinear temperature dependency of entropy change of the system during the heat transfer does not allow us to factor out the heat from the integral, and as a result, the solution to this integral is not straightforward in contrast to the one that we use the constant temperature of infinitely sized heat bath for calculating the surrounding entropy change. Since the temperature of the system is lower than the surrounding during a spontaneous heat transfer to the system, the entropy change of the system has a higher magnitude than the entropy change of the surrounding. \|∆Ssys\| > \|∆Ssur\| (21) (21) \|∆Ssys\| > \|∆Ssur\| 4 Central role of entropy in thermodynamics This means that there is no net entropy flow between the system and the surrounding: ∆Suni = T∆Ssur + T∆Ssys = 0 (30) ∆Suni = T∆Ssur + T∆Ssys = 0 (30) (30) Similarly, the net work and the Gibbs energy change in a reversible cyclic process is zero: (31) ∆Gcyc = T∆Ssur −T∆Ssys + T∆Ssys −T∆Ssur = 0 (31) In a hypothetical reversible process, the absolute value of the entropic term for the surrounding and system are equal but they can be negative or positive individually. In a nearly impossible reversible process when the system is in the absolute equilibrium state, the exchange of heat between the system and surrounding is zero. In another word, the net entropy from both the system and surrounding perspective is zero and therefore the universe is zero. We can make hypothetical examples of a perfect system to maintain the energy of the system the same during a cyclic process like a simple pendulum or harmonic oscillator when the opposing forces such as friction and air resistance are neglected. Nevertheless, this contrasts with the second law of thermodynamics in a currently expanding universe where a natural process accompanies an increase in total entropy. In a hypothetical reversible process, the absolute value of the entropic term for the surrounding and system are equal but they can be negative or positive individually. In a nearly impossible reversible process when the system is in the absolute equilibrium state, the exchange of heat between the system and surrounding is zero. In another word, the net entropy from both the system and surrounding perspective is zero and therefore the universe is zero. We can make hypothetical examples of a perfect system to maintain the energy of the system the same during a cyclic process like a simple pendulum or harmonic oscillator when the opposing forces such as friction and air resistance are neglected. Nevertheless, this contrasts with the second law of thermodynamics in a currently expanding universe where a natural process accompanies an increase in total entropy. ∆Suni = T∆Ssur + T∆Ssys > 0 (32) (32) ∆Suni = T∆Ssur + T∆Ssys > 0 In nature where we face reality, the processes are not reversible. In an irreversible cyclic process that takes place spontaneously in the system (\|∆Ssys\| > \|∆Ssur\|), the process can be forced to reverse its direction by external energy. Central role of entropy in thermodynamics The same amount of energy that is released from the system should return to the system in a specific form to reverse the process. For this to happen, we need to compensate the energy of the system to the same amount that it was before the forward process began at the initial equilibrium state. From thermodynamic equations, one perceives in a spontaneous process part of the energy is lost as dissipated work. During the transfer of energy to the system from surrounding, the entropy of the system increases more than the heat that is absorbed by the system such that in the system Q −T∆S < 0. The increase of the entropy of the system that is rooted in the increase in the chaosity of the system leads to the expansion work of −p∆V in the system when the degrees of freedom colloid with the system’s boundaries. In an irreversible process, we should compensate for the dissipated heat and useful work to reverse the direction of the process. Therefore, in a cyclic irreversible process as the system returns to its initial state, the net change of entropy of the system is zero. On the other hand, the total entropy change of the universe in an irreversible process is larger than zero. This means even though the entropy of the system stays constant, the entropy of the surrounding, and therefore universe in a real system increases. It is clear how the total entropy of the universe increases in a spontaneous process through the mobility of particles during the interaction between the system and its surrounding. For instance, in an exothermic combustion reaction, a massive amount of entropic energy that is stored in a highly ordered structure is released to the surrounding when activated energy is provided to the system. It is to some extent counterintuitive to understand how an irreversible non-spontaneous process leads to an increase in the entropy of the universe as it seems some part of the transferred heat is being lost in the system and surrounding. Also, according to free energy formulas, a spontaneous process from the system’s perspective may seem non-spontaneous from the surrounding’s perspective. For instance, through a heat transfer from a hot surrounding to a cold system, the system’s Gibbs free energy decreases, but it increases in the surrounding. Central role of entropy in thermodynamics In this regard, the total free energy of the universe is: ∆Auni = ∆Asur + ∆Asys (26) (26) ∆Auni = ∆Asur + ∆Asys where p∆Vsur = −p∆Vsys. Therefore, for a single process we write: where p∆Vsur = −p∆Vsys. Therefore, for a single process we write: ∆Auni = ∆Guni = {T∆S}sur −{T∆S}sys + {T∆S}sys −{T∆S}sur = 0 (27 (27) With a closer look at this equation for a single process, it is shown that the total entropy change of the universe can be extracted from this relationship as a sum of the entropy change of surrounding in the surrounding and the entropy change of the system in the system from an ether’s perspective: ∆Suni = ∆Ssur + ∆Ssys (28) (28) ∆Suni = ∆Ssur + ∆Ssys To derive the free energy change of the universe, no assumption about the reversibility of the process is made. We solely concluded that the total free energy change of the universe during a single process is always zero because the net entropic energies of the surrounding and system balance each other out. However, we do not provide any information regarding whether the system stays in the final equilibrium configuration or not. In another word, we only calculated the total free energy change of the universe by looking at a single process from the system’s and surrounding’s perspectives to show how the universe preserves its energy in a process with no judgment on the total entropy change of the universe with an arrow of time in either a reversible or irreversible process. Nevertheless, we still believe any system ultimately reaches thermodynamic equilibrium with its surrounding. 5 Central role of entropy in thermodynamics A PREPRINT Central role of entropy in thermodynamics A PREPRINT Central role of entropy in thermodynamics A PREPRINT To convey our conjecture we discuss three cases: a) a reversible cyclic process, b) an irreversible cyclic process, and c) a general form of irreversible process that can be continued by sequential processes of successive incomplete cycles. In a reversible cycle where, (29) ∆Acyc = T∆Ssur,f −T∆Ssys,f −p∆Vsys,f + T∆Ssys,r −T∆Ssur,r −p∆Vsur,r = 0 (29) ∆Acyc = T∆Ssur,f −T∆Ssys,f −p∆Vsys,f + T∆Ssys,r −T∆Ssur,r −p∆Vsur,r = 0 the exchange of heat is zero. This imaginary cyclic process can be roughly manifested in reality through an infinitesimally slow process where the system is in quasistatic equilibrium with its surrounding. Central role of entropy in thermodynamics Central role of entropy in thermodynamics A PREPRINT external force. However, the very chaotic behavior of particles in a dynamical system creates a net repulsive force during the exchange of heat and dissipation of energy in an irreversible process that results in the enlargement of the universe. The repetition of a cyclic process in real heat engines results in a turbulent of these chaotic interactions through back-and-forth processes and dissipation of energies in the system and surrounding which increases the size of the universe constantly with an arrow of time. external force. However, the very chaotic behavior of particles in a dynamical system creates a net repulsive force during the exchange of heat and dissipation of energy in an irreversible process that results in the enlargement of the universe. The repetition of a cyclic process in real heat engines results in a turbulent of these chaotic interactions through back-and-forth processes and dissipation of energies in the system and surrounding which increases the size of the universe constantly with an arrow of time. This is concluded if we deal with an irreversible cyclic process where the external energy force the system to return to its initial equilibrium state. But what if we have a natural irreversible process like a pendulum or oscillator in presence of friction and air resistance? We want to know what will happen if an incomplete cycle repeats successively in an irreversible manner through time. The non-equilibrium state dissipates its energy to the system and surrounding over time in repeated cycles until it reaches an equilibrium state of zero driving entropic energy. In this regard, for the free energy of successive processes in n number of quasi-cycles, according to thermodynamics we write: ∆Gsuc = ∞ X n=1 {T∆S}sur,2n−1 −{T∆S}sys,2n−1 + {T∆S}sys,2n −{T∆S}sur,2n = 0 (33) (33) therefore, the entropic energy of forward and reverse processes eventually become identical in classical thermodynamics, and we can write down lim n→∞ {T∆S}sur,n −{T∆S}sys,n = 0 (34) (34) During a cyclic process, this relation conforms with all four potential thermodynamic energies of ∆Usuc, ∆Hsuc, ∆Gsuc, and ∆Asuc, whether the process is reversible or irreversible as the ultimate final state is in equilibration. During a cyclic process, this relation conforms with all four potential thermodynamic energies of ∆Usuc, ∆Hsuc, ∆Gsuc, and ∆Asuc, whether the process is reversible or irreversible as the ultimate final state is in equilibration. Central role of entropy in thermodynamics A principal example of the physical meaning of this equation could be the following: The universe’s size reaches a maximum where the attraction forces overcompete the repulsion forces and the size of the universe start to decrease. Under this condition, the second law of thermodynamics is reversed and the entropy of the universe decreases in every single natural process (∆Suni < 0). (5) The decrease in size of the universe continues until the particles are very close to each other where the size of the universe is minimum and this time repulsive forces between them overcompetes the attraction forces. This time, the size of the universe starts to grow again (∆Suni > 0). These incomplete cycles continue for an adequately long time until the size of the universe damps to an equilibrium state similar to a real pendulum or harmonic oscillator, except there may remain only small thermal fluctuations in a reversible manner in the universe where ∆Suni = 0. In this case, microscopic particles in the universe would perpetually fluctuate around an absolute equilibrium state reversibly in accessible microscopic states through the undamped oscillation of free energies that we present in Figure 2. Given this mindset, we can also say that a cyclic process with large modes of periodic oscillation in the free energy change is reversible (Figure 2: left graph). On the other hand, in a non-equilibrium system depending on how far it is driven from the equilibrium and what other forces are on the system, the damping time can vary from one to another. Furthermore, the relative sizes of the surrounding and the system affect the trend in periodic cycles of energy dissipation. Nonetheless, the system eventually reaches an equilibrium state with the surrounding. In Figure 2: middle and right graphs, we show two examples of energy dissipation through damping in irreversible processes within a system that is driven away from equilibrium. 0 10 20 t (a.u.) −10 −5 0 5 10 ∆G (a.u.) reversible process 0 10 20 t (a.u.) −10 −5 0 5 10 ∆G (a.u.) slow dissipation 0 10 20 t (a.u.) −10 −5 0 5 10 ∆G (a.u.) fast dissipation Figure 2: Simple examples of a reversible process and two irreversible processes with fast and slow dissipation rates. Central role of entropy in thermodynamics Therefore, we should look at the total Gibbs free energy change or total entropy change of the universe to evaluate the spontaneity of a process. It is intuitively understandable how total negative net entropic energy goes along with an increase in the size and therefore entropy of the universe. However, what if heat is transferred from the cold system to the surrounding? To do this, external work needs to be done on the system which in turn causes an overall increase in the total net entropic energy of the universe. From a mechanistic point of view, the collective forces of microscopic degrees of freedom with intrinsic random Brownian motions at a specific temperature within a medium can act like resistance against an 6 Central role of entropy in thermodynamics 0 10 20 t (a.u.) −10 −5 0 5 10 slow dissipation ∆G (a u ) 0 10 20 t (a.u.) −10 −5 0 5 10 ( ) fast dissipation 0 10 20 t (a.u.) −10 −5 0 5 10 ∆G (a.u.) reversible process ∆G (a.u.) Figure 2: Simple examples of a reversible process and two irreversible processes with fast and slow dissipation rates. 7 Central role of entropy in thermodynamics A PREPRINT As we discussed, an irreversible process in an out-of-equilibrium system dissipates its energy to the surrounding over time. Figure 2 (middle and right graphs) shows how a system initially with non-negligible net entropic energy dissipates its energy. While the dissipation of energy can be gradual (Figure 2: middle graph) but it can occur very quickly with critical damping behavior (Figure 2: right graph). It was illustrated that in a successive repeating non-cyclic process the net entropic energy dissipates through the system and surrounding with an arrow of time. Here we call the total dissipated energy (Wdiss) the net entropic energy (−T∆Ssys,net = T∆Ssur,net) between the system and its surrounding as is shown in Eq 19. Using this definition, Helmholtz’s energy change will be: (35) ∆Asys = T∆Ssys,net −Wsys (35) equivalently the relationship between the Helmholtz energy and the work that is done by surrounding on the system from the system’s perspective will be ∆Asys = T∆Ssys,net + Wsur (36) ∆Asys = T∆Ssys,net + Wsur (36) ∆Asys = T∆Ssys,net + Wsur (36) According to this equation in an equilibrium state of energy where "T∆Ssys,net = 0" the work that is performed reversibly on the system will be equal to the Helmholtz free energy of the system: ∆Asys = Wsur (37) (37) ∆Asys = Wsur In another word, during a reversible process, the net entopic energy that is absorbed and released from the system is zero. However, in a spontaneous process in a system, the net entropy for the system is negative as \|T∆Ssur\| < \|T∆Ssys\|. In such a non-equilibrium state of energy, the process is irreversible and the dissipated energy is positive (Wdiss > 0). As a result, the work that is done on the system is larger than the Helmholtz energy of the system. Central role of entropy in thermodynamics ∆Asys < Wsur (38) (38) ∆Asys < Wsur From thermodynamics, it is perceived that in a real system where the process is spontaneous and irreversible, part of the available potential energy of the system that is supposed to do macroscopic work is lost through dissipation as a result of entropy production.(6; 7) To understand where this energy goes, we should look at the system from a microscopic perspective and track the motions of the system’s constituents precisely in accessible microscopic states. The random Brownian motion of particles creates chaos in the system. The chaotic collisions of particles oppose them to move in an orderly fashion toward a specific direction of the driving force. Under these circumstances, entropic force does not transfer intactly between the particles through these collective collisions. This is analogically similar to friction as an opposition force to the work on the macroscopic object. In statistical mechanics, the energy that is lost through the entropy production as a consequence of random displacements and collisions of microscopic degrees of freedom on the way of dissipating out the heat and performing useful work is called the dissipated work. If we account for the displacement of each particle in the calculation of the total work that is done by the system, then one can see that all the energy is preserved and we receive the equality (∆Asys = Wsur) regardless of the irreversibility of the process in macroscopic scale. To clarify this, we choose a path to take us toward considering the microscopic works in our calculations with the aid of statistical mechanics. To do this, we use Boltzmann’s definition of entropy to statistically calculate the net entropy of a microcanonical ensemble of N independent particles where all the states are equally probable: ∆Snet = Nk(ln wf −ln wi) (39) (39) where wf and wi are the multiplicities of N independent particles in the final and initial configurations of the system respectively. By dividing the multiplicity of wn in configuration n with the total multiplicity of the system in all the available states, wtot, we return: ∆Snet = Nk(ln Pf −ln Pi) = Nk ln Pf Pi (40) (40) this is the Shannon entropy of N independent particles for a single event. Shannon entropy is a general version of Boltzmann’s entropy where the probability of microstates is not necessarily equal. Central role of entropy in thermodynamics If we substitute the above equation into the Gibbs free energy relation of the net entropic energy of a system, the following equation returns: 8 Central role of entropy in thermodynamics A PREPRINT Central role of entropy in thermodynamics A PREPRINT Central role of entropy in thermodynamics Central role of entropy in thermodynamics A PREPRINT T∆Ssur −T∆Ssys = NkT ln Pf Pi (41) (41) To avoid any confusion, we imagined the entropy change is calculated for the surrounding since the heat bath is assumed to be infinitely large. As a result, the equilibrium temperature would be equal to both the initial and final temperatures of the heat bath. As ∆G = ∆Ssys,net, we can write: To avoid any confusion, we imagined the entropy change is calculated for the surrounding since the heat bath is assumed to be infinitely large. As a result, the equilibrium temperature would be equal to both the initial and final temperatures of the heat bath. As ∆G = ∆Ssys,net, we can write: Pf Pi = exp(∆Gsys NkT ) (42) (42) the probability of final and initial configurations can interchangeably be used for forward and reverse process probabili- ties respectively. Because during a forward and reverse process, sequentially, the final and initial configurations are obtained for a system. Since the above equation for Gsys is written for a forward process, therefore: the probability of final and initial configurations can interchangeably be used for forward and reverse process probabili- ties respectively. Because during a forward and reverse process, sequentially, the final and initial configurations are obtained for a system. Since the above equation for Gsys is written for a forward process, therefore: Pf Pr = exp(∆Gsys NkT ) (43) as Pf Pr = exp(∆Gsys NkT ) Pf Pr = exp(∆Gsys NkT ) (43) (43) as ∆Gsys = ∆Asys + p∆Vsys (44) ∆Gsys = ∆Asys + p∆Vsys (44) using this equation, we can rewrite Eq. 42 of the energy production (8) as follows: using this equation, we can rewrite Eq. Central role of entropy in thermodynamics Therefore: exp(−∆Asys NkT ) = NkT exp(p∆Vsys NkT ) = NKT exp(−p∆Vsur NkT ) (49) (49) since all the work that is done by the surrounding is assumed to be the expansion work of p∆V , w ll the work that is done by the surrounding is assumed to be the expansion work of p∆V , we derive: exp(−∆Asys NkT ) = exp(−Wsur NkT ) (50) (50) ∆Asys = −NKT ln exp(−Wsur NkT ) (51) (51) 9 Central role of entropy in thermodynamics A PREPRINT Central role of entropy in thermodynamics A PREPRINT this equation shows that the change of Helmholtz’s energy is equal to the ensemble averaging of the microscopic works done on the system by the surrounding regardless that whether the process is reversible or irreversible.(9; 10) While we reach a general equation that relates a state function of Helmholtz free energy change to the path function of the work, we notice an interesting equation for Gibbs energy change where exp(∆Gsys NkT ) = 1 (52) exp(∆Gsys NkT ) = 1 (52) This equation has an infinite number of solutions as follows: This equation has an infinite number of solutions as follows: This equation has an infinite number of solutions as follows: This equation has an infinite number of solutions as follows: ∆Gsys NkT = 2πiκ where 0 ≤κ < ∞ (53) ∆Gsys NkT = 2πiκ where 0 ≤κ < ∞ (53) for κ = 0, ∆Gsys NkT = 2πiκ where 0 ≤κ < ∞ (53) (53) for κ = 0, ∆Gsys = 0 ∆Gsys = 0 (54) (54) this is the Gibbs free energy change for a reversible process in equilibrium. On the other hand, the lowest nonzero bound of ∆Gsys is when κ = 1 which we return this is the Gibbs free energy change for a reversible process in equilibrium. On the other hand, the lowest nonzero bound of ∆Gsys is when κ = 1 which we return ∆Gsys = 2πiNkT (55) (55) This may be the free energy required for the single displacement of N independent particles resulting from random fluctuations of particles in a medium. This leads to an imaginary version of the lowest bound of entropy change during the displacement of a single particle (11) as follows: ∆Ssys,net = 2πik (56) (56) 2πik is the energy required for the smallest displacement of a single particle in the system. Central role of entropy in thermodynamics 42 of the energy production (8) as follows: Pr Pf = exp(−∆Asys + p∆Vsys NkT ) (45) Pr Pf = exp(−∆Asys + p∆Vsys NkT ) (45) Pr Pf = exp(−∆Asys + p∆Vsys NkT ) (45) where (45) where where ∆Asys + p∆Vsys NkT = σ ∆Asys + p∆Vsys NkT = σ (46) t f th f ll i ti ∆Asys + p∆Vsys NkT = σ (46) to form the following equation: (46) NkT σ (46) to form the following equation: to form the following equation: to form the following equation: Z +∞ −∞ Pr dσ = Z +∞ −∞ Pf exp(−σ) dσ (47) Z +∞ −∞ Pr dσ = Z +∞ −∞ Pf exp(−σ) dσ (47) (47) We assume a stochastic behavior of the Gibbs energy change in infinitely large ensemble measurements to receive a normal distribution. Consequently, we can derive the following: We assume a stochastic behavior of the Gibbs energy change in infinitely large ensemble measurements to receive a normal distribution. Consequently, we can derive the following: 1 = exp⟨−∆Asys + p∆Vsys NkT ⟩ (48) (48) as the Helmholtz free energy is a state function and it does not depend on the path of a process, it can be taken out of the ensemble averaging. Therefore: as the Helmholtz free energy is a state function and it does not depend on the path of a process, it can be taken out of the ensemble averaging. Central role of entropy in thermodynamics Central role of entropy in thermodynamics A PREPRINT energy flow between the heat bath as a hidden entity and the system to regulate the system’s temperature. Under this assumption, we will end up explaining the information with deterministic classical mechanics by which information loses its abstract interpretation and would simply is resulted from interaction energies between particles. Information is the entropic energy required for all the possible combinations of the configuration of particles throughout the available states at possibly the Planck scale of the system in a unit of temperature. energy flow between the heat bath as a hidden entity and the system to regulate the system’s temperature. Under this assumption, we will end up explaining the information with deterministic classical mechanics by which information loses its abstract interpretation and would simply is resulted from interaction energies between particles. Information is the entropic energy required for all the possible combinations of the configuration of particles throughout the available states at possibly the Planck scale of the system in a unit of temperature. 3 Conclusions During entropy production of an irreversible process where a net heat flow of entropic energy is detected in a system regardless of its spontaneity, the total net entropic energy of the universe increases owing to the net repulsive forces. The collision of heated particles with stochastic Brownian motions as an impedance to useful work forms a turbulent flow of chaotic interactions between constituents of the universe which results in a net repulsive force. The enlargement of the universe in an irreversible process in a non-equilibrium state continues until the net attractive forces overcome the net repulsive forces, and this process continues until the universe reaches an equilibrium. Using the entropy production relation of the statistical mechanics, we show a simplistic version of converting the inequality between Helmholtz free energy and the work for macroscopic objects through an irreversible process of non-equilibrium thermodynamics to the equality of the work of reversible microscopic fluctuations with the free energy of the very collective irreversible process. There, we further found a mathematical solution to the Gibbs free energy using the entropy production relation which led us to Newton’s second law of motion. The extraction of Newton’s law of motion from the lowest nonzero bound of entropy for the displacement of a single particle through a microscopic state in space may be implying about entropic energy foundation from an unknown entity. Central role of entropy in thermodynamics If the Eq. 56 is a valid solution to the change of entropy, then using the entropic force concept (12; 13), Newton’s second law of motion is derived which measures the force on a single particle of mass m and acceleration a during the displacement as follows: F = ma (57) (57) F = ma (57) F = ma F = ma This makes us think that entropy is the amount of information that is needed to build a system with all possible combinations of stochastic particles in the accessible states. The change of entropy or information is more comprehendible than its absolute definition. By rearranging Eq. 36, we see that the net entropic energy as the lost dissipated work (Wdiss) is the difference in the Helmholtz free energy change (maximum useful work) and the expansion work (useful work) of the surrounding. (58) Wdiss = Wsur −∆Asys (58) Wdiss is the amount of energy that is wasted for the random displacements of stochastic particles in the system during a macroscopic process. Inferring from Eq. 50 and 58, we think that the sum of dissipated and useful works is equal to the total force for the displacement of particles from initial to final configuration as below: Wdiss + Wsys = F∆x T (59) (59) This is equivalent to the external force on basis of Newton’s third law of motion. When we apply force on the system the entropy of the system increases as the constituting degrees of freedom of the system are mobilized further and find a higher number of accessible states through expansion with time’s arrow. Based on the above equation, we can tell that information change is the emergent entropic force for the displacement of the system’s constituents from an initial to the final configuration at a given temperature. The source of this information either is a projection of another entity or it is coming from underlying degrees of freedom.(14; 15; 16; 11; 17) An interesting analogy to these underlying degrees of freedom is the heat bath in computer simulations where there is an 10 References [1] P. Bak, How Nature Works: the Science of Self-Organized Criticality. New York, NY: Springer, 2013. OCLC: 1059402712. [2] S. Cushman, “Editorial: Entropy in Landscape Ecology,” Entropy, vol. 20, p. 314, Apr. 2018. [3] E. Pranzini and A. T. Williams, “The Equilibrium Concept, or. . . (Mis)concept in Beaches,” Geosciences, vol. 11, p. 59, Jan. 2021. [4] B. B. Mandelbrot, The fractal geometry of nature. Brattleboro, Vermont: Echo Point Books & Media, 2021. OCLC: 1295176876. [5] L. Susskind, “Three Impossible Theories,” 2021. [5] L. Susskind, “Three Impossible Theories,” 2021. [6] D. J. Evans and D. J. Searles, “The Fluctuation Theorem,” Advances in Physics, vol. 51, pp. 1529–1585, Nov. 2002. [7] D. J. Searles and D. J. Evans, “Fluctuation theorem for stochastic systems,” Physical Review E, vol. 60, pp. 159– 164, July 1999. [8] G. E. Crooks, “Entropy production fluctuation theorem and the nonequilibrium work relation for free energy differences,” Physical Review E, vol. 60, pp. 2721–2726, Sept. 1999. [9] C. Jarzynski, “Nonequilibrium Equality for Free Energy Differences,” Physical Review Letters, vol. 78, pp. 2690– 2693, Apr. 1997. [10] C. Jarzynski, “Equalities and Inequalities: Irreversibility and the Second Law of Thermodynamics at the Nanoscale,” Annual Review of Condensed Matter Physics, vol. 2, pp. 329–351, Mar. 2011. [11] F. Mohsen, “Quantum Mechanics Emerging from Complex Brownian Motions,” preprint, Mar. 2022. 12] E. Verlinde, “On the origin of gravity and the laws of Newton,” Journal of High Energy Physics Apr. 2011. 13] R. A. Konoplya, “Entropic force, holography and thermodynamics for static space-times,” The E Journal C, vol. 69, pp. 555–562, Oct. 2010. [14] L. 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W4250688374.txt	https://www.nomos-elibrary.de/10.5771/0344-9777-2006-3-342.pdf?download_full_pdf=1	de		Mitarbeiter des Heftes	Zeitschrift für öffentliche und gemeinwirtschaftliche Unternehmen	2,006	cc-by	317	Mitarbeiter des Heftes Prof. Dr. Günther E. Braun, Universität der Bundeswehr München, Institut für Betriebswirtschaftslehre des öffentlichen Bereichs und Gesundheitswesens, Werner-Heisenberg-Weg 39, D-85577 Neubiberg Prof. Dr. Dieter Brenzke, Westsächsische Hochschule Zwickau (FH), Fachbereich Wirtschaftswissenschaften, Management für Unternehmen mit öffentlichen Aufgaben, Postfach 201037, D-08056 Zwickau Prof. Dr. Reinhard Busse, Technische Universität Berlin, Lehrstuhl für Management im Gesundheitswesen, Straße des 17. Juni 145, D-10623 Berlin Prof. Dr. Werner W. Engelhardt, Hochwaldstraße 38, D-50935 Köln Birgit Grüb, Universität Hamburg, Fakultät Wirtschafts- und Sozialwissenschaften, Arbeitsbereich Public Management, Rentzelstraße 7, D-20146 Hamburg Prof. Dr. Andrea Hausmann, Europa-Universität Viadrina Frankfurt (Oder), Juniorprofessur für Kulturmanagement, Große Scharnstraße 59, D-15230 Frankfurt (Oder) Prof. Dr. Bernd Helmig, University of Fribourg, Chair of Nonprofit-Management & Marketing, Verbandsmanagement Institut (VMI)/Institute for Research on Management of Associations and other Nonprofit.Organizations, Boulevard de Pérolles 90, CH-1700 Fribourg Prof. Dr. Hans-Georg Napp, Bankdirektor, Landesbank Hessen-Thüringen Girozentrale, Neue Mainzer Straße 52-58, D-60311 Frankfurt am Main Prof. Dr. Wolfgang Ossadnik, Universität Osnabrück, Fachgebiet Rechnungswesen und Controlling, Postfach 44 69, D-49069 Osnabrück Katja Roth, Universität zu Köln, Seminar für Genossenschaftswesen, Albertus-Magnus-Platz, D-50923 Köln Manuela Rottmann, Dunckerstraße 27, D-10439 Berlin Prof. Dr. Kuno Schedler, Universität St. Gallen, Public Management Center of Excellence, Institut für Öffentliche Dienstleistungen und Tourismus, Dufourstraße 40a, CH-9000 St. Gallen Rico Schlösser, Universität Osnabrück, Fachbereich Wirtschaftswissenschaften, Finanzierung und Banken, Katharinenstraße 7, D-49069 Osnabrück Dr. Jonas Schreyögg, Technische Universität Berlin, Lehrstuhl für Management im Gesundheitswesen, Straße des 17. Juni 145, D-10623 Berlin André Schumann, Universität der Bundeswehr München, Institut für Betriebswirtschaftslehre des öffentlichen Bereichs und Gesundheitswesens, Werner-Heisenberg-Weg 39, D-85577 Neubiberg Prof. Dr. Jan Ziekow, Deutsche Hochschule für Verwaltungswissenschaften Speyer, Lehrstuhl für Öffentliches Recht, insbesondere allgemeines und besonderes Verwaltungsrecht, Postfach 1409, D-67324 Speyer Redaktion Anke Reile, Universität Mannheim, L 5,4 am Schloss, D-68131 Mannheim, Tel. 0621/181-1725, e-mail: zoegu@bwl.uni-mannheim.de 342 https://doi.org/10.5771/0344-9777-2006-3-342 Generiert durch IP '51.159.168.146', am 14.07.2024, 19:05:58. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. ZögU, Band 29, Heft 3, 2006
https://openalex.org/W4321644369	https://zenodo.org/records/7670072/files/19.pdf	Hindi	null	बुजुर्गों के मानसिक स्वास्थ्य की देखभाल के लिए विशेष बिंदु	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	801	February, 2023; 3 (02), 201-203 रूपल हुड्डा रूपल हुड्डा पारिवारिक संसाधन प्रबंधन ववभाग, गृह ववज्ञान महाववद्यालय, चौधिी चिण वसंह हरियाणा कृवि ववश्वववद्यालय- 125004 February, 2023; 3(02), 201-203 बुजुर्ों के मानलिक स्वास्थ्य की देखभाल के ललए लवशेष लबोंदु Popular Article February, 2023; 3 (02), 201-203 रूपल हुड्डा रूपल हुड्डा पारिवारिक संसाधन प्रबंधन ववभाग, गृह ववज्ञान महाववद्यालय, चौधिी चिण वसंह हरियाणा कृवि ववश्वववद्यालय- 125004 February, 2023; 3(02), 201-203 बुजुर्ों के मानलिक स्वास्थ्य की देखभाल के ललए लवशेष लबोंदु Popular Article रूपल हुड्डा पारिवारिक संसाधन प्रबंधन ववभाग, गृह ववज्ञान महाववद्यालय, चौधिी चिण वसंह हरियाणा कृवि ववश्वववद्यालय- 125004 रूपल हुड्डा पारिवारिक संसाधन प्रबंधन ववभाग, गृह ववज्ञान महाववद्यालय, चौधिी चिण वसंह हरियाणा कृवि ववश्वववद्यालय- 125004 बढ़ते तनाव के बीच आजकल ज्यादाति लोग वबगड़ते मानवसक स्वास्थ्य से बुिी तिह से प्रभाववत हो िहे हैं वजसके कािण लोग मानवसक बीमारियों का भी विकाि आसानी से हो जाते हैं। नेिनल काउंवसल ऑन एवजंग (National Council on Aging) के अनुसाि, हि चाि में से एक वयस्क को अवसाद, वचंता औि मनोभ्रंि जैसे मानवसक ववकाि का अनुभव होता है। इसी को ध्यान में िखते हुए अंदाजा लगाया जा िहा है वक यह संख्या साल 2030 तक बढ़कि 15 वमवलयन तक पहुंचेगी। ऐसे में खासकि बुजुगों को इस स्थिवत से वनपटना जरूि आना चावहए। यही वजह है वक ववश्व स्वास्थ्य संगठन (WHO) हि साल 10 अक्टूबि को ववश्व मानवसक स्वास्थ्य वदवस को मान्यता देता है औि पूिी दुवनया में ववश्व मानवसक स्वास्थ्य वदवस मनाया जाता है। इसका सीधा लक्ष्य है वक जो लोग मानवसक रूप से अस्वथि होते हैं उन्हें इसके प्रवत जागरूक वकया जाए औि उन्हें इस स्थिवत से बाहि वनकाला जाए। इस ववश्व मानवसक स्वास्थ्य वदवस को देखते हुए हम उन बुजुगग लोगों के वलए कुछ आसान तिीके लेकि आएं हैं जो लोग अक्सि अपनी बीमािी के दौिान मेंटल हेल्थ से प्रभाववत हो िहे हैं। यानी हम आज आपको इस लेख के जरिए बताएंगे वक बुजुगों के मानवसक स्वास्थ्य को वकसी भी बीमािी के दौिान कैसे स्वथि िखा जाए। बुजुर्ग लोर्ोों के ललए तनाव के मुख्य कारण: 201 Published 23.2.2023 Official Website www.thescienceworld.net thescienceworldmagazine@gmail.com बुजुर्ग लोर्ोों के ललए तनाव के मुख्य कारण: • िािीरिक स्वास्थ्य से जुड़ी समस्याएं • उम्र के साि ििीि की गवतवववध कम होना 201 Published 23.2.2023 रूपल हुड्डा February, 2023; 3 (02), 201-203 आहाि िािीरिक स्वास्थ्य के कई पहलु जैसे की वजन, हृदय िोग औि मधुमेह जैसी बीमारियों को प्रभाववत किता है। लेवकन आहाि न केवल आपको िािीरिक रूप से स्वथि किने का काम किता है, बस्ि ये वरिष्ठ मानवसक स्वास्थ्य को भी प्रभाववत किता है औि स्वथि िहने में आपकी मदद किता है। इसके वलए बुजुगों को हमेिा अपनी डाइट में साबुत अनाज, फलों औि सस्ियों, प्रोटीन औि स्वथि वसा को िावमल किना चावहए। एक स्वथि,संतुवलत आहाि को अपनाने से मस्स्ष्क स्वास्थ्य को बढ़ावा देने में आसानी होती है। इसके साि ही ये अवसाद को कम किने औि मनोभ्रंि के खतिे को कम किता है। फोवलक एवसड, मैग्नीवियम, वजंक, आयिन, औि ववटावमन बी-6, बी-12, ववटावमन-डी औि ओमेगा-3 फैटी एवसड जैसे कुछ पोिक तत्ों का मानवसक स्वास्थ्य के साि मजबूत संबंध है। मानलिक स्वास्थ्य और शारीररक व्यायाम: हालांवक अप्रत्यक्ष,लेवकन िािीरिक गवतवववधयों का भी मानवसक स्वास्थ्य पि लाभकािी प्रभाव पड़ता है। बीमाि िहने के दौिान अक्सि बुजुगग कुछ भी किने में सक्षम नहीं होते, लेवकन अगि वो अपने ििीि की मूवमेंट अच्छी तिह से किें तो वो अपने मानवसक स्वास्थ्य को भी बेहति िख सकते हैं। व्यायाम ज्यादाति अपने िािीरिक फायदों के वलए जाना जाता है, यह एक जरूिी मानवसक स्वास्थ्य गवतवववध भी है जो आपके मूड को बेहति किता है औि तनाव हामोन को कम किने का काम किता है। इसवलए आप कुछ देि व्यायाम या टहलने की आदत जरूि डालें। मानलिक स्वास्थ्य को बनाए रखने के ललए पयागप्त नीोंद की भूलमका: मानलिक स्वास्थ्य को बनाए रखने के ललए पयागप्त नीोंद की भूलमका: नींद मानवसक स्वास्थ्य का एक अलग वहस्सा है औि अमेरिकन साइकोलॉजी एसोवसएिन के अनुसाि, ज्यादा नींद सुखी, स्वथि, लम्बी औि सुिवक्षत जीवन का लाभ देती है। वहीं, बुजुगों को नींद की गड़बड़ी औि अवनद्रा का खतिा बढ़ जाता है, वजससे अवसाद औि दूसिी पुिानी बीमारियों का खतिा बढ़ने लगता है। इसवलए आप िोजाना अपनी नींद बेहति तिीके से पूिी किें। 202 Published 23.2.2023 Official Website www.thescienceworld.net thescienceworldmagazine@gmail.com 202 Published 23.2.2023 Published 23.2.2023 रूपल हुड्डा February, 2023; 3 (02), 201-203 अच्छे मानलिक स्वास्थ्य के ललए कुछ महत्वपूणग िुझाव: अच्छे मानलिक स्वास्थ्य के ललए कुछ महत्वपूणग िुझाव: अच्छे मानलिक स्वास्थ्य के ललए कुछ महत्वपूणग िुझाव: • पौविक औि स्वच्छ भोजन • पयागप्त नींद • िािीरिक रूप से वफट िहने के वलए वनयवमत व्यायाम • अकेलेपन से बचने के वलए रिश्ों को बनाए िखना • मनोिंजक गवतवववधयों के वलए समय वनकालें • आध्यास्िक औि धावमगक प्रिाओं में िावमल हों • घि में एक पालतू जानवि िखें • डॉक्टि के पास वनयवमत रूप से जाएँ। • पौविक औि स्वच्छ भोजन 203 Published 23.2.2023 Official Website www.thescienceworld.net thescienceworldmagazine@gmail.com 203 Official Website www.thescienceworld.net thescienceworldmagazine@gmail.com 203 Published 23.2.2023 Published 23.2.2023
https://openalex.org/W2021051258	https://www.revistas.usp.br/rcf/article/download/34242/36974	Portuguese	null	Análise das práticas de evidenciação de informações obrigatórias, não-obrigatórias e avançadas nas demonstrações contábeis das sociedades anônimas no Brasil: um estudo comparativo dos exercícios de 2002 e 2005	Revista Contabilidade & Finanças	2,007	cc-by	10,115	ANÁLISE DAS PRÁTICAS DE EVIDENCIAÇÃO DE INFORMAÇÕES OBRIGATÓRIAS, NÃO-OBRIGATÓRIAS E AVANÇADAS NAS DEMONSTRAÇÕES CONTÁBEIS DAS SOCIEDADES ANÔNIMAS NO BRASIL: UM ESTUDO COMPARATIVO DOS EXERCÍCIOS DE 2002 E 2005* ANALYSIS OF MANDATORY, NON-MANDATORY AND ADVANCED INFORMATION DISCLOSURE PRACTICES IN FINANCIAL STATEMENTS OF COMPANIES IN BRAZIL: A COMPARATIVE STUDY BETWEEN 2002 AND 2005 50 VERA MARIA RODRIGUES PONTE Professora Titular do Mestrado em Administração de Empresas da Universidade de Fortaleza – CE E-mail: pontecaminha@pontecaminha.com VERA MARIA RODRIGUES PONTE Professora Titular do Mestrado em Administração de Empresas da Universidade de Fortaleza – CE E-mail: pontecaminha@pontecaminha.com MARCELLE COLARES OLIVEIRA Professora do Mestrado em Administração e da Graduação em Ciências Contábeis da Universidade de Fortaleza Professora da Graduação em Ciências Contábeis da Universidade Federal do Ceará – CE Doutora em Controladoria e Contabilidade – USP E-mail: marcellecolares@unifor.br HÉBER MOURA Professor Titular do Curso de Mestrado em Administração da Universidade de Fortaleza – UNIFOR – CE E-mail: heberm@unifor.br HÉBER MOURA Professor Titular do Curso de Mestrado em Administração da Universidade de Fortaleza – UNIFOR – CE E-mail: heberm@unifor.br RENATA COELHO DE ALMEIDA DO CARMO Graduanda pela Universidade de Fortaleza – CE E-mail: renatacac@hotmail.com RESUMO Em todo o mundo se discute sobre a transparência e a qualidade na divulgação das informações contábeis. No sentido de contribuir para esse debate, o presente estudo procura responder à seguinte questão de pesquisa: Quais as mudanças percebidas na evidenciação de informações obrigatórias, não-obrigatórias e avançadas praticada pelas sociedades anônimas no Brasil? Trata-se de uma pesquisa exploratória-descritiva, cujas amostras são de natureza não-probabilística acidental. Foram analisadas as demonstrações contábeis de 95 empresas, referentes ao exercício de 2002, e 119 alusivas ao exercício de 2005. No tocante aos itens recomendados pelos pareceres nos 15/87, 17/89 e 19/90 da CVM, a pesquisa revela a não-ocorrência de melhoria das práticas de disclosure das companhias estudadas. Com relação às informações contábeis de natureza avançada e não-obrigatória propugnadas pelas práticas de governança corporativa, veriﬁ ca-se um avanço na sua evidenciação pelas empresas analisadas, que dispensam atenção especial à divulgação de suas práticas de responsabilidade social e do Balanço Social, das Demonstrações do Fluxo de Caixa (DFC) e do Valor Adicionado (DVA). Palavras-chave: Evidenciação. Demonstrações Contábeis. Sociedades Anônimas no Brasil. Recebido em 16.12.2006 • Aceito em 18.04.2007 • 2a versão aceita em 09.08.2007 * artigo originalmente apresentado no 7º Congresso USP de Controladoria e Contabilidade, 26 e 27, julho 2007 Segundo Iudícibus (2000, p. 121), a evidenciação: Segundo Iudícibus (2000, p. 121), a evidenciação: Segundo Iudícibus (2000, p. 121), a evidenciação: [...] é um compromisso inalienável da Contabilida- de com seus usuários e com os próprios objetivos. As formas de evidenciação podem variar, mas a essência é sempre a mesma: apresentar informação quantitativa e qualitativa de maneira ordenada, deixando o menos possível para ﬁ car de fora dos demonstrativos formais, a ﬁ m de propiciar uma base adequada de informação para o usuário. Com o objetivo de contribuir para o debate sobre dis- closure, a presente pesquisa busca responder ao seguinte questionamento: Quais as mudanças percebidas na evi- denciação de informações de natureza obrigatória, não- obrigatória e avançada praticada pelas sociedades anôni- mas no Brasil? Para orientar e balizar o presente estudo formularam-se as seguintes hipóteses: H0: A média de informações contábeis de natureza obrigatória, não-obrigatória e avançada eviden- ciadas pelas empresas coincide nos anos 2002 e 2005; Para Dantas, Zendersky e Niyama (2004), dado o obje- tivo maior da Contabilidade, uma atenção especial deve ser dispensada ao papel desempenhado pela evidenciação ou disclosure. Para ser consideradas úteis, as demonstrações contábeis devem conter as informações necessárias para uma adequada interpretação da situação econômico-ﬁ nan- ceira da entidade. H1: A média de informações contábeis de natureza obrigatória, não-obrigatória e avançada evidencia- das pelas empresas não coincide nos anos 2002 e de 2005. Estudos cientíﬁ cos têm revelado a preocupação com o disclosure de informações contábeis convergentes e amplas, envolvendo aspectos econômicos, ﬁ nanceiros, sociais, ambientais, de produtividade e de gestão, entre outros. A análise da evidenciação dessas informações e o acompa- nhamento de sua evolução estão cada vez mais presentes nas discussões tanto acadêmicas, quanto empresariais e de órgãos, sejam públicos ou privados, ligados ao mercado de capitais e aos proﬁ ssionais que interagem direta ou in- diretamente com esse mercado. Assim, a presente pesquisa tem por objetivo geral veri- ﬁ car se as demonstrações contábeis das sociedades anôni- mas brasileiras referentes ao exercício de 2005 apresentam um maior nível de evidenciação de informações conside- radas de natureza obrigatória, não-obrigatória e avançada quando confrontadas com as do ano de 2002. Para alcance dos objetivos da pesquisa, o trabalho foi segmentado em cinco tópicos. No primeiro, comentam-se as recentes pesquisas brasileiras sobre disclosure. ABSTRACT All over the world, there have been discussions on transparency and quality in the disclosure of accounting information. Aiming at contributing towards this debate, this study seeks to answer the following research question: What are the perceived changes in the disclosure of mandatory, non-mandatory and advanced accounting reporting experienced by companies in Brazil? Financial statements from 95 companies were assessed, referring to corporate annual reports of 2002 and from 119 companies referring to corporate annual reports of 2005. Concerning items recommended in rules numbers 15/87, 17/89 and 19/90 by the Brazilian Securities and Exchange Commission, this research reveals that there was no improvement in the disclosure practices of the companies under analysis. As to advanced and non-mandatory accounting information, proposed by corporate governance practices, an increase in their disclosure was observed, with companies giving special attention to the publication of their social responsibility practices and Corporate Social Reporting, Cash Flow Statements and Statements of Value Added. Keywords: Disclosure. Financial Statements. Companies in Brazil. Recebido em 16.12.2006 • Aceito em 18.04.2007 • 2a versão aceita em 09.08.2007 * artigo originalmente apresentado no 7º Congresso USP de Controladoria e Contabilidade, 26 e 27, julho 2007 R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 RCF-45-USP_A4-Analise.indd 50 RCF-45-USP_A4-Analise.indd 50 5/12/2007 17:36:17 5/12/2007 17:36:17 51 ANÁLISE DAS PRÁTICAS DE EVIDENCIAÇÃO DE INFORMAÇÕES OBRIGATÓRIAS, NÃO-OBRIGATÓRIAS E AVANÇADAS NAS DEMONSTRAÇÕES CONTÁBEIS... 51 1 INTRODUÇÃO No Brasil, ainda avança lentamente a adesão à divulgação de informações internacionalmente já instituídas e reco- nhecidas como relevantes para o eﬁ caz processo decisório dos stakeholders. Segundo Iudícibus (2000, p. 20), o objetivo da Con- tabilidade praticamente permaneceu inalterado ao longo dos anos; as mudanças substanciais veriﬁ caram-se nos tipos de usuário e nas formas de informação que têm sido demandadas. O cumprimento da missão da Contabilidade tornou-se mais difícil de ser alcançado, já que cada usuá- rio deseja um conjunto especíﬁ co de informações capaz de suprir seu modelo decisório. No Brasil, inserem-se iniciativas pela maior evidencia- ção de informações econômico-ﬁ nanceiras e por alterações na forma de reconhecimento e mensuração de determina- dos eventos e transações, privilegiando-se a essência so- bre a forma e o ﬁ el cumprimento aos princípios contábeis – esses fazendo parte do próprio arcabouço teórico da con- tabilidade. Segundo Oliveira, Gomes e Costa (2004), a partir da se- gunda metade do século XX muitos dos usuários da Con- tabilidade passaram a exigir das empresas a evidenciação de informações nas demonstrações contábeis diferentes das tradicionais, razão pela qual o tema disclosure ganhou destaque na pauta de debates da ciência contábil. Tais iniciativas envolvem orientações referentes à nor- matização contábil brasileira editadas pelos mais variados organismos, como o Conselho Federal de Contabilidade (CFC), o Instituto dos Auditores Independentes do Brasil (IBRACON), a Comissão de Valores Mobiliários (CVM) e aquelas editadas por entidades reguladoras de segmentos especíﬁ cos da economia que adotam normatização con- tábil própria, como o Banco Central do Brasil, a Secretaria de Previdência Complementar (SPC), a Superintendência de Seguros Privados (SUSEP), Agência Nacional de Ener- gia Elétrica (ANEEL), bem como orientações emanadas de órgãos como a Bolsa de Valores de São Paulo (BOVESPA), o Instituto Brasileiro de Governança Corporativa (IBGC), o Instituto Brasileiro de Análises Sociais e Econômicas (IBA- SE), entre outros. Niyama e Gomes (1996, p. 65) aﬁ rmam que: Disclosure [...] diz respeito à qualidade das informa- ções de caráter ﬁ nanceiro e econômico, sobre as ope- rações, recursos e obrigações de uma entidade, que se- jam úteis aos usuários das demonstrações contábeis, entendidas como sendo aquelas que de alguma forma inﬂ uenciem na tomada de decisões, envolvendo a en- tidade e o acompanhamento da evolução patrimonial, possibilitando o conhecimento das ações passadas e a realização de inferências em relação ao futuro. 2 PESQUISAS EMPÍRICAS BRASILEIRAS SOBRE PRÁTICAS DE DISCLOSURE Em muitos países, pesquisas têm sido realizadas in- vestigando a evidenciação de informações contábeis obri- gatórias e voluntárias. À guisa de exemplo, cite-se Leuz (2000), que estudou os estímulos das empresas alemãs para evidenciação voluntária da Demonstração dos Fluxos de Caixa e observou que, embora ela não fosse uma infor- mação obrigatória na Alemanha até 1998, quando passou a ser requerida para as empresas listadas em Bolsa, um crescente número de empresas a divulgou e o autor asso- cia isso às recomendações das associações de proﬁ ssionais alemães, à IAS – 7 (Internacional Accounting Standards) e aos padrões contábeis de outros países. O autor faz a análise usando ferramentas estatísticas investigando a di- vulgação da DFC em 1992, 1994 e 1996, tendo em vista as recomendações de órgãos nacionais (alemães) e/ou inter- nacionais emitidas nesses anos sugerindo sua elaboração, e conclui que o mercado de capitais foi a mola propulsora da divulgação da DFC. manter um bom relacionamento com seus stakeholders, via transparência das informações contábeis e da gestão em- presarial. Pesquisas no Brasil também têm investigado a divulgação de informações contábeis obrigatórias e volun- tárias. Nossa e Carvalho (2003) realizaram estudo com o objetivo de investigar o nível de disclosure de informações ambientais apresentado pelas empresas do setor de papel e celulose. Os autores concluíram que o disclosure de in- formações ambientais apresentado pelas empresas diverge com relação aos respectivos portes, aos países de locali- zação e aos tipos de relatório (ﬁ nanceiro ou especíﬁ co), mostrando-se, ainda, incipiente e frágil em relação ao nível de conﬁ abilidade e comparabilidade das informações. Cardoso et al. (2003) realizaram estudo exploratório com o objetivo de investigar o grau de transparência com que as informações sobre riscos ﬁ nanceiros e não-ﬁ nan- ceiros foram abordadas nos relatórios 20F das empresas de telecomunicações, nos anos 2000 e 2001. Os autores con- cluem que as empresas selecionadas na amostra cumprem rigorosamente o estabelecido na norma expedida pela Secu- rity Exchange Commission (SEC). ç Verrecchia (2001, p. 97-98) estudou a literatura con- tábil e classiﬁ cou as pesquisas sobre evidenciação em con- tabilidade em 3 categorias: pesquisas que investigam os efeitos do disclosure no comportamento dos investidores (association-based disclosure); pesquisas que investigam os incentivos dos gestores ou empresas para evidenciar determinadas informações (discretionary-based disclosure) e pesquisas que visam a descobrir os arranjos de eviden- ciação preferíveis (efﬁ ciency-based disclosure). 2 PESQUISAS EMPÍRICAS BRASILEIRAS SOBRE PRÁTICAS DE DISCLOSURE Segundo o autor, nas duas primeiras categorias de pesquisas sobre evi- denciação, a coleta de dados ocorre após a divulgação das informações contábeis e, na última, ocorre antes. Nessas pesquisas, os autores utilizam modelos matemáticos para explicar o disclosure de informações obrigatórias e volun- tárias. Verrecchia (2001) realiza uma análise crítica desses modelos e apresenta um modelo de sua autoria sugerindo a redução da assimetria informacional como um ponto de partida para a integração da eﬁ ciência da escolha da eviden- ciação, dos incentivos do disclosure e da endogeneidade do mercado de capitais. Alves e Oliveira (2003) pesquisaram sobre a evolução da evidenciação da DVA e a geração e distribuição de ri- queza nas demonstrações contábeis de uma amostra de 88 empresas brasileiras. Concluíram que cresceu o número de empresas que publicam a DVA, que não havia um con- senso acerca da sua localização em meio às demais infor- mações contábeis, nem da utilização de um modelo único para sua evidenciação, embora preponderasse o modelo da Fipecaﬁ . Oliveira e Ribeiro (2003) analisaram a evolução da evi- denciação das informações de natureza ambiental em uma seqüência de três anos da empresa Petrobrás, concluindo que a cada ano a empresa vem evidenciando mais infor- mações, embora todas em notas explicativas, ou seja, não passando pela mensuração e reconhecimento de seus efei- tos no patrimônio da empresa. Segundo Dantas, Zendersky e Niyama (2004), diversos estudos têm evidenciado os benefícios, para as organiza- ções, de um maior nível de evidenciação contábil, e vários outros demonstram a relutância das empresas em aumen- tar o nível de disclosure. Diante dessa realidade, os autores realizaram estudo com o objetivo de discutir essa aparen- te dualidade, procurando destacar os diversos conceitos relacionados com o tema e utilizando as conclusões de pesquisas empíricas desenvolvidas em âmbitos nacional e internacional para dar suporte às argumentações teóricas. Concluem que um maior nível de disclosure representa uma via de mão dupla, pois beneﬁ cia os usuários com as infor- Dye (2001) realizou uma análise crítica do trabalho de Verrecchia (2001) e da literatura por ele utilizada. Segundo Iudícibus (2000, p. 121), a evidenciação: Em segui- da, trata-se das orientações contidas na Lei das Sociedades por Ações e em Pareceres de Orientação da CVM sobre a divulgação de informações não contempladas nas demons- A tônica dessas discussões, entre outras coisas, está voltada para a deﬁ nição de quais e de quantas informa- ções contábeis evidenciar, como e por que evidenciá-las. R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 RCF-45-USP_A4-Analise.indd 51 RCF-45-USP_A4-Analise.indd 51 5/12/2007 17:36:20 5/12/2007 17:36:20 52 Vera Maria Rodrigues Ponte • Marcelle Colares Oliveira • Héber Moura • Renata Coelho de Almeida do Carmo trações contábeis tradicionais, consideradas de natureza avançada e não-obrigatória. No terceiro, são apresentados os métodos e técnicas relativos à amostragem utilizada e ao tratamento dos dados consubstanciados nas demons- trações contábeis. No quarto tópico, apresentam-se os re- sultados da pesquisa documental. No último tópico, são relatadas as conclusões do estudo. 2 PESQUISAS EMPÍRICAS BRASILEIRAS SOBRE PRÁTICAS DE DISCLOSURE O autor apresenta discussão sobre a premissa central da literatura a respeito do disclosure e as questões conceituais envolven- do os vários modelos matemáticos e apresenta uma avalia- ção das tendências recentes na literatura sobre disclosure: a maioria está baseada em velhos modelos ou assertivas, que não correspondem à concepção do mundo real e têm uso limitado para estudos empíricos. Devido ao processo de globalização e desenvolvimento do mercado de capitais, tornou-se fundamental a empresa R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 RCF-45-USP_A4-Analise.indd 52 RCF-45-USP_A4-Analise.indd 52 RCF-45-USP_A4-Analise.indd 52 RCF-45-USP_A4-Analise.indd 52 5/12/2007 17:36:21 5/12/2007 17:36:21 ANÁLISE DAS PRÁTICAS DE EVIDENCIAÇÃO DE INFORMAÇÕES OBRIGATÓRIAS, NÃO-OBRIGATÓRIAS E AVANÇADAS NAS DEMONSTRAÇÕES CONTÁBEIS... 53 mações de que necessitam para o seu processo decisório, o que reﬂ ete na valorização da entidade, aumentando a con- ﬁ abilidade da organização perante credores, investidores e demais agentes, contribuindo para o aumento da liquidez de suas ações e a redução do custo de capital. presa, procurando veriﬁ car sua relação com a participação de investidores não-controladores, em especial acionistas minoritários e credores, na estrutura de capital da compa- nhia. O autor encontrou elementos diretamente relacio- nados com a escolha da estrutura de capital e que podem inﬂ uenciar o nível de divulgação de uma empresa, como, por exemplo, a sujeição da emissão de títulos à regulamen- tação e à sua listagem nos mercados de capitais. Darós e Borba (2004) realizaram estudo sobre as for- mas de evidenciação de instrumentos ﬁ nanceiros derivati- vos nas demonstrações contábeis divulgadas no Brasil. Os autores constataram que a grande maioria das empresas não atende às determinações da CVM, nem evidencia de forma clara, concisa e objetiva as informações referentes às suas operações envolvendo instrumentos ﬁ nanceiros derivativos. Carvalho et al. (2004) analisaram os reﬂ exos da ado- ção dos conceitos e práticas de governança corporativa na evidenciação das informações contábeis por empresas do setor de papel e celulose, concluindo que apesar de as em- presas desse setor estarem sujeitas a uma maior exigência do mercado em evidenciar de maneira transparente suas informações contábeis e de governança, ainda não alcan- çaram o nível ideal de divulgação de acordo com os concei- tos nacionais e internacionais. 2 PESQUISAS EMPÍRICAS BRASILEIRAS SOBRE PRÁTICAS DE DISCLOSURE Dutra e Busato (2004) procuraram aferir o grau de trans- parência das companhias abertas brasileiras no que se re- fere aos programas de remuneração com uso de opções. Os resultados obtidos indicaram que as normas em vigor no Brasil encontravam-se muito aquém daquelas em vigor nos EUA. Demonstraram, ainda, que não há uniformidade de conteúdo nas divulgações realizadas pelas empresas, al- gumas delas disponibilizando volume de dados insuﬁ ciente para a avaliação do impacto dos programas de opções. Dalmácio e Paulo (2004) investigaram a forma de evidenciação de aspectos socioambientais e econômico- ﬁ nanceiros nas demonstrações contábeis de empresas industriais, sociedades anônimas, do Estado do Espírito Santo, constatando que as empresas componentes da amostra utilizam o Relatório da Administração e as Notas Explicativas para tornar públicas essas informações. Silva, Rodrigues e Abreu (2004) realizaram pesquisa com o objetivo de veriﬁ car se os Relatórios da Adminis- tração divulgados pelas empresas representam fonte de in- formação sobre a organização. Os resultados encontrados mostraram que os Relatórios da Administração possuem informação relevante sobre as companhias analisadas. Segundo os autores, o estudo demonstra que os relató- rios otimistas apresentam maior volume de frases sobre reforma administrativa, enquanto os relatórios pessimistas centram sua atenção na conjuntura econômica. Medeiros e Quinteiro (2005) avaliaram o efeito da evi- denciação de informações contábeis relevantes na mobilida- de de capitais internacionais em um conjunto de 22 países, incluindo o Brasil, concluindo que a evidenciação dessas informações afeta positivamente, e que a inﬂ uência da Con- tabilidade Fiscal sobre a Contabilidade Financeira afeta nega- tivamente essa mobilidade, e que esse efeito se sobrepõe ao primeiro na decisão de aplicação do investidor. Com o objetivo de veriﬁ car o nível de utilização dos conceitos de evidenciação do passivo pelas empresas brasi- leiras, segundo os padrões introduzidos pelo Intergoverna- mental Working Group of Experts on International Standards of Accounting and Reporting (UN-ISAR), Santana, Luiz e Ricardinho Filho (2004) analisaram as demonstrações contábeis das dez primeiras empresas classiﬁ cadas pelo Prêmio Transparência ANEFAC/FIPECAFI/SERASA 2003. Os autores concluiram que as empresas brasileiras ainda evidenciam o passivo de forma tradicional, ou seja, levando preponderantemente em consideração o passivo referente às obrigações legais. 2 PESQUISAS EMPÍRICAS BRASILEIRAS SOBRE PRÁTICAS DE DISCLOSURE Borba, Alves e Rover (2005) investigaram as diferen- tes práticas de evidenciação ambiental nas demonstrações contábeis de companhias brasileiras submetidas à CVM e à SEC, concluindo que nos informes publicados segundo as normas da SEC o objetivo é mensurar e reconhecer o impacto de determinados fatos contábeis sobre o patrimô- nio da companhia e que nas publicações junto à CVM tais evidenciações têm caráter publicitário. Como se pode observar, as pesquisas no Brasil tratam não só das informações contábeis tradicionais, mas tam- bém de outras que têm sido consideradas importantes para o processo decisório dos stakeholders, tanto internos quanto externos. Pereira (2004) estudou as causas da variação do vo- lume de divulgação de informações por parte de uma em- 3 INFORMAÇÕES COMPLEMENTARES DE NATUREZA AVANÇADA E NÃO-OB Algumas empre- sas apresentam descrição e análise por segmento ou linha de produto, quando relevantes para a sua compreensão e avaliação; c) maior ênfase às demonstrações ﬁ nanceiras consoli- dadas, de maneira que as demonstrações individua- lizadas da companhia controladora sejam apresenta- das em separado e em menor destaque, contendo as contas e seus respectivos valores exigidos em lei; b) comentários sobre a conjuntura econômica geral: concorrência nos mercados, atos governa- mentais e outros fatores exógenos relevantes sobre o desempenho da companhia; d) maior ênfase às demonstrações com correção in- tegral, de maneira que aquelas elaboradas na for- ma societária, quando publicadas, sejam também apresentadas em separado, contendo as contas e valores legalmente exigidos. c) recursos humanos: número de empregados no término dos dois últimos exercícios e turnover nos dois últimos anos; segmentação da mão-de-obra segundo a localização geográﬁ ca; nível educacional ou produto; investimento em treinamento; fundos de seguridade e outros planos sociais; Iniciativas voltadas para a adoção das normas interna- cionais são as grandes mudanças de natureza contábil na Lei no 6.404, propostas pelo Projeto de Lei no 3.741/2000. As alterações propostas no Projeto de Lei têm como arca- bouço teórico básico as recomendações do Internacional Accounting Standards Board (IASB), órgão que congrega as entidades proﬁ ssionais de Contabilidade de quase todos os países, inclusive o Brasil. Dentre as mudanças propostas, destacam-se: a substituição da Demonstração das Origens e Aplicações de Recursos pela DFC e a obrigatoriedade de elaboração da DVA. d) investimentos: descrição dos principais investi- mentos realizados, objetivos, montantes e origens dos recursos alocados; e) pesquisa e desenvolvimento: descrição sucinta dos projetos, recursos alocados, montantes aplica- dos e situação dos projetos; e) pesquisa e desenvolvimento: descrição sucinta dos projetos, recursos alocados, montantes aplica- dos e situação dos projetos; f) novos produtos e serviços: descrição de novos produtos, serviços e expectativas a eles relacionadas; f) novos produtos e serviços: descrição de novos produtos, serviços e expectativas a eles relacionadas; g) proteção ao meio-ambiente: descrição e objetivos dos investimentos efetuados e montante aplicado; ç Em junho de 2002, a CVM apresentou uma cartilha de governança corporativa tipicamente brasileira. A Cartilha da CVM dá especial atenção aos aspectos relacionados à evidenciação contábil, como a adoção de padrões interna- h) reformulações administrativas: descrição das mudanças administrativas, reorganizações societá- rias e programas de racionalização; h) reformulações administrativas: descrição das mudanças administrativas, reorganizações societá- rias e programas de racionalização; R. Cont. Fin. 3 INFORMAÇÕES COMPLEMENTARES DE NATUREZA AVANÇADA E NÃO-OB Com o objetivo de atender às necessidades dos usuá- rios externos, a Lei no 6.404/76 deﬁ ne um conjunto mínimo de informações que devem ser disponibilizadas pelas orga- nizações de capital aberto. Nesse sentido, a Contabilidade Financeira é responsável por fornecer as demonstrações contábeis tradicionais – Balanço Patrimonial, Demonstra- ção do Resultado do Exercício, Demonstração de Lucros ou Prejuízos Acumulados ou Demonstração das Mutações do Patrimônio Líquido e Demonstração das Origens e Aplica- ções de Recursos – e informações suplementares através do Relatório da Administração e das Notas Explicativas. Ocorre que as informações divulgadas nas demons- trações tradicionais parecem não atender às necessidades dos usuários. Daí a evidenciação de informações adicio- R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 RCF-45-USP_A4-Analise.indd 53 RCF-45-USP_A4-Analise.indd 53 5/12/2007 17:36:21 5/12/2007 17:36:21 54 Vera Maria Rodrigues Ponte • Marcelle Colares Oliveira • Héber Moura • Renata Coelho de Almeida do Carmo i) investimentos em controladas e coligadas: indicação dos investimentos efetuados e objetivos das inversões; nais via Notas Explicativas ou Relatório da Administração. Preocupados em garantir o fornecimento de informações contábeis capazes de atender às exigências dos usuários externos, com freqüência os órgãos reguladores da prática contábil têm expedido normas tratando da divulgação das demonstrações contábeis. j) direitos dos acionistas e dados de mercado: políticas relativas à distribuição de direitos, desdo- bramentos e agrupamentos; valor patrimonial por ação, negociação e cotação das ações em bolsas de valores; A Lei no 6.404/76 determina a obrigatoriedade de in- cluir, no Relatório da Administração: a aquisição de de- bêntures de própria emissão da empresa (art. 55, § 2.º); as disposições sobre a política de reinvestimento de lucros e distribuição de dividendos constantes de acordo de acio- nistas (art. 118, § 5.º); e a relação dos investimentos em sociedades coligadas e controladas e as modiﬁ cações ocor- ridas durante o exercício (art. 243). 3 INFORMAÇÕES COMPLEMENTARES DE NATUREZA AVANÇADA E NÃO-OB k) perspectivas e planos para o exercício em curso e os vindouros: poderá ser divulgada a expectativa da administração quanto ao exercício corrente, baseada em premissas e fundamentos explicitamente citados, sendo que essa informação não se confunde com projeções, por não ser quan- tiﬁ cada; k) perspectivas e planos para o exercício em curso e os vindouros: poderá ser divulgada a expectativa da administração quanto ao exercício corrente, baseada em premissas e fundamentos explicitamente citados, sendo que essa informação não se confunde com projeções, por não ser quan- tiﬁ cada; A CVM entende que o Relatório da Administração cons- titui elemento poderoso de comunicação da companhia junto aos acionistas e à comunidade em que está inserida, devendo ser redigido em linguagem simpliﬁ cada, para ser acessível ao maior número de pessoas e empresas. l) em se tratando de companhia de participações, o relatório deve contemplar as informações acima mencionadas, ainda que de forma mais sintética, relativas às empresas investidas. A CVM manifestou-se acerca do Relatório da Administra- ção em seus Pareceres de Orientação nos 15/87, 17/89 e 19/90. De acordo com esses pareceres, o relatório deverá conter as informações contempladas na Lei no 6.404/76. Entretanto, a título de recomendação e exemplo, a CVM apresenta a relação dos itens que constituem informações que atendam às linhas gerais retrocitadas, já apresentadas por muitas companhias no Brasil (sendo comuns em alguns outros países): O Parecer de Orientação no 24/92 da CVM trata dos avanços na qualidade da informação e divulgação das de- monstrações contábeis, apoiando e estimulando iniciati- vas nesse sentido, apontando como exemplos de formas de enriquecimento da informação levada ao público: a) apresentação de demonstrações complementares, como a DFC e a DVA; a) descrição dos negócios, produtos e serviços: histórico das vendas físicas dos últimos dois anos e vendas em moeda de poder aquisitivo da data do encerramento do exercício social. Algumas empre- sas apresentam descrição e análise por segmento ou linha de produto, quando relevantes para a sua compreensão e avaliação; b) apresentação de Notas Explicativas sobre: valor de mercado dos estoques, ouro e ações de alta liquidez e resultados por linha de produtos ou negócios, em especial referentes às demonstrações consolidadas; a) descrição dos negócios, produtos e serviços: histórico das vendas físicas dos últimos dois anos e vendas em moeda de poder aquisitivo da data do encerramento do exercício social. 4 METODOLOGIA DA PESQUISA O presente estudo constitui uma pesquisa exploratória- descritiva, compreendendo uma investigação bibliográﬁ ca, para levantamento das orientações legais e regulamenta- res especíﬁ cas sobre divulgação de informações contábeis avançadas e não-obrigatórias aplicadas às sociedades anô- nimas no Brasil, ou seja, revisão da literatura disponível sobre o assunto; bem como uma pesquisa documental, para levantamento e análise das informações contidas nas demonstrações contábeis publicadas. Em seguida, deu-se início à pesquisa documental, para levantamento e análise das informações registradas nas demonstrações contábeis publicadas pelas socieda- des anônimas no Brasil. Foram analisadas as demonstra- ções contábeis de 95 empresas referentes ao exercício encerrado em 31/12/2002, e de 119 referentes ao exer- cício encerrado em 31/12/2005. Quanto à composição e dimensionamento das amostras utilizadas, trata-se de amostras não-probabilísticas acidentais, de demonstra- ções contábeis de sociedades anônimas, publicadas na Gazeta Mercantil, no período de janeiro a março de 2003, e na Gazeta Mercantil e no Valor Econômico, em igual período de 2006. A pesquisa documental compreendeu: a) a delimitação do universo e seleção das empresas a serem pesquisadas; b) a coleta das demonstrações contábeis referentes aos exercícios sociais ﬁ ndos em 31/12/2002 e 31/12/2005 e efetivamente publicadas; c) a análise da conformidade dos itens divulgados nas demonstrações contábeis coletadas, com as orientações legais e regulamentares especíﬁ cas so- bre divulgação de informações contábeis avançadas e não- obrigatórias aplicadas às sociedades anônimas no Brasil e d) tabulação, consolidação e análise dos dados. A justiﬁ cativa para a amostragem utilizada está rela- cionada à abrangência, linha editorial e credibilidade dos jornais em que foram publicadas as demonstrações con- tábeis pesquisadas, tratando-se de periódicos com pene- tração nacional, especializados em matérias relacionadas ao mundo dos negócios, detentores de grande credibi- lidade, com bastante utilidade em diversos segmentos. Portanto, com os dados colhidos e analisados, foi possí- vel retratar a evidenciação contábil das sociedades anô- nimas segundo os parâmetros escolhidos, embora não se possa generalizar as constatações para esse universo de empresas. No contexto econômico atual, de um mundo sem fronteiras, com operações cada vez mais soﬁ sticadas e velozes, considerou-se que o intervalo de três anos com- preendendo desde a criação em 2002 dos níveis diferen- ciados de governança corporativa pela Bovespa, até os dias atuais, seria um período razoável para se averiguar as mudanças ocorridas no nível de evidenciação de in- formações obrigatórias, não-obrigatórias e avançadas por empresas brasileiras. 3 INFORMAÇÕES COMPLEMENTARES DE NATUREZA AVANÇADA E NÃO-OB • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 5/12/2007 17:36:21 5/12/2007 17:36:21 RCF-45-USP_A4-Analise.indd 54 RCF-45-USP_A4-Analise.indd 54 ANÁLISE DAS PRÁTICAS DE EVIDENCIAÇÃO DE INFORMAÇÕES OBRIGATÓRIAS, NÃO-OBRIGATÓRIAS E AVANÇADAS NAS DEMONSTRAÇÕES CONTÁBEIS... 55 cionais de contabilidade (IAS/USGAAP); elaboração e di- vulgação da Demonstração dos Fluxos de Caixa e da DVA. çada e não-obrigatória. Devido ao período de investigação deste estudo, as orientações da Deliberação no 488 não são contempladas na presente pesquisa, limitada ao exame das orientações contidas nos Pareceres de Orientação nos 15/87, 17/89, 19/90 e 24/92. Em dezembro de 2002, a Bovespa criou o Novo Merca- do, o Nível 1 e o Nível 2 de governança corporativa, que, dentre outras práticas, prevê: melhoria nas informações prestadas trimestralmente, entre as quais a exigência de demonstrações contábeis consolidadas e da demonstração dos ﬂ uxos de caixa; disponibilização de balanço anual de acordo com as normas do US GAAP ou do IAS GAAP, para empresas do Nível 2 e Novo Mercado. Em janeiro de 2006, a Bovespa criou o BOVESPA MAIS, segmento do mercado de balcão organizado, em que po- dem ser listadas companhias abertas que possuam registro na CVM. Para participar desse segmento, as empresas de- vem assumir o compromisso com regras de transparência como: Balanço Patrimonial, Demonstração de Resultado e Comentários de Desempenho Consolidados e a Demons- tração dos Fluxos de Caixa em Notas Explicativas. Dada a importância e a necessidade de as práticas con- tábeis brasileiras convergirem com as práticas contábeis internacionais, a CVM aprovou a Deliberação no 488, de 3 de Outubro de 2005, orientando as companhias a divulgar um conjunto adicional de informações de natureza avan- 5 RESULTADOS DA PESQUISA O referencial teórico que subsidiou a análise das de- monstrações contábeis das companhias pesquisadas, no tocante a informações complementares, de natureza obri- gatória, não-obrigatória e avançada, compreendeu: signiﬁ cativas as diferenças entre as médias de respostas das empresas nos dois períodos. p p Consiste, portanto, na avaliação das hipóteses: p p Consiste, portanto, na avaliação das hipóteses: H0: A média de informações contábeis de natureza obrigatória, não-obrigatória e avançada eviden- ciadas pelas empresas coincide nos anos 2002 e 2005; a) os conteúdos abordados no Pareceres de Orienta- ção da CVM nos 15/87, 17/89 e 19/90, à guisa de exemplo dos itens que constituem informações que atendem às exigências da Lei no 6.404/76 sobre Re- latório da Administração; H1: A média de informações contábeis de natureza obrigatória, não-obrigatória e avançada evidencia- das pelas empresas não coincide nos anos 2002 e 2005. H1: A média de informações contábeis de natureza obrigatória, não-obrigatória e avançada evidencia- das pelas empresas não coincide nos anos 2002 e 2005. b) o conteúdo abordado no Parecer de Orientação da CVM no 24/92, à guisa de exemplo dos itens que constituem informações contábeis avançadas; Aceitar a hipótese H0 signiﬁ ca considerar que a propor- ção das empresas que evidenciaram o particular item não se alterou substancialmente, mercê do estímulo realizado pelos órgãos reguladores e pela sociedade como um todo no sentido de uma maior transparência por parte das em- presas brasileiras. Por outro lado, a rejeição da hipótese nula indica a existência de diferença signiﬁ cativa nos resul- tados das empresas nos dois exercícios considerados. Vale ressaltar que referido teste foi precedido pelo de Levene, para veriﬁ car a igualdade das variâncias nas duas popula- ções, tendo sido feitos os ajustes necessários no teste das médias, dependendo de as variâncias populacionais terem sido ou não consideradas iguais. 5 RESULTADOS DA PESQUISA c) o conteúdo da cartilha de governança corporativa e do Projeto de Lei no 3.741, no que se referem à evi- denciação de informações adicionais, como proje- tos sociais, EBITDA (lucro antes dos juros, tributos sobre o lucro, depreciação, amortização e exaustão) e Balanço Social, que se alinham às idéias de trans- parência sobre informações pertinentes ao desen- volvimento dos negócios; d) demais orientações recentes da CVM e da Bovespa, sobre evidenciação da DFC, demonstrações conso- lidadas e em padrões internacionais; e) e orientações da Lei no 6.404 no que tange à obri- gatoriedade de apresentação de informações espe- cíﬁ cas no Relatório da Administração. g A Tabela 1 informa os índices das empresas que evi- denciaram as informações em 2002 e 2005, considerando o total de 95 no ano 2002 e de 119 em 2005. Também são apresentadas as signiﬁ câncias estatísticas na forma de p-values, correspondentes à probabilidade de ocorrên- cia do Erro Tipo I do teste de hipótese, representando a probabilidade de se rejeitar erroneamente a hipótese H0. Para o presente estudo, foram considerados signiﬁ cativos apenas valores inferiores a 0,10. Deve ser observado que os resultados gerados pelo teste aqui empregado são os mes- mos obtidos para o teste não-paramétrico χ2 de indepen- dência estatística, no sentido de veriﬁ car se as freqüências observadas diferem signiﬁ cativamente nos dois exercícios observados. Conforme mencionado no tópico Metodologia da Pes- quisa, as amostras para os anos 2002 e 2005 foram cons- truídas de forma aleatória, não havendo obrigatoriedade de as empresas incluídas na amostra de um exercício ser incluída também na do outro. Assim, vale ressaltar que 27 empresas integrantes da amostra de 2002 também fazem parte da de 2005. Dessa maneira, a análise dos dados é apresentada em dois tópicos, um abordando todo o con- junto de empresas e outro reunindo apenas o das empresas incluídas nas duas amostras. 4 METODOLOGIA DA PESQUISA Nessa fase da pesquisa, procedeu-se à leitura completa das demonstrações e foram elaboradas tabelas que identi- ﬁ cavam as evidenciações praticadas pelas empresas inte- grantes da amostra nos anos 2002 e 2005. Os dados reu- nidos nas tabelas foram trabalhados no software Statistical Package for Social Sciences SPSS, versão 12.0, e em seguida submetidos a testes estatísticos. Assim, foi realizada aná- lise qualitativa dos dados, por meio de discussão crítica, além de se recorrer à análise quantitativa. Partindo-se do confronto do referencial teórico com os dados colhidos, foi também possível elucidar o problema de pesquisa e erigir conclusões. O parâmetro para análise das demonstrações contábeis pesquisadas são as informações consideradas de natureza obrigatória, não-obrigatória e avançada conforme referen- cial teórico, que podem constar do Relatório da Adminis- tração e das Notas Explicativas. Sendo assim, na primeira etapa de desenvolvimento deste estudo teórico-empírico, buscou-se conhecer as orientações legais e regulamentares especíﬁ cas sobre di- vulgação de informações contábeis aplicadas às sociedades anônimas no Brasil. R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 RCF-45-USP_A4-Analise.indd 55 RCF-45-USP_A4-Analise.indd 55 5/12/2007 17:36:21 5/12/2007 17:36:21 56 Vera Maria Rodrigues Ponte • Marcelle Colares Oliveira • Héber Moura • Renata Coelho de Almeida do Carmo 5.1 Análise dos dados considerando todas as Empresas Como muitas empresas que prestaram informações em 2002 não o ﬁ zeram em 2005, e vice-versa, há o risco da ocorrência de distorções relativamente à variação veriﬁ cada entre os dois exercícios. Entretanto, contando com a pos- sibilidade da existência de aleatoriedade nos dois períodos e considerando que se trata de um estudo exploratório so- bre o assunto, é válido empreender a comparação entre as diferentes amostras nos dois anos. A Tabela 1 apresenta os dados levantados para os anos 2002 e 2005, no tocante às orientações dos Pareceres nos 15/87, 17/89 e 19/90 da CVM. Apesar de as instruções da CVM determinarem a evidenciação das informações no corpo do Relatório da Administração, considerou-se atendida a recomendação mesmo quando efetuada em Notas Explicativas, já que várias empresas adotam essa prática. A Tabela 2 resume as mudanças de prática ocorri- das de 2002 para 2005. A análise dos dados revela que do conjunto de onze itens investigados, sete não apresentam alteração ou seja, a proporção de empresas que estão evi- denciando esses tópicos em 2005 não é signiﬁ cativamente diferente da registrada em 2002. Em quatro itens – C, F, H e K – veriﬁ cou-se uma redução na proporção de empresas que vêm observando as orientações da CVM. Os tópicos Veriﬁ ca-se, na Tabela 1, que para cada item recomen- dado pelos pareceres da CVM, de “A” até “K”, foi levantado o número de empresas que o evidenciou nos anos 2002 e 2005. Com o propósito de avaliar a signiﬁ cância das va- riações observadas, realizou-se para cada tópico um teste de hipótese de diferença de médias entre os dois anos. Ba- seado na estatística t-student, referido teste avalia se são R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 RCF-45-USP_A4-Analise.indd 56 RCF-45-USP_A4-Analise.indd 56 5/12/2007 17:36:22 5/12/2007 17:36:22 RCF-45-USP_A4-Analise.indd 56 RCF-45-USP_A4-Analise.indd 56 ANÁLISE DAS PRÁTICAS DE EVIDENCIAÇÃO DE INFORMAÇÕES OBRIGATÓRIAS, NÃO-OBRIGATÓRIAS E AVANÇADAS NAS DEMONSTRAÇÕES CONTÁBEIS... 5.1 Análise dos dados considerando todas as Empresas 57 NÁLISE DAS PRÁTICAS DE EVIDENCIAÇÃO DE INFORMAÇÕES OBRIGATÓRIAS, NÃO-OBRIGATÓRIAS E AVANÇADAS NAS DEMONSTRAÇÕES CONTÁBE Tabela 1 Comportamento do grupo de empresas pesquisado no tocante à divulgação, no Relatório da Administração e Notas Explicativas, dos itens recomendados nos Pareceres de Orientação da CVM Item 2002 2005 Signiﬁ cância Estatística nº % Nº % A) descrição dos negócios, produtos e serviços: histórico das vendas físicas dos últimos dois anos e vendas em moeda corrente da data do encerramento do exercício Social 85 90,0 112 94,0 0,228 B) comentários sobre a conjuntura econômica geral: concorrência nos mercados, atos governamentais e outros fatores exógenos relevantes sobre o desempenho da companhia 54 57,0 58 49,0 0,240 C) recursos humanos: número de empregados no término dos dois últimos exercícios e turnover nos dois últimos anos; segmen- tação da mão-de-obra segundo a localização geográﬁ ca; nível educacional ou produto; investimento em treinamento; fundos de seguridade e outros planos sociais 55 58,0 45 38,0 0,003 D) investimentos: descrição dos principais investimentos realizados, objetivos, montantes e origens dos recursos alocados 62 65,0 73 61,0 0,557 E) pesquisa e desenvolvimento: descrição sucinta dos projetos, recursos alocados, montantes aplicados e situação dos projetos 23 24,0 28 24,0 0,908 F) novos produtos e serviços: descrição de novos produtos, serviços e expectativas a eles relacionadas 30 33,0 12 10,0 0,000 G) proteção ao meio-ambiente: descrição e objetivo dos investimen- tos efetuados e montante aplicado 30 32,0 36 30,0 0,836 H) reformulações administrativas: descrição das mudanças adminis- trativas, reorganizações societárias e programas de racionaliza- ção 46 48,0 30 25,0 0,000 I) investimentos em controladas e coligadas: indicação dos investi- mentos efetuados e objetivos pretendidos com as inversões 57 60,0 81 68,0 0,225 J) direitos dos acionistas e dados de mercado: políticas relativas à distribuição de direitos, desdobramentos e grupamentos; valor patrimonial por ação, negociação e cotação das ações em Bolsas de Valores 69 73,0 89 75,0 0,723 K) perspectivas e planos para o exercício em curso e os futuros: poderá ser divulgada a expectativa da administração quanto ao exercício corrente, baseada em premissas e fundamentos explicitamente formulados, sendo que essa informação não se confunde com projeções, por não ser quantiﬁ cada 33 35,0 26 22,0 0,039 Fonte: Elaboração dos autores Tabela 1 Comportamento do grupo de empresas pesquisado no tocante à divulgação, no Relatório da Administração e Notas Explicativas, dos itens recomendados nos Pareceres de Orientação da CVM Fonte: Elaboração dos autores Tabela 2 Comportamento de todas as empresas em 2002 e 2005 Ocorrência Número de itens Discriminação de itens Itens que não apresentaram alteração de comportamento 7 A, B, D, E, G, I, J Itens que apresentaram menor evidenciação em 2005 4 C, F, H, K Itens que apresentaram maior evidenciação em 2005 0 Fonte: Elaboração dos autores Tabela 2 Comportamento de todas as empresas em 2002 e 2005 Fonte: Elaboração dos autores exercício em curso e futuro (K), pois para esses as empre- sas sempre têm informações a evidenciar. 5.1 Análise dos dados considerando todas as Empresas Com relação às informações de natureza avançada, observa-se uma efetiva mudança de comportamento, quando comparados os anos 2002 e 2005 (Tabela 4 ). Em 2005 nenhuma empresa deu ênfase às demonstrações com correção integral, reﬂ exo da Lei no 9.249, que eliminou a adoção de qualquer sistema de correção monetária de balan- ço, tanto para ﬁ ns ﬁ scais quanto para ﬁ ns societários. A Tabela 4 revela que para os itens N (notas sobre valor de mercado dos estoques, ouro e ações de alta liquidez) e P (maior ênfase às demonstrações ﬁ nanceiras consolidadas) veriﬁ cou-se uma redução na proporção de empresas que os evidenciam. No ano 2002, as empresas dispensaram atenção espe- cial à divulgação de seus projetos sociais, tendo sido esse o item que apresentou a maior freqüência de evidenciação, atingindo 44%. Em 2005, esse item continuou a receber ênfase das empresas, repetindo-se a freqüência de 44% re- gistrada anteriormente. Outros itens, contudo, passaram a receber maior atenção das empresas. É o caso da DFC, da DVA e do EBITDA. Observa-se, também, uma mudança de comportamento com relação ao tópico Balanço Social, que em 2005 registrou freqüência de 29%, quase dobrando o desempenho de 2002 (17%). 5.1 Análise dos dados considerando todas as Empresas referentes a novos produtos e serviços (F) e reformulações administrativas (H) podem ter apresentado esse comporta- mento em função da não-ocorrência de eventos relaciona- dos aos itens, não se registrando, portanto a não-obser- vância maior dos pareceres da CVM em 2005. O mesmo não pode ser aﬁ rmado com relação aos itens relacionados com recursos humanos (C) e perspectivas e planos para o Com relação à divulgação das informações contábeis avançadas, a Tabela 3 apresenta os dados levantados para os anos 2002 e 2005, no tocante às orientações do Parecer no 24/92 da CVM, da Cartilha de Governança Cor- porativa e do Projeto de Lei no 3.741. R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 RCF-45-USP_A4-Analise.indd 57 RCF-45-USP_A4-Analise.indd 57 5/12/2007 17:36:22 5/12/2007 17:36:22 58 Vera Maria Rodrigues Ponte • Marcelle Colares Oliveira • Héber Moura • Renata Coelho de Almeida do Carmo Tabela 3 Comportamento do grupo de empresas pesquisado no tocante à divulgação dos itens recomendados para evidenciação, conforme Parecer de Orientação no 24/92 da CVM, Cartilha de Governança Corporativa e anteprojeto de reforma da Lei das S/A Item 2002 2005 Signiﬁ cância Estatística nº % nº % L) Demonstração de Fluxo de Caixa 27 28,0 71 60,0 0,000 M) Demonstração do Valor Adicionado 26 27,0 56 47,0 0,003 N) notas sobre valor de mercado dos estoques, ouro e ações de alta liquidez 29 31,0 13 11,0 0,001 O) notas sobre resultados por linha de produtos ou negócios, em espe- cial referentes às demonstrações consolidadas. 26 27,0 34 29,0 0,847 P) maior ênfase às demonstrações ﬁ nanceiras consolidadas, de manei- ra que as demonstrações individualizadas da companhia controlado- ra sejam apresentadas num quadro separado, em menor destaque, contendo as contas e seus respectivos valores exigidos em lei 33 35,0 16 13,0 0,000 Q) maior ênfase às demonstrações com correção integral, de maneira que aquelas elaboradas na forma societária, quando publicadas, sejam também apresentadas em separado, contendo as contas e valores legalmente exigidos 3 3,0 0 0,0 0,083 R) projetos sociais 42 44,0 52 44,0 0,940 S) EBITDA 35 37,0 67 56,0 0,004 T) Balanço Social 16 17,0 34 29,0 0,040 Fonte: Elaboração dos autores T) Balanço Social Fonte: Elaboração dos autores O tratamento dos dados evidenciados na Tabela 3 foi o mesmo para os da Tabela 1. 5.2 Análise dos dados das empresas que apresentaram demonstrações em 2002 e 2005 Do conjunto de empresas estudadas, 27 fazem parte da amostra de 2002 e de 2005. As demonstrações des- sas companhias foram estudadas em separado, recebendo tratamento estatístico semelhante àquele adotado para o universo estudado. A Tabela 5 apresenta as informações levantadas no tocante aos anos 2002 e 2005 relativamente às 27 empresas, no que se refere às orientações dos Parece- res nos 15/87, 17/89 e 19/90 da CVM. As freqüências registradas para a DFC (28% em 2002 e 60% em 2005) e da DVA (27% em 2002 e 47% em 2005), comprovam o efetivo reconhecimento da relevância dessas demonstrações para a gestão e avaliação econômico-ﬁ nan- ceira das organizações. Tabela 4 Comportamento das empresas de 2002 a 2005 Ocorrência Número de itens Discriminação de itens Itens que não apresentaram alteração de comportamento 3 O, Q, R Itens que apresentaram menor evidenciação em 2005 2 N, P Itens que apresentaram maior evidenciação em 2005 4 L, M, S, T Fonte: Elaboração dos autores Tabela 4 Comportamento das empresas de 2002 a 2005 R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 RCF-45-USP_A4-Analise.indd 58 RCF-45-USP_A4-Analise.indd 58 5/12/2007 17:36:22 5/12/2007 17:36:22 ANÁLISE DAS PRÁTICAS DE EVIDENCIAÇÃO DE INFORMAÇÕES OBRIGATÓRIAS, NÃO-OBRIGATÓRIAS E AVANÇADAS NAS DEMONSTRAÇÕES CONTÁBEIS... 59 E DAS PRÁTICAS DE EVIDENCIAÇÃO DE INFORMAÇÕES OBRIGATÓRIAS, NÃO-OBRIGATÓRIAS E AVANÇADAS NAS DEMONSTRAÇÕES CONTÁBEIS... 5.2 Análise dos dados das empresas que apresentaram demonstrações em 2002 e 2005 Tabela 5 Comportamento do grupo de 27 empresas pesquisado no tocante à divulgação dos itens recomendados para evidenciação, conforme Pareceres de Orientação da CVM Item 2002 2005 Signiﬁ cância Estatística nº % nº % A) descrição dos negócios, produtos e serviços: histórico das vendas físicas dos últimos dois anos e vendas em moeda corrente da data do encerramento do exercício social 27 100,0 25 93,0 0,161 B) comentários sobre a conjuntura econômica geral: concorrência nos mercados, atos governamentais e outros fatores exógenos relevantes sobre o desempenho da companhia 20 74,0 20 74,0 1,000 C) recursos humanos: número de empregados no término dos dois últimos exercícios e turnover nos dois últimos anos; segmentação da mão-de-obra segundo a localização geográﬁ ca; nível educacional ou produto; investimento em treinamento; fundos de seguridade e outros planos sociais 22 82,0 15 56,0 0,041 D) investimentos: descrição dos principais investimentos realizados, objetivos, montantes e origens dos recursos alocados 21 78,0 19 70,0 0,543 E) pesquisa e desenvolvimento: descrição sucinta dos projetos, recursos alocados, montantes aplicados e situação dos projetos 9 33,0 10 37,0 0,781 F) novos produtos e serviços: descrição de novos produtos, serviços e expectativas a eles relacionadas 11 41,0 4 15,0 0,034 G) proteção ao meio-ambiente: descrição e objetivo dos investimentos efetuados e montante aplicado 11 41,0 11 41,0 1,000 H) reformulações administrativas: descrição das mudanças administrati- vas, reorganizações societárias e programas de racionalização 18 67,0 10 37,0 0,029 I) investimentos em controladas e coligadas: indicação dos investimen- tos efetuados e objetivos pretendidos com as inversões 20 74,0 24 89,0 0,167 J) direitos dos acionistas e dados de mercado: políticas relativas à distri- buição de direitos, desdobramentos e grupamentos; valor patrimo- nial por ação, negociação e cotação das ações em Bolsas de Valores 24 89,0 23 85,0 0,692 K) perspectivas e planos para o exercício em curso e os futuros: poderá ser divulgada a expectativa da administração quanto ao exercício cor- rente, baseada em premissas e fundamentos explicitamente formula- dos, sendo que essa informação não se confunde com projeções, por não ser quantiﬁ cada 13 48,0 11 41,0 0,592 Fonte: Elaboração dos autores a 5 Comportamento do grupo de 27 empresas pesquisado no tocante à divulgação dos itens recomendados para evidenciação, conforme Pareceres de Orientação da CVM Tabela 5 Fonte: Elaboração dos autores As Tabelas 7 e 8 apresentam os resultados da aná- lise dos dados das 27 empresas no tocante aos tópicos relacionados com a divulgação de informações contábeis avançadas. 5.2 Análise dos dados das empresas que apresentaram demonstrações em 2002 e 2005 Os resultados são diferentes dos apresentados nas Tabelas 3 e 4 para os tópicos L, M, S, T. Para esse nú- mero menor de companhias não se observa em 2005 uma maior evidenciação das demonstrações do Fluxo de Caixa e do Valor Adicionado, do EBITDA e do Balanço Social. Essa A Tabela 6 possibilita uma avaliação da mudança de prática ocorrida entre 2002 e 2005 nas 27 empresas. Ob- serva-se que o grupo agora analisado apresenta comporta- mento similar ao do conjunto anteriormente investigado, havendo comportamento diferenciado apenas para o tó- pico “K” (perspectivas e planos para o exercício em curso e os futuros), que nesse caso não apresentou redução na proporção de empresas. Tabela 6 Comportamento das 27 empresas nos anos 2002 e 2005 Ocorrência Número de itens Discriminação de itens Itens que não apresentaram alteração de comportamento 8 A, B, D, E, G, I, J, K Itens que apresentaram menor evidenciação em 2005 3 C, F, H Itens que apresentaram maior evidenciação em 2005 0 Fonte: Elaboração dos autores Tabela 6 Comportamento das 27 empresas nos anos 2002 e 2005 R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 5.2 Análise dos dados das empresas que apresentaram demonstrações em 2002 e 2005 2007 RCF-45-USP_A4-Analise.indd 59 RCF-45-USP_A4-Analise.indd 59 RCF-45-USP_A4-Analise.indd 59 RCF-45-USP_A4-Analise.indd 59 5/12/2007 17:36:23 5/12/2007 17:36:23 60 Vera Maria Rodrigues Ponte • Marcelle Colares Oliveira • Héber Moura • Renata Coelho de Almeida do Carmo Tabela 7 Comportamento do grupo de 27 empresas pesquisado no tocante à divulgação dos itens recomendados para evidenciação, conforme Parecer de Orientação no 24/92 da CVM, Cartilha de Governança Corporativa e anteprojeto de reforma da Lei das S/A Tabela 7 Comportamento do grupo de 27 empresas pesquisado no tocante à divulgação dos itens recomendados para evidenciação, conforme Parecer de Orientação no 24/92 da CVM, Cartilha de Governança Corporativa e anteprojeto de reforma da Lei das S/A Item 2002 2005 Signiﬁ cância Estatística nº % nº % L) Demonstração de Fluxo de Caixa 11 41,0 17 63,0 0,106 M) Demonstração do Valor Adicionado 9 33,0 14 52,0 0,175 N) notas sobre valor de mercado dos estoques, ouro e ações de alta liquidez 11 41,0 3 11,0 0,013 O) notas sobre resultados por linha de produtos ou negócios, em especial referentes às demonstrações Consolidadas 13 48,0 9 33,0 0,277 P) maior ênfase às demonstrações ﬁ nanceiras consolidadas, de maneira que as demonstrações individualizadas da companhia controladora se- jam apresentadas num quadro separado, em menor destaque, contendo as contas e seus respectivos valores exigidos em lei 11 41,0 4 15,0 0,034 Q) maior ênfase às demonstrações com correção integral, de maneira que aquelas elaboradas na forma societária, quando publicadas, sejam tam- bém apresentadas em separado, contendo as contas e valores legalmen- te exigidos 1 4,0 0 0,0 0,322 R) projetos sociais 18 67,0 17 63,0 0,781 S) EBITDA 15 56,0 18 67,0 0,412 T) Balanço Social 6 22,0 7 26,0 0,756 Fonte: Elaboração dos autores Fonte: Elaboração dos autores Tabela 8 Comportamento das 27 empresas nos anos 2002 e 2005 Ocorrência Número de itens Discriminação de itens Itens que não apresentaram alteração de comportamento 7 L, M, O, Q, R, S, T Itens que apresentaram menor evidenciação em 2005 2 N, P Itens que apresentaram maior evidenciação em 2005 0 Fonte: Elaboração dos autores Tabela 8 Comportamento das 27 empresas nos anos 2002 e 2005 Fonte: Elaboração dos autores denciação diferente em relação ao grupo das 95 compa- nhias estudadas. diferença deve-se ao fato de esse grupo já apresentar em 2002 uma elevada evidenciação desses itens. Na verdade, em 2002, essas empresas já adotavam uma prática de evi- 6 CONSIDERAÇÕES FINAIS No que diz respeito aos itens recomendados pelos pa- receres nos 15/87, 17/89 e 19/90 da CVM, a pesquisa revela que apesar da pressão da sociedade por maior transparên- cia nos negócios, não se veriﬁ ca uma melhoria nas práticas de disclosure das companhias brasileiras, quando compara- dos os anos 2002 e 2005. Também causa espécie que a evidenciação de informa- ções sobre recursos humanos e perspectivas e planos para o exercício em curso e os futuros tenha sido objeto de me- nor evidenciação no ano 2005, quando essas são informa- ções que toda empresa deveria interessar-se em divulgar para o público em geral. Os itens mais evidenciados pelas empresas, em 2005, dizem respeito à descrição dos negócios, produtos e ser- viços, à conjuntura econômica, a investimentos realizados e direitos dos acionistas. Com relação a esse último item, causa estranheza que apenas 73% das empresas, em 2002, e 75%, em 2005, o tenham apresentado no corpo das de- monstrações contábeis, quando a política de reinvesti- mento de lucros e distribuição de dividendos com base em acordo de acionistas constitui exigência expressa na Lei no 6.404/76, art.118, § 5.º. Com relação às informações contábeis de natureza avança- da e não-obrigatória propugnadas pelas práticas de governan- ça corporativa sugeridas pelo IBGC, pela Bovespa e pela CVM, veriﬁ ca-se um avanço na prática de disclosure pelas empresas brasileiras. As empresas estão dispensando atenção especial à divulgação de seus projetos sociais, das demonstrações do Fluxo de Caixa e do Valor Adicionado, do EBITDA e do Balanço Social. Esse fato comprova que as companhias já reconhecem como diferencial competitivo a inclusão de informações não exigidas pela legislação em suas demonstrações contábeis. R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 5/12/2007 17:36:23 5/12/2007 17:36:23 RCF-45-USP_A4-Analise.indd 60 RCF-45-USP_A4-Analise.indd 60 RCF-45-USP_A4-Analise.indd 60 RCF-45-USP_A4-Analise.indd 60 61 NÁLISE DAS PRÁTICAS DE EVIDENCIAÇÃO DE INFORMAÇÕES OBRIGATÓRIAS, NÃO-OBRIGATÓRIAS E AVANÇADAS NAS DEMONSTRAÇÕES CONTÁBE Além das 27 empresas que divulgaram as demonstra- ções em 2002 e 2005 nos meios de comunicação utili- zados na pesquisa serem todas sociedades abertas, ob- servou-se que 12 delas pertencem a um dos três níveis de governança corporativa da Bovespa (Nível 1, Nível 2 e Novo Mercado), o que auxilia na compreensão do elevado nível de evidenciação das informações avançadas e não- obrigatórias sugeridas pela Bovespa, CVM e IBGC. ricano. 6 CONSIDERAÇÕES FINAIS Segundo o autor, as empresas soﬁ sticaram os seus balanços apresentando informações apreciadas pelas boas práticas de relacionamento com o mercado. Os resultados do estudo levam a concluir que as em- presas brasileiras ainda têm muito a evoluir no sentido da transparência e qualidade da divulgação das demonstra- ções contábeis. Contudo, indicam, também, que o apelo da sociedade no sentido da adoção de boas práticas de governança corporativa parece estar inﬂ uenciando o com- portamento das organizações, que já incluem no corpo das suas demonstrações contábeis publicadas maior volume de informações contábeis de natureza avançada e não- obrigatória. A presente pesquisa reforça em parte os resultados da pesquisa de Soares (2001), que, investigando a qualidade das demonstrações contábeis das empresas brasileiras, as- sinala uma mudança de comportamento das empresas de capital aberto, que passaram a dar nova roupagem a seus balanços depois de negociar ações no mercado norte-ame- Referências ALVES, J. F. V.; OLIVEIRA, M. C.A evolução da evidenciação da demonstração do valor adicionado no Brasil e a geração e distribuição de riqueza por empresas brasileiras. In: CONGRESSO USP DE CONTROLADORIA E CONTABILIDADE, 3., 2003, São Paulo. Anais... São Paulo: FEA/USP, 2003. CD-ROM. BORBA, J. A.; ALVES, J. L.; ROVER, S. Análise do conteúdo ambiental das demonstrações contábeis publicadas no Brasil e nos Estados Unidos: um estudo nas companhias com ADR nível III. In: CONGRESSO USP DE CONTROLADORIA E CONTABILIDADE, 5, 2005, São Paulo. Anais... São Paulo: FEA/USP, 2005. CD-ROM. CARDOSO, R. L.; RICCIO, É. L.; MENDONÇA NETO, O. R.; MANTOVANI, F. A evolução recente da transparência dos fatores de risco nas informações contábeis: uma análise de empresas brasileiras de telecomunicações. In: ENCONTRO NACIONAL DE PESQUISA EM ADMINISTRAÇÃO, EnANPAD, 2003; Atibaia, Anais... Atibaia, 2003, CD-ROM, CCG 811. CARVALHO, F. A. et al. 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Disponível em: <http://www.cvm.gov.br/>. Acesso em: 20 dez. 2006. LEUZ, C. The development of voluntary cash ﬂ ow statements in German and the inﬂ uence of international reporting standards. Schmalenbach Business Review, v. 52, n. 2, Apr. 2000. MEDEIROS, O. R.; QUINTEIRO, L. G. L. Ambiente de evidenciação contábil e mobilidade de capitais internacionais. In: CONGRESSO USP DE CONTROLADORIA E CONTABILIDADE, 5., 2005, São Paulo. Anais... São Paulo: FEA/USP, 2005. CD-ROM. R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 RCF-45-USP_A4-Analise.indd 61 RCF-45-USP_A4-Analise.indd 61 5/12/2007 17:36:23 5/12/2007 17:36:23 62 Vera Maria Rodrigues Ponte • Marcelle Colares Oliveira • Héber Moura • Renata Coelho de Almeida do Carmo NIYAMA, J. K.; GOMES, A. L. O. Contribuição ao aperfeiçoamento dos procedimentos de evidenciação contábil aplicáveis às demonstrações ﬁ nanceiras de bancos e instituições assemelhadas. 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Disponível em: <http://www.cvm.gov.br/>. Acesso em 20 dez. 2006. PARECER DE ORIENTAÇÃO n. 17/89. Disponível em: <http://www.cvm.gov.br/>. Acesso em 20 dez. 2006. PARECER DE ORIENTAÇÃO n. 19/90. Disponível em: <http://www.cvm.gov.br/>. Acesso em 20 dez. 2006. PARECER DE ORIENTAÇÃO n. 24/92. Disponível em: <http://www.cvm.gov.br/>. Acesso em 20 dez. 2006. PEREIRA, M. A. Estudo do nível de divulgação e sua relação com a estrutura de capital em empresas brasileiras. In: ENCONTRO NACIONAL DE PESQUISA EM ADMINISTRAÇÃO, EnANPAD, 2004; Curitiba, Anais... Curitiba, 2004, CD-ROM. TO DE LEI n. 3.741/2000. Disponível em: <http://www.planalto.gov.br/ccivil_03/Projetos/Quadros/quadro_PL/2000.htm>. Acesso em 2006. SILVA, C. A. T.; RODRIGUES, F. F.; ABREU, R. L. Análise dos relatórios de administração das companhias abertas brasileiras: um estudo do exercício social de 2002. In: ENCONTRO NACIONAL DE PESQUISA EM ADMINISTRAÇÃO, EnANPAD, 2004; Curitiba, Anais... Curitiba, 2004, CD-ROM, CCG 2500. SOARES, A. H. 2001. Balanços se soﬁ sticaram para atrair investidor estrangeiro. Gazeta Mercantil, São Paulo, 20 set. Caderno Finanças & Mercados, p. B-2. VERRECCHIA, R. E. Essays on disclosure. Journal of Accounting and Economics, n. 32, p. 97-180, 2001. R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 NOTA – Endereço dos autores Universidade de Fortaleza Rua Washington Soares, 1321 Edson Queiroz – Fortaleza – CE 60811-341 -USP_A4-Analise.indd 62 -USP_A4-Analise.indd 62 5/12/2007 17:36:24 5/12/2007 17:36:24 NOTA – Endereço dos autores NOTA – Endereço dos autores Universidade de Fortaleza Rua Washington Soares, 1321 Edson Queiroz – Fortaleza – CE 60811-341 R. Cont. Fin. • USP • São Paulo • v. 18 • n. 45 • p. 50 - 62 • set./dez. 2007 RCF-45-USP_A4-Analise.indd 62 RCF-45-USP_A4-Analise.indd 62 5/12/2007 17:36:24 5/12/2007 17:36:24
https://openalex.org/W3021741243	https://www.research.ed.ac.uk/files/155199170/gps.5324.pdf	English	null	The Observational Scale of Level of Arousal: A brief tool for assessing and monitoring level of arousal in patients with delirium outside the <scp>ICU</scp>	International journal of geriatric psychiatry	2,020	cc-by	6,683	General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Digital Object Identifier (DOI): 10.1002/gps.5324 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: International Journal of Geriatric Psychiatry Edinburgh Research Explorer The Observational Scale of Level of Arousal: a brief tool for assessing and monitoring level of arousal in patients with delirium outside the ICU Citation for published version: Hall, R, Stiobhairt, A, Allerhand, M, Maclullich, A & Tieges, Z 2020, 'The Observational Scale of Level of Arousal: a brief tool for assessing and monitoring level of arousal in patients with delirium outside the ICU', International Journal of Geriatric Psychiatry. https://doi.org/10.1002/gps.5324 Citation for published version: Hall, R, Stiobhairt, A, Allerhand, M, Maclullich, A & Tieges, Z 2020, 'The Observational Scale of Level of Arousal: a brief tool for assessing and monitoring level of arousal in patients with delirium outside the ICU', International Journal of Geriatric Psychiatry. https://doi.org/10.1002/gps.5324 R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Funding information Results: A total of 44 patients (40.7%) were diagnosed with delirium. OSLA scores were higher in delirium (pooled median = 3, InterQuartile Range [IQR] = 2-5) com- pared to no delirium (pooled median = 1, IQR = 1-2; P-values <.05 to <.001). The Area under the Receiver Operating Characteristic curve was 0.82 (95% Confidence Interval (CI) = 0.77-0.86). OSLA scores were responsive to change in delirium status (ß = −3.09. SE = 1.41, P < .03). Conclusions: This study provides preliminary evidence supporting use of the OSLA as an instrument for identifying abnormal level of arousal associated with delirium and monitoring this longitudinally. Further validation in larger cohorts with blinded raters is required. K E Y W O R D S arousal, attention, cognition, delirium, orthopaedic surgery Roanna Hall and Antaine Stíobhairt contributed equally to this study. Alasdair M. J. MacLullich and Zoë Tieges contributed equally to this study. 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. © 2020 The Authors. International Journal of Geriatric Psychiatry published by John Wiley & Sons Ltd Int J Geriatr Psychiatry. 2020;1–7. wileyonlinelibrary.com/journal/gps 1 Roanna Hall and Antaine Stíobhairt contributed equally to this study. Alasdair M. J. MacLullich and Zoë Tieges contributed equally to this study. 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. © 2020 The Authors. International Journal of Geriatric Psychiatry published by John Wiley & Sons Ltd Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Received: 12 October 2019 Accepted: 25 April 2020 DOI: 10.1002/gps.5324 Received: 12 October 2019 Accepted: 25 April 2020 wileyonlinelibrary.com/journal/gps 1 Int J Geriatr Psychiatry. 2020;1–7. Roanna Hall1,2,3 \| Antaine Stíobhairt1 \| Mike Allerhand2 \| Alasdair M. J. MacLullich1,2,3 \| Zoë Tieges1,2 1Edinburgh Delirium Research Group, University of Edinburgh, Edinburgh, UK 2Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Edinburgh, UK 1Edinburgh Delirium Research Group, University of Edinburgh, Edinburgh, UK Objectives: Altered level of arousal, encompassing drowsiness and hyper- vigilance, affects at least 10% of acutely unwell patients. Existing scales provide limited coverage of milder changes in level of arousal. We devised the Observa- tional Scale of Level of Arousal (OSLA) to enable more detailed arousal assess- ment. Here, we provide a preliminary case-control study of performance of the OSLA in assessing abnormal level of arousal associated with delirium outside the ICU. 3Medicine of the Elderly Department, Royal Infirmary of Edinburgh, Edinburgh, UK Correspondence Zoë Tieges, Edinburgh Delirium Research Group, University of Edinburgh, Room S1642, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh EH16 4SA, UK. Email: zoe.tieges@ed.ac.uk Methods: Hip fracture patients (N = 108, median age = 82 years) were assessed for delirium pre- and post-operatively using the Confusion Assessment Method and the Delirium Rating Scale-Revised-98. The OSLA has four graded items assessing eye opening, eye contact, posture, and movement (score range 0 [normal arousal]-15). We assessed the psychometric and diagnostic characteristics of the OSLA. Adjusted linear mixed effects models were used to explore responsiveness of the OSLA to within-patient change in delirium status. Funding information Research into Ageing (AgeUK) and the British Geriatrics Society, Grant/Award Number: 342 Key points Outside the field of delirium, abnormal level of arousal is increas- ingly seen as a crucial marker of illness severity and predictor of mor- tality in hospitalised patients.8 In United Kingdom hospitals, level of arousal is routinely assessed using the AVPU scale (A, alert; V, responds to voice; P, responds to pain; U, unresponsive) as one of six indicators as a National Early Warning Score.9 Yet level of arousal in the specific context of delirium remains relatively understudied compared to its key cognitive symptom of attention deficits and other features.10-13 TABLE 1 Diagnostic and Statistical Manual of Mental Disorders (DSM) and International Classification of Diseases (ICD) diagnostic criteria descriptions of arousal disturbance in delirium In delirium, level of arousal is often reduced but there is a wide range of severities, from mild drowsiness to only being able to pro- duce a basic motor response to a verbal stimulus. Conversely, patients may have heightened arousal and appear agitated and hyperalert. The Richmond Agitation-Sedation Scale (RASS),14 which was originally developed to assess agitation or sedation levels in Intensive Care Unit (ICU) patients, has recently been modified for use as a delirium screen by including assessment of attention (mRASS).7 The RASS is the most studied arousal scale in delirium.4,15 However, a RASS score of +1 or −1 does not provide detailed information on the degree to which level of arousal is abnormal. More generally, an overall lack of granularity and operationalisation to capture this important feature of delirium in both arousal-specific and general delirium scales suggests that there would be value in having an instrument that provides a more detailed assessment. Key points Delirium is a severe, acute neurocognitive disorder characterised by disturbances in attention, level of arousal and other mental functions. It affects at least one in eight hospitalised older patients and is inde- pendently associated with multiple adverse outcomes.1-3 • Assessment of level of arousal is a core part of the evalu- ation of delirium, and arousal measurements could be useful in clinical practice as a strong indicator of delirium. • Assessment of level of arousal is a core part of the evalu- ation of delirium, and arousal measurements could be useful in clinical practice as a strong indicator of delirium. • The Observational Scale of Level of Arousal (OSLA) was developed as a brief observational instrument to charac- terise the abnormalities of level of arousal associated with delirium (score range 0 [normal arousal]-15). It comprises four items evaluating different aspects of arousal: eye opening, eye contact, posture, and movement. Alterations in level of arousal are common in delirium, with many patients showing hypo- or hyperarousal.4 The hypoactive subtype, characterised by drowsiness or somnolence, is the most common form of delirium.5 The arousal component of delirium has been described variably in standard diagnostic criteria (Table 1). In DSM-5, severely reduced level of arousal precluding cognitive testing or interview but above the level of coma is considered to indicate severe inat- tention. Assessment of level of arousal is therefore a core part of the evaluation of the features of delirium, and arousal measure- ments appear to be useful in clinical practice as a strong indicator of delirium.4,6,7 • The area under the Receiver Operating Characteristic curve for the OSLA for detecting delirium was 0.82. OSLA scores were responsive to within-patient change in delir- ium status and severity over time. • This study provides support for the utility of the OSLA as a brief, accurate instrument for measuring level of arousal in delirium and for monitoring change in arousal in non- ICU patients over time. Further validation studies are necessary to establish the clinical utility of the OSLA. K E Y W O R D S arousal, attention, cognition, delirium, orthopaedic surgery arousal, attention, cognition, delirium, orthopaedic surgery Roanna Hall and Antaine Stíobhairt contributed equally to this study. Alasdair M. J. MacLullich and Zoë Tieges contributed equally to this study. 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. © 2020 The Authors. International Journal of Geriatric Psychiatry published by John Wiley & Sons Ltd Int J Geriatr Psychiatry. 2020;1–7. 1 \| INTRODUCTION 2 HALL ET AL. HALL ET AL. 2 Key points • Assessment of level of arousal is a core part of the evalu- ation of delirium, and arousal measurements could be useful in clinical practice as a strong indicator of delirium. • The Observational Scale of Level of Arousal (OSLA) was developed as a brief observational instrument to charac- terise the abnormalities of level of arousal associated with delirium (score range 0 [normal arousal]-15). It comprises four items evaluating different aspects of arousal: eye opening, eye contact, posture, and movement. • The area under the Receiver Operating Characteristic curve for the OSLA for detecting delirium was 0.82. OSLA scores were responsive to within-patient change in delir- ium status and severity over time. • This study provides support for the utility of the OSLA as a brief, accurate instrument for measuring level of arousal in delirium and for monitoring change in arousal in non- ICU patients over time. Further validation studies are necessary to establish the clinical utility of the OSLA. 2.2 \| Participants A total of 108 community dwelling patients were recruited from orthopaedic wards at the Royal Infirmary of Edinburgh, Scotland. Patients were eligible if they were aged over 60 years and had an acute hip fracture and spinal anaesthesia. Patients were not eligible if they were nursing home residents; had taken oral or inhaled steroids in the last 10 weeks; had significant Parkinson's disease or other comorbid diseases with a prognosis of less than 1 year; or had major communication difficulties such as aphasia or where English was not their first language. 2 \| MATERIALS AND METHODS TABLE 2 The Observational Scale of Level of Arousal Observational Scale of Level of Arousal (OSLA) Eye opening Score Description 0 Open on arrival and remain so, under patient's control, outlasts stimulus 1 Open on arrival but close if stimulus removed 1 Open to voice but then outlasts stimulus 2 Open to voice but close if stimulus removed 3 Open to gentle physical stimulation (squeezing hand, gently shaking shoulder) 4 Open to pain only 5 No eye opening Eye contact Score Description 0 Spontaneously makes and holds eye contact appropriately 1 Drowsy and makes eye contact to command but cannot hold it for very long 1 Alert but eyes wandering, some appropriate eye contact 2 Alert but eyes wandering, little or no appropriate eye contact 2 Drowsy but makes brief eye contact 3 Eyes will/are open but no eye contact Posture (NB take into account weakness due to stroke or neurological disease, etc.) Score Description 0 Sitting out in chair or up in bed, holding appropriate posture 1 Slumped in chair or bed but attempts to sit upright and sustain posture on request 2 Slumped in chair or bed and unable to sustain posture 3 Lying in bed and unable or no response to request to sustain posture Movement Score Description 0 Moves spontaneously and purposefully with no restless or agitated movements 1 Occasional or mild restless or fidgety movements, no aggressive or vigorous movements 1 Reduced frequency of movement, mildly slowed up 2 Frequent restless or fidgety movements, no aggressive or vigorous movements 2 Moderately reduced frequency and speed of movement, interfering with assessment or self-care 3 Aggressive or vigorous, recent pulling out of lines 4 Overtly combative, violent 4 Severely reduced frequency and speed of movement, few spontaneous movements Score (0-15) This was a secondary analysis of data from a prospective cohort study in older adults with acute hip fracture with and without delirium.17 Participants were assessed in the 24 hours prior to their surgery, repeatedly up to 14 days post-operatively, and at 3, 6, and 12 months post-operatively. Data up to day 14 are reported here. The study was approved by the Scotland A Research Ethics Committee and written consent from patients or legal proxies was obtained. HALL ET AL. Key points DSM/ICD Description DSM-III Clouding of consciousness (reduced clarity of awareness of the environment), with reduced capacity to shift, focus, and sustain attention to environmental stimuli (Criterion A) Disturbance of sleep-wakefulness cycle, with insomnia or daytime drowsiness (one of four features under Criterion B, of which at least two must be present) DSM-III-Revised Reduced level of consciousness, for example, difficulty keeping awake during examination (one of six features under Criterion C, of which at least two must be present) DSM-IV A disturbance of consciousness, that is, reduced clarity of awareness of the environment, with reduced ability to focus, sustain, or shift attention (Criterion A) DSM-V A disturbance of attention (that is, reduced ability to direct, focus, sustain, and shift attention) and awareness (reduced orientation to the environment) ICD-10 Clouding of consciousness, that is, reduced clarity of awareness of the environment, with reduced ability to focus, sustain, or shift attention (Criterion A) To address this, we developed a new scale entitled the Obser- vational Scale of Level of Arousal (OSLA; Table 2). It was designed for research use, to characterise the abnormalities of level of arousal associated with delirium, complementing assessments of attention and other features of delirium. Individual item scores characterise the profile of abnormalities while the single overall score provides an index of severity. The OSLA was used in a small study examining the relationship between altered arousal and inat- tention.4 OSLA scores were strongly associated with delirium diag- nosis. Another study reported good diagnostic accuracy of the 4 delirium diagnosis. These analyses fitted a within-person fluctuation model to test if changes in OSLA over time (of the order of days) were predicted by changes in each of the two time-varying predictors. Two models were fitted, one with delirium diagnosis as time-varying predic- tor (mean-centred) and all covariates (model 1), and one with delirium severity as time-varying predictor and all covariates (model 2) (R function lmer24). The models included the following time-invariant covariates: age, sex, IQCODE, CCI, and APACHE II score. The depen- dent variable was OSLA score. make regarding level of arousal in practice. The second stage involved editing the list of potential items to construct a scale which would be: (a) rapid and simple to score, (b) based on observation after a brief encounter alone, (c) operationalised using brief behavioural descrip- tions, and (d) allow grading of severity of the arousal disturbance for each item. The four items have different numbers of grades of sever- ity based on what grades could readily be distinguished on brief observation. A total score (range 0-15) is obtained by summation of scores on each of the four items, with higher scores indicating greater abnormality. The OSLA is scored after a brief interaction with the patient and based on behavioural observations rather than cognitive testing. It generally takes under 1 minute to complete and does not require a verbal response from the patient. All time-varying variables were standardised into units of the SD at baseline (the pre-operation assessment) with all means measured from the baseline mean. Continuous covariates were centred on the respective sample median: age (82 years), CCI (score of 1) and APACHE II (score of 8). Sex and IQCODE (scores ≥3.44 indicating dementia) were represented by a dummy variable. All models included the main effects of the covariates and their interaction with time. All tests were administered by R. J. H. in the same order at the participants' hospital bedside. The two time-varying predictors were decomposed into two vari- ables: a person-mean (PM) variable and a within-person (WP) centred variable. In both models the WP variable entered the model at level-1, and the PM variable at level-2. Here we only report results of the WP variables. The ‘WPseverity’ effect represents how variation in OSLA over time fluctuates in step with variation in delirium severity. Study sample Participants were aged 61 to 95 (median = 82, IQR = 75-87) years. Baseline and demographic characteristics are provided in Table 3. There was some attrition of participants during the perioperative period, with seven participant withdrawals, three exclusions (two due to non-operative management of the fracture, and one due to an unpredictable and significant complication) and one death. The num- ber of patients who provided data for the OSLA varied across assess- ments: pre-operative = 108, day 1 = 96, day 2 = 86, day 3 = 47, day 4 = 98, day 7 = 77, day 14 = 48 (reasons of missing values are pres- ented in Table S1). The overall rate of delirium was 40.7% of patients. We assessed the suitability of the data for exploratory factor analysis (EFA) using Bartlett's test of sphericity, the determinant of the correlation matrix and the Kaiser-Meyer-Olkin measure of sam- pling adequacy. Horn's parallel analysis (with 10 000 iterations) was used to empirically determine the number of factors to retain. Factors with eigenvalues greater >1 were assumed to be meaningful. We then conducted minimum residuals EFA without rotation—as parallel analy- sis suggested only a single factor—and used factor loadings of 0.40 or greater in the factor designation. OSLA scores were compared between groups with and without delirium at each assessment. A receiver operating characteristic (ROC) analysis was conducted on OSLA scores with delirium diag- nosis as a reference to assess the ability of the OSLA to detect delirium, for data collapsed across assessments and also separately for each assessment. The relationship between scores on the OSLA and the DRS-R98 severity scale was examined using Kendal-Tau correlations. Like- wise, the ‘WPdiagnosis’ effect represents how variation in OSLA over time fluctuates in step with delirium diagnosis. 2.4 \| Statistical analysis Analysis was carried out using R version 3.0.1.24 Cases were excluded pairwise where data were missing. A threshold of P < .05 was taken to denote statistical significance. Demographics and test scores for each group are presented as medians (interquartile range [IQR]) unless otherwise specified. Com- parisons of OSLA scores and all other data were made between groups with and without delirium using Mann-Whitney U tests with continuity correction, separately for each assessment and also pooled assessments. Estimates of effect size r were calculated by dividing z- scores by the square-root of n.25 Pearson's chi-squared tests were used for categorical data where appropriate. Correlations were calcu- lated using Kendall's Tau due to frequent ties in the data. Holm cor- rections were applied to multiple comparisons. HALL ET AL. HALL ET AL. \| Measurements and procedures The diagnosis of delirium was made by a geriatrician (RJH), aided by the use of the Confusion Assessment Method (CAM)18 and Delirium Rating Scale-Revised-98(DRS-R98),19 and supplemented with assess- ments of level of consciousness (Richmond Agitation-Sedation Scale (RASS)14 and cognition (Mini-Mental State Examination).20 Delirium was considered present when CAM scores were positive or the total DRS-R98 score was over 17.75. Assessments took place preopera- tively, daily from post-operative days 1-4, on day 7 and once between days 10-14 or until transfer to a rehabilitation unit or discharge from hospital. Participants were assessed as frequently as possible, includ- ing once per weekend, although this was dependent on researcher availability and with the aim of not becoming burdensome for partici- pants. Illness severity and comorbidity were measured using the Acute Physiology Age and Chronic Health Evaluation (APACHE) II score,21 the Charlson Comorbidity Index (CCI)22 and the number of regular medications taken on admission. The Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE) was used to assess pre- existing cognitive impairment.23 OSLA for detecting delirium on its own, and in combination with an attention task.16 Here, we provide preliminary evaluation of the OSLA as a brief instrument to identify abnormal levels of arousal associated with delir- ium in patients with acute hip fracture. First, we assessed the psycho- metric characteristics of the OSLA, using exploratory factor analysis. Second, we assessed the diagnostic performance of the OSLA for delirium detection, because acute onset altered level of arousal is con- sidered a strong indicator of delirium.4 Third, to explore the potential utility of the OSLA in detecting changes in delirium longitudinally, we assessed its ability to detect within-person fluctuations in delirium status and symptom severity over several test occasions. The OSLA comprises four items, each assessing a different fea- ture of arousal: eye opening, eye contact, posture, and movement (Table 2). The items were developed by R. J. H. and A. M. J. M. through a two-stage process. The first stage involved generating potential items through informal observations in routine clinical prac- tice and reviewing items in existing arousal scales. The goal of this stage was to capture and operationalise the judgements that clinicians 3.1.3 \| Utility of the OSLA for measuring longitudinal changes in level of arousal associated with delirium 3.1.3 \| Utility of the OSLA for measuring longitudinal changes in level of arousal associated with delirium Further, scores on the OSLA correlated significantly with scores on the DRS-R98 severity sub-scale at each assessment point and when data were collapsed across assessments (Table 5). Thus, higher OSLA scores reflecting a greater degree of arousal abnormalities were associated with higher delirium symptom severity. A change in diagnosis from delirium to no delirium was associated with a within-person decrease in the patient's OSLA score of 3.09 (Model 1: β = −3.09, SE = 1.41, P < .03). Further, a within-person increase in DRS-R98 delirium severity score of 1 unit (SD) relative to The Area under the ROC Curve for the OSLA for detecting delir- ium when data were pooled across assessments was 0.82 (P < .001, 95% CI [0.77, 0.86]). 3.1.2 \| Association between OSLA and delirium status and severity Scores on the OSLA were consistently higher in patients with delirium than those without delirium at each assessment, and also when data were collapsed across assessments (Table 4). A delirium diagnosis was always accompanied by an OSLA score greater than 0. accounted for 31% of the variance using minimum residuals factor analysis (Table S2). from individual assessment days yielded similar results to those of the pooled analysis (Table S3). Using an OSLA cutoff score ≥2, sensitivity and specificity for delirium were 0.87 (95% CI [0.84, 0.93]) and 0.53 (95% CI [0.48, 0.58]), respectively. A higher cutoff score of ≥3 was associated with a decline in sensitivity to 0.65 (95% CI [0.56, 0.74]) with an increased specificity of 0.85 (95% CI [0.81, 0.88]). The previ- ously suggested OSLA cutoff score of ≥4 for delirium detection4 resulted in a sensitivity of 0.42 (95% CI [0.33, 0.51]) and specificity of 0.95 (95% CI [0.93, 0.97]). 3.1.3 \| Utility of the OSLA for measuring longitudinal changes in level of arousal associated with delirium The Area under the ROC Curve analyses for data TABLE 3 Patient demographic characteristics according to the presence of delirium at a minimum of one assessment point or the absence of delirium at all assessment points during hospital stay raphic characteristics according to the presence of delirium at a minimum of one assessment point or the absence nts during hospital stay TABLE 3 Patient demographic characteristics according to the presence of delirium at a minimum of one ass delirium at all assessment points during hospital stay TABLE 3 Patient demographic characteristics according to the presence of delirium at a minimum of one assessment point or the absence of delirium at all assessment points during hospital stay Delirium No delirium Comparison Male:Female ratio (% Female) 20:24 (36) 22:42 (64) P = .25, 95% CI [0.67, 3.75], X2(1) = 1.35 Age (years) 83 (77-88) 81 (71-86) P = .163, 95% CI [−6, 1], U = 1185, z = −1.4 n 44 64 Length of hospital stay (days) 62 (28-81) 16 (11-30) P < .001, 95% CI [17, 48], U = 529, z = −4.6 n 49 59 Charlson Comorbidity Index 1 (0–2) 1 (0-1) P = .064, 95% CI [−0, 0], U = 1124, z = −1.9 n 44 64 Frailty Index on admission 0 (0–2) 0 (0-0) P = .007, 95% CI [0, 1], U = 1045, z = −2.7 n 44 64 IQCODE 3 (3-3) 3 (3-4) P < .001, 95% CI [0, 0], U = 666, z = −3.7 n 40 57 APACHE II 9 (7-11) 8 (6-10) P = .007, 95% CI [−2, 0], U = 977, z = −2.7 n 44 64 Note: Age, Acute Physiology and Chronic Health Evaluation (APACHE) II score range 0 to 71, higher score indicating greater illness severity. Charlson Comorbidity Index score range 0 to 31, higher score indicating greater comorbidity. Frailty index range 0 to 3, higher score indicating greater frailty. Infor- mant Questionnaire of Cognitive Decline in the Elderly (IQCODE) average score range 1 to 5, higher score indicating greater cognitive decline. Descriptive statistics for continuous variables are expressed as medians (interquartile range). Ratios for categorical variables are expressed as frequencies (%). Note: Age, Acute Physiology and Chronic Health Evaluation (APACHE) II score range 0 to 71, higher score indicating greater illness severity. Charlson Comorbidity Index score range 0 to 31, higher score indicating greater comorbidity. 3.1.1 OSLA scores ranged between 0 and 9 (median = 2). Individual item score ranges were 0 to 3 (Eye Opening), 0 to 3 (Eye Contact), 0 to 2 (Posture), and 0 to 3 (Movement). The four items of the OSLA were suitable for EFA, as indicated by Bartlett's test (X2[6] = 166.4, P < .001), the determinant of the correlation matrix (0.74) and the overall (0.67) and item (all 0.67) Kaiser-Meyer-Olkin measure of sampling adequacy. A single factor, as indicated by a scree plot (Figure S1) and parallel analysis (Adjusted Eigenvalue = 1.76, Unadjusted Eigenvalue = 1.91, Estimated bias = 0.15) Exploratory linear mixed effects models were used to evaluate responsiveness of the OSLA to change in delirium status and severity over time, to provide additional information on its performance and also to explore the importance of level of arousal in contributing to a HALL ET AL. 4 \| DISCUSSION The present study provides preliminary support for the utility of the OSLA as a brief, accurate instrument for measuring level of arousal in delirium. The OSLA showed gradations in scores and was responsive to a change in delirium diagnosis within patients over time. Factor analysis of the eye opening, eye contact, movement, and posture items confirmed that it is appropriate to group these four features under a single factor (ie, arousal). This study has several limitations. Delirium diagnosis and arousal assessments were done by a single rater, hence estimations of inter- rater reliability were not possible, and the diagnostic accuracy of the OSLA for delirium may have been inflated. Formal assessments of interrater reliability and validity using independent, blinded raters are required to supplement the current findings. Further, a number of patients were lost to follow-up in the first 14 days post-surgery. In the pre-fracture and immediate post-operative stages, scoring of the Movement item may have been confounded by the patient's fracture, which could restrict movement. Also, the different numbers of grading levels for each item might mean that some items carry greater weight on the scale. A wide range of OSLA scores was seen in the present study (0-9) and a delirium diagnosis was always accompanied by an OSLA score greater than 0. The OSLA therefore appears to be sensitive to the gra- dations in level of arousal seen in delirium in this non-ICU patient population. Importantly, the OSLA was responsive to fluctuations in delirium status and severity over time within individual patients. This further supports the utility of the OSLA for assessing an aspect of delirium severity, and provides initial support for the utility of the OSLA in monitoring change in arousal as part of delirium in patients over time. Chester et al7 report a similar sensitivity for the mRASS (0.64) compared to the OSLA (0.65 using a cutoff ≥3) but with higher speci- ficity (mRASS: 0.93, OSLA: 0.85). The latter finding may partly have resulted from incorporating attention assessments into the mRASS. Interestingly, the OSLA in our study performed broadly comparably while assessing level of arousal alone, without any items specifically assessing attention. 6 HALL ET AL. HALL ET AL. TABLE 5 Correlation of DRS-R98 severity scores with the Observational Scale of Level of Arousal Pre-operative Day 1 Day 2 Day 3 Day 4 Day 7 Day 14 Overall Correlation coefficient 0.35 0.45 0.46 0.44 0.50 0.47 0.58 0.46 n 107 90 81 39 94 76 46 533 P-value, 95% Confidence Intervals (CI) P < .001, 95% CI [0.17, 0.50] P < .001, 95% CI [0.26, 0.60] P < .001, 95% CI [0.26, 0.61] P < .001, 95% CI [0.14, 0.44] P < .001, 95% CI [0.33, 0.64] P < .001, 95% CI [0.27, 0.63] P < .001, 95% CI [0.35, 0.74] P < .001, 95% CI [0.39, 0.46] Note:DRS-R98, Delirium Rating Scale-Revised-98. Calculated using Kendall's Tau with Holm correction. TABLE 5 Correlation of DRS-R98 severity scores with the Observational Scale of Level of Arousal Note:DRS-R98, Delirium Rating Scale-Revised-98. Calculated using Kendall's Tau with Holm correction. populations. Specifically, the OSLA allows arousal features to be scored independently of one another, enabling observers to charac- terise a patient's level of arousal in some detail, while retaining its brevity and providing a measure of arousal in a single severity score. As such, it might prove useful as a brief, standardised delirium classifi- cation method (ie, hypo- vs hyperactive delirium) based on level of arousal alone.15 Although the OSLA was developed as a stand-alone test, it could complement existing delirium assessment batteries. Of note, a brief combined arousal-attention assessment using OSLA and SAVEAHAART has been shown to have high diagnostic accuracy for detecting delirium even in a subgroup of patients with dementia, and thus could have clinical utility for diagnosing delirium superimposed on dementia.16,26 its average was associated with an increase in OSLA score of 0.5 (Model 2: ß = 0.50, SE = 0.13, P < .001). its average was associated with an increase in OSLA score of 0.5 (Model 2: ß = 0.50, SE = 0.13, P < .001). 4 \| DISCUSSION Of note, the present findings suggest a lower cut- off point of ≥2 or ≥3 compared to the previously suggested optimal cutoff of ≥4 for the OSLA.4 Future studies are needed to evaluate the utility of OSLA in dif- ferent populations (eg, palliative care, emergency departments) and to assess the prognostic value of higher OSLA scores for unfavourable outcomes in prospective cohort studies. A recent study provided pre- liminary support for the utility of the OSLA as a tool for detailed mea- surement of level of arousal in ICU patients,27 though the OSLA itself has yet to be formally validated in this population. In conclusion, this study provides promising evidence in support of the OSLA as a method for arousal assessment in the context of delirium. The OSLA may usefully complement existing measures of delirium where additional detail is desirable. Scores on the OSLA and the DRS-R98 severity scores were asso- ciated at each assessment point, even though the latter does not mea- sure level of arousal explicitly. This likely reflects the hierarchical relationship between arousal and cognition, whereby level of arousal must be sufficient before cognition can be reasonably tested. This finding suggests that level of arousal may provide a useful, practical marker for grading severity of delirium. 3.1.3 \| Utility of the OSLA for measuring longitudinal changes in level of arousal associated with delirium Frailty index range 0 to 3, higher score indicating greater frailty. Infor- mant Questionnaire of Cognitive Decline in the Elderly (IQCODE) average score range 1 to 5, higher score indicating greater cognitive decline. Descriptive statistics for continuous variables are expressed as medians (interquartile range). Ratios for categorical variables are expressed as frequencies (%). E 4 Results of the Observational Scale of Level of Arousal according to the presence or absence of delirium TABLE 4 Results of the Observational Scale of Level of Arousal according to the presence or absence of delirium Pre-operative Day 1 Day 2 Day 3 Day 4 Day 7 Day 14 Overall Delirium 3 (2–4) 4 (2-5) 4 (2-4) 3 (3-5) 3 (2-4) 3 (3-6) 4 (4-6) 3 (2-5) n 13 26 17 14 22 8 3 103 No delirium 2 (1–2) 2 (1-3) 2 (1-2) 1 (1-2) 1 (1-2) 1 (1-2) 1 (0-2) 1 (1-2) n 94 70 69 34 75 68 44 454 Comparison P < .001, 95% CI [1, 2], r = 0.36 P < .001 95% CI [1, 3], r = 0.46 P < .001 95% CI [1, 2], r = 0.37 P < .001 95% CI [1, 3], r = 0.54 P < .001 95% CI [1, 2], r = 0.50 P < .001 95% CI [1, 4], r = 0.39 P = .005 95% CI [1, 6], r = 0.41 P < .001 95% CI [1, 2], r = 0.44 REFERENCES 1. Inouye SK, Westendorp RG, Saczynski JS. Delirium in elderly people. Lancet. 2014;383(9920):911-922. 1. Inouye SK, Westendorp RG, Saczynski JS. Delirium in elderly people. Lancet. 2014;383(9920):911-922. 20. 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Biological Sciences Research Council, the Engineering and Physical Sciences Research Council, the Economic and Social Research Council, and the Medical Research Council is gratefully acknowledged. 14. Sessler CN, Gosnell MS, Grap MJ, et al. The Richmond Agitation- Sedation Scale: validity and reliability in adult intensive care unit patients. Am J Respir Crit Care Med. 2002;166(10):1338-1344. 15. Han JH, Brummel NE, Chandrasekhar R, et al. Exploring Delirium's heterogeneity: association between arousal subtypes at initial presen- tation and 6-month mortality in older emergency department patients. Am J Geriatr Psychiatry. 2017;25(3):233-242. Zoë Tieges https://orcid.org/0000-0002-3820-3917 Zoë Tieges https://orcid.org/0000-0002-3820-3917 19. Trzepacz PT, Mittal D, Torres R, Kanary K, Norton J, Jimerson N. Vali- dation of the Delirium Rating Scale-revised-98: comparison with the delirium rating scale and the cognitive test for delirium. J Neuropsychiatry Clin Neurosci. 2001;13(2):229-242. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available onrequest from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. 17. Hall RJ, Ferguson KJ, Andrews M, et al. Delirium and cerebrospinal fluid S100B in hip fracture patients: a preliminary study. Am J Geriatr Psychiatry. 2013;21(12):1239-1243. 18. Inouye SK, van Dyck CH, Alessi CA, Balkin S, Siegal AP, Horwitz RI. Clarifying confusion: the confusion assessment method. A new method for detection of delirium. Ann Intern Med. 1990;113(12):941-948. REFERENCES J Hosp Med. 2012;7(5):450-453. 26. Quispel-Aggenbach DWP, Holtman GA, Zwartjes H, Zuidema SU, Luijendijk HJ. Attention, arousal and other rapid bedside screening instruments for delirium in older patients: a systematic review of test accuracy studies. Age Ageing. 2018;47(5):644-653. 8. Bleyer AJ, Vidya S, Russell GB, et al. Longitudinal analysis of one mil- lion vital signs in patients in an academic medical center. Resuscitation. 2011;82(11):1387-1392. 27. Tang E, Laverty M, Weir A, et al. Development and feasibility of a smartphone-based test for the objective detection and monitoring of attention impairments in delirium in the ICU. J Crit Care. 2018;48: 104-111. 9. Royal College of Physicians. National Early Warning Score (NEWS): Standardising the assessment of acute illness severity in the NHS. Report of a working party. London; 2017. 10. Brown LJ, Fordyce C, Zaghdani H, Starr JM, MacLullich AM. Detecting deficits of sustained visual attention in delirium. J Neurol Neurosurg Psychiatry. 2011;82(12):1334-1340. ACKNOWLEDGEMENTS This work was supported by a Clinical Research Training Fellowship to R. J. H. from Research into Ageing (AgeUK) and the British Geriat- rics Society (grant number 342). Funding from the Biotechnology and The OSLA could have utility in operationalising the subtler arousal changes that appear to indicate delirium in general hospital HALL ET AL. SUPPORTING INFORMATION Additional supporting information may be found online in the Additional supporting information may be found online in the Supporting Information section at the end of this article. 11. Lowery DP, Wesnes K, Brewster N, Ballard C. Quantifying the associ- ation between computerised measures of attention and confusion assessment method defined delirium: a prospective study of older orthopaedic surgical patients, free of dementia. Int J Geriatr Psychia- try. 2008;23(12):1253-1260. pp g y Supporting Information section at the end of this article. Supporting Information section at the end of this article. How to cite this article: Hall R, Stíobhairt A, Allerhand M, MacLullich AMJ, Tieges Z. The Observational Scale of Level of Arousal: A brief tool for assessing and monitoring level of arousal in patients with delirium outside the ICU. Int J Geriatr Psychiatry. 2020;1–7. https://doi.org/10.1002/gps.5324 12. Tieges Z, Brown LJ, Maclullich AM. Objective assessment of attention in delirium: a narrative review. Int J Geriatr Psychiatry. 2014;29(12): 1185-1197. 13. Tieges Z, Evans JJ, Neufeld KJ, MacLullich AMJ. The neuropsychology of delirium: advancing the science of delirium assessment. Int J Geriatr Psychiatry. 2018;33(11):1501-1511.
https://openalex.org/W3123847856	https://wrap.warwick.ac.uk/139779/1/WRAP-Switching-energy-suppliers-money-market-Loomes-2020.pdf	English	null	Switching Energy Suppliers: It's Not All about the Money	Social Science Research Network	2,017	cc-by	18,209	abstract Many consumers do not take advantage of lower energy prices available in lib eralized retail markets. We provide evidence to explain why consumers may leave substantial amounts of “money on the table” in this way. We observe real decisions made by over 7,000 consumers in a collective switching auction, supplemented by their responses to a survey. We identify factors which may inhibit switching and show that expectations of high switching rates in an unregulated market may be unrealistic. Our findings have important implica tions for the design and regulation of energy markets, including imposition of price caps on “default” retail tariffs in 2019 in the UK and parts of Australia. Keywords: Retail energy market, Switching suppliers, Probit models, Behavioral consumers Keywords: Retail energy market, Switching suppliers, Probit models, Behavioral consumers https://doi.org/10.5547/01956574.42.3.ddel 1. INTRODUCTION The UK government has introduced price caps on “default” retail energy prices,1 sixteen years after removing price regulation from the market. Its primary declared objective is fairness to consumers who “leave money on the table”.2 This price cap policy follows a decade of intervention from governments and regulators to address consumer “inertia” which has yielded disappointing results, as many consumers seem to remain “disengaged”. There are particular concerns for house holds in hardship who pay more than necessary for a commodity that absorbs a significant propor tion of their income. The regulator has seen low switching rates as problematic since its Energy Supply Probe (Ofgem, 2008), and the Competition and Markets Authority (2016) found an Adverse Effect on Competition from weak customer response. Such disengagement does not sit easily with 1. See “Draft Domestic Gas and Electricity (Tariff Cap) Bill’, Department for Business, Energy and Industrial Strategy, Cm 9516, October 2017, available at: https://www.gov.uk/government/publications/draft-domestic-gas-and-electricity-tariff- cap-bill 1. See “Draft Domestic Gas and Electricity (Tariff Cap) Bill’, Department for Business, Energy and Industrial Strategy, Cm 9516, October 2017, available at: https://www.gov.uk/government/publications/draft-domestic-gas-and-electricity-tariff- cap-bill 2. However capping prices raises additional challenges for the long-term development of a competitive market, particu larly if reduced potential savings lower consumers’ incentive to engage with the market. For a discussion of the effects of this price cap, see Deller et al. (2017a), in response to a government consultation about the enabling legislation. 2. However capping prices raises additional challenges for the long-term development of a competitive market, particu larly if reduced potential savings lower consumers’ incentive to engage with the market. For a discussion of the effects of this price cap, see Deller et al. (2017a), in response to a government consultation about the enabling legislation. a Centre for Competition Policy, University of East Anglia. b School of Business and Economics, University of Loughborough. d Corresponding author. Centre for Competition Policy, University of East Anglia. E-mail: C.Waddams@uea.ac.uk. e School of Economics, Universidad de Malaga. Corresponding author. Centre for Competition Policy, University of East Anglia. E-mail: C.Waddams@uea.ac. S h l f E i U i id d d M l d Corresponding author. Centre for Competition Policy, University of East Anglia. E-mail: C.Wad e School of Economics, Universidad de Malaga. f Department of Economics, University of Reading. The Energy Journal, Vol. 42, No. 3. 1. See “Draft Domestic Gas and Electricity (Tariff Cap) Bill’, Department for Business, Energy and Industrial Strategy, Cm 9516, October 2017, available at: https://www.gov.uk/government/publications/draft-domestic-gas-and-electricity-tariff- cap-bill The Energy Journal, Vol. 42, No. 3. This is an open access article under the terms of the Creative Commons Attribution License (CC-BY), which permits use, distribution and reproduction in any medium, provided the original work is properly cited. All rights reserved. p 2. However capping prices raises additional challenges for the long-term development of a competitive market, particu larly if reduced potential savings lower consumers’ incentive to engage with the market. For a discussion of the effects of this price cap, see Deller et al. (2017a), in response to a government consultation about the enabling legislation. The following article is a preprint of a scientiﬁc paper that has completed the peer-review process and been accepted for publication within The Energy Journal. While the International Association for Energy Economics (IAEE) makes every effort to ensure the veracity of the material and the accuracy of the data therein, IAEE is not responsible for the citing of this content until the article is actually printed in a ﬁnal version of The Energy Journal. For example, preprinted articles are often moved from issue to issue affecting page numbers, and actual volume and issue numbers. Care should be given when citing Energy Journal preprint articles. a Centre for Competition Policy, University of East Anglia. Switching Energy Suppliers: It’s Not All About the Money David Deller,a Monica Giulietti,b Graham Loomes,c Catherine Waddams Price,d Anna Moniche,e and Joo Young Jeonf 1. INTRODUCTION This is an open access article under the terms of the Creative Commons Attribution License (CC-BY), which permits use, distribution and reproduction in any medium, provided the original work is properly cited. All rights reserved. 95 96 / The Energy Journal naïve utility-maximising models where consumers are expected to purchase a homogeneous product at a lower price. We explore why one group of active and apparently well-motivated consumers did not accept offers of lower energy prices and reduced bills, even though it seemed easy for them to do so. Despite the evidence of this kind of inaction, policymakers have sometimes relied on a rather narrow view of the behavior of rational consumers. An example of this approach can be found in a call for evidence issued by the UK Department of Business Innovation and Skills (2015). “If you knew you had won £200 on the lottery, would you forget to claim it? Probably not. Yet consumers across the UK are effectively ignoring significant savings every year when they stick with their current providers of essential but routine services.” Our investigation allows us to consider a range of non-monetary factors which are often ignored when devising policies to address consumer “in ertia”. Indeed, we find a number of non-monetary factors which seem influential and which help to explain the apparently weak consumer response to savings opportunities in this context, suggesting that price competition for this apparently homogeneous product may have less power than is often assumed by policymakers. The energy sector is not only important in the individual budgets of millions of households, but, as current UK policy demonstrates, is politically sensitive and represents significant value in the overall economy. These findings therefore have important implications both for the optimal design and regulation of such markets and for the management strategies of firms operating in the energy and other industries. Consumer inaction in the face of extensive potential energy savings is widely observed, both in US States which have opened retail energy markets (Hortaçsu et al., 2017) and in the UK, where energy markets are relatively mature (Competition and Markets Authority, 2016). In the Spring of 2012, Which? (a subscription-based consumer organisation3) and 38 Degrees (a campaigning group) advertised an open invitation for consumers to join The Big Switch (TBS), the largest collective energy switching exercise ever conducted in the UK. 3. Which? is the trading name of the British Consumers’ Association. 4. About 85% of British households (Ofgem, 2015), and 88% of our samples, use both gas and electricity. f 7. The geographically uniform Co-op bid was not always cheaper than some local tariffs. Open Access Article 3. Which? is the trading name of the British Consumers’ Association. 4. About 85% of British households (Ofgem, 2015), and 88% of our samples, use both gas and electricity. 5. This information mirrored that required by price comparison websites to identify the best offer for a consumer. 6. In the UK market there are regional variations in tariffs, but the rules of the auction required the same price to be of fered throughout the UK. 7. The geographically uniform Co-op bid was not always cheaper than some local tariffs. q y p p yf 6. In the UK market there are regional variations in tariffs, but the rules of the auction required the same price red throughout the UK. 1. INTRODUCTION Participants provided infor mation about their energy (electricity and, where relevant, gas4) consumption5 which was passed on in aggregate form to the energy companies bidding in the auction. On the supply side, the auction eers provided an open invitation to bidders, but imposed some restrictions, including geographical uniformity,6 which deterred some suppliers. Some established providers expressed concern about how participation in the auction might affect the regulator’s views on prices charged to other cus tomers. In the event, five companies, including three of the six leading providers, joined the auction. Each participating consumer then received a personalised offer based on the bid made by the win ning company (Cooperative Energy—henceforth, Co-op) and was invited to accept it, but with no obligation to do so. If, for any particular consumer, there happened to be a cheaper deal available from another company on the Which? price comparison website, the consumer was shown that cheaper deal as well as the offer from Co-op.7 A small number of participants in TBS already had a deal which TBS could not improve upon: these observations have been excluded from the present analysis as we are interested in the behavior of consumers who had an opportunity to save on their existing bills but did not take up the offer. Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 97 When presented with the offer(s), participants had to take little further action to complete a switch.8 Yet only just over a quarter of those who were presented with positive savings took the small step necessary to accept the offer. Even for savings of over £300 per year (around a third of the average bill), fewer than half switched, despite the fact that these participants had already actively opted into TBS, faced no additional search costs and often had characteristics which are usually associated with market engagement. We explore why so many consumers chose not to switch even when offered substantial savings in a benign switching context. Our analysis combines energy characteristics and decision data from nearly 87,000 house holds with survey data for a subset of just under 7,500 participants who provided additional infor mation about their personal characteristics and attitudes. Linking these sources of data provides a unique opportunity to observe on a large scale the decisions which consumers made about whether or not to switch. 1. INTRODUCTION Our data allow us to investigate switching decisions separately from the search process which consumers often face when contemplating changing supplier.9 Low rates of switching are often attributed to the deterrent effects of having to search: even with online price comparison sites available, it requires some determination to set time aside to search among many somewhat complex tariffs when, ex ante, the benefits of the search are uncertain. In the present study, the focus upon the “accept or decline offer” stage of TBS enables the isolation of a “pure” switching decision, since very little extra effort was required to accept the offer.10il We find that a wide range of factors influence a consumer’s decision about whether or not to switch. The offer of a substantial monetary saving alone is often insufficient to ensure switching, even for those who said they had opted into the auction with monetary savings as a motivating factor. Other broad factors which influence the switching decision include uncertainty about vari ous aspects of the offer(s), preferences over non-price characteristics, concerns about the switching process itself and time pressures. Many of the factors identified can be located within a rational deci sion-making framework, suggesting that the perceived net benefit from switching may be much less than solely the magnitude of potential monetary savings. Consequently, switching rates are likely to be substantially lower than we might initially expect, even in favourable conditions. An important policy implication is that energy markets need to be designed with such barriers in mind and in the knowledge that switching rates may be difficult to raise above a modest level. While much of the behavior might be understood within a rational choice framework, TBS also provided some evidence of responses which may not fit rationality assumptions so well. For example, some participants saw two offers: the one from the Co-op and another (cheaper) offer that was the best from any other company on the Which? price comparison website. While more choice is conventionally regarded as desirable, in this case simply being shown two offers rather than one reduced the probability of switching, all other things being equal. The paper proceeds as follows. Section 2 provides an overview of the literature regarding consumer behavior in energy markets. Section 3 gives a detailed description of the data and a range of descriptive statistics. Section 4 explains the econometric methods used. 8. This was simple personal information such as name, address and date of birth; and bank details so that payment could be arranged. 8. This was simple personal information such as name, address and date of birth; and bank details so that payment could be arranged. 9. The importance of distinguishing between search costs and switching costs is emphasised by Wilson (2012). 9. The importance of distinguishing between search costs and switching costs is emphasised by Wilson (20f 8. This was simple personal information such as name, address and date of birth; and bank details so that payment could be arranged. 9. The importance of distinguishing between search costs and switching costs is emphasised by Wilson (2012). 10. See Klemperer (1987, 1995) for additional detail regarding different types of switching cost. 2. LITERATURE REVIEW The importance of consumer switching for the healthy functioning of markets has long been recognised (for example, see Waterson, 2003; and McFadden, 2006). In the energy market, the increasing emphasis given to consumer behavior and aggregate switching rates by regulators and politicians is evidenced by the escalating number of policy reports and initiatives on the topic: for example, Competition and Markets Authority (2016), Department of Business, Energy and Indus trial Strategy (2017) and Council of European Energy Regulators (2017). Consumer switching behavior in energy markets has been investigated in a number of survey-based academic studies. For example, Ek and Söderholm (2008), Juliusson et al. (2007), Gamble et al. (2009) and Weber et al. (2009) report survey data for parts of continental Europe, while in the UK, survey based papers include Waddams Price and Bennett (1999), Waddams Price (2004), Giulietti et al. (2005), Waddams Price and Zhu (2016), He and Reiner (2017) and Flores and Waddams Price (2018). These studies each identify anticipated monetary gains from switching as a key driver of search and switching, consistent with a rational model of consumer choice, where indi viduals allocate their time to different tasks according to the expected gains available. Nevertheless, these studies also find that factors beyond monetary savings influence the likelihood of switching. For example, Flores and Waddams Price (2018) and Waddams Price and Zhu (2016) report that the experience of switching in other markets positively influenced switching behavior in the electricity market. He and Reiner (2017) confirmed that non-price factors, particularly consumers’ attitudes to energy, which are related both to psychological factors and political allegiance, may hinder con sumers from switching even when it appears rational. While such surveys explore consumer moti vations and expectations, they often rely on respondents’ ability to recall and report accurately their thoughts and actions at a previous switching event. In the present study, recall issues are reduced as the switching decision is directly observed and billing information, switching behavior and the offers received by participants are recorded contemporaneously in the switching dataset. In terms of combining billing information with socio-economic information, Kleit et al. (2012) and Hortaçsu et al. (2017) are the closest papers to the current study. Kleit et al. investigate switching behavior in Pennsylvania following the removal of residential rate caps in 2010. 1. INTRODUCTION In section 5, we present results. Section 6 concludes and suggests some implications of our results for managers and policy makers. The importance of distinguishing between search costs and switching costs is emphasised by Wilson (2012). 0. See Klemperer (1987, 1995) for additional detail regarding different types of switching cost. Open Access Article 98 / The Energy Journal Open Access Article 2. LITERATURE REVIEW They find that households are more likely to switch, and do so faster, in areas with a more educated population, lower unemployment rates and higher median household incomes. However the authors rely on area level socio-economic information, rather than combining billing information with individual-level data. A similar approach is employed by Hortaçsu et al. (2017) for the Texan electricity market be tween 2002 and 2006. Hortaçsu et al. find that the percentage of potential energy savings realised by consumers is positively related to an area’s education level and negatively related to its level of poverty. Using data from the Belgian electricity market from 2012 to 2016, and combining market share, price and advertising data with consumer surveys, Dressler and Weiergraber (2017) identify several sources of inertia. Over 65s show a strong preference for the incumbent; some consumers are prepared to pay a premium for green energy; switching costs amount to a significant proportion of annual electricity expenditure; and supplier advertising significantly affects consumer awareness, as measured by use of price comparison websites. Several papers estimate search and switching costs using aggregate price data rather than the decisions of individual consumers, including Giulietti et al. (2010) and Salies (2005). Giulietti et al. (2014) use a sequential search model to estimate how far price dispersion in the marketplace can be explained by search costs. Wilson and Waddams Price (2010) show that consumers may struggle to make “good” decisions in the UK electricity market, i.e. switching to the cheapest supplier; while Switching Energy Suppliers: It’s Not All About the Money / 99 Zhu (2013) cautions that non-switching in the presence of monetary savings can still be consistent with rational behavior if consumers have a preference for their existing suppliers. As markets have matured, collective switching schemes have been introduced. In the UK, the Department for Energy and Climate Change (2013) provides a broad overview of the perfor mance of such schemes, detailing the outcomes of 31 projects which received funding from the Cheaper Energy Together fund at the end of 2012. However, the data reported are mainly descrip tive, with no quantitative analysis of the reasons for the considerable variation in the switching rates achieved (from 5.5% to 23.1%). Deller et al. (2017b) find a similarly low typical response to opt-in collective switching schemes, which themselves are mainly small scale, in their more thorough international review. 2. LITERATURE REVIEW The European Commission (2016) reports some success with collec tive switching schemes, particularly in Portugal, but unfortunately further details are not provided. Direct comparison of switching rates between collective and individual mechanisms is difficult because of the absence of a clearly defined control group; and because of the need to distinguish between a switching rate among the whole population of consumers and those who have already expressed their interest in switching by opting into a scheme such as that described in this paper. However, the British regulator (Ofgem, 2019) showed in a series of randomised controlled trials that collective switching schemes could increase switching rates amongst previously “disengaged” customers by more than five times compared with a control group.11 The most successful of these trials resulted in switching rates of around 30% for those invited to participate, compared to 5% in the control group. While this rate seems similar to the levels reported in this paper, the Ofgem rates may be more remarkable, since they relate to consumers who were previously disengaged, while the consumers whose decisions are analysed in this paper often had some prior experience of switching and had taken active steps to opt in to TBS. Regarding consumer aggregation exercises in the US, Littlechild (2008) reviews the per formance of a municipal aggregation scheme in Ohio, while Loxley and Salant (2004) describe the choice of an auction mechanism used to select the default service provider in New Jersey. Opt-out switches,12 as occur in some US municipalities, gain much higher participation rates than opt-in schemes, and effectively operate as competition for a sector of the market, rather than focusing on competing for individual accounts. Ofgem13 is exploring such schemes as a longer term solution to non-engagement; however, they raise issues of privacy and default rules, and, like the government’s existing price caps, are likely to require primary legislation (see Deller et al., 2017b). Our analysis of TBS opt-in campaign provides the first econometric investigation of consumer switching behav ior as part of a collective switching/consumer aggregation exercise, in conjunction with substantial complementary individual survey data. 11. The trials sent disengaged energy customers a variety of letters, testing whether highlighting potential savings, sign posting to an exclusive tariff, and offering support with switching can increase rates of customers choosing to switch tariff. The intervention was designed to make the process of switching as simple as possible. 12. An opt-out scheme involves collectively switching consumers to an alternative deal, unless they explicitly state that they do not agree to an automatic change of supplier; this is in contrast to an opt-in collective switch where consumers them selves need to undertake some action, even if only by agreeing to the switch’s terms, in order to be included. 13. Dermot Nolan’s speech to Energy UK 19th October 2017 https://www.ofgem.gov.uk/system/files/docs/2017/10/ euk_final_19.10_v2.pdf 13. Dermot Nolan’s speech to Energy UK 19th October 2017 https://www.ofgem.gov.uk/system/files/docs/2017/10/ euk_final_19.10_v2.pdf 11. The trials sent disengaged energy customers a variety of letters, testing whether highlighting potential savings, sign posting to an exclusive tariff, and offering support with switching can increase rates of customers choosing to switch tariff. The intervention was designed to make the process of switching as simple as possible. 12 A t t h i l ll ti l it hi t lt ti d l l th li itl t t th t 3. DATA Our data combine observations of actual switching decisions from TBS with additional survey responses from a large sample of TBS participants who were contacted about nine months 13. Dermot Nolan’s speech to Energy UK 19th October 2017 https://www.ofgem.gov.uk/system/files/docs/2017/10/ euk_final_19.10_v2.pdf Open Access Article 100 / The Energy Journal later. Complete records of energy bill details and the offer(s) each person received as part of TBS in May 2012 were obtained for 139,644 people. Then in Spring 2013, half of this group, randomly selected, were sent a follow up survey to elicit information about factors which might have affected the probability of each individual switching energy supplier, generating 15,329 complete responses. As our research questions focus on the (non) response to financial savings, we chose to consider only those individuals who have been offered monetary savings as a result of TBS. In the discussion of our analysis we therefore focus on 119,125 TBS participants (May 2012 sample) who had received an of fer of positive savings and 12,750 participants with complete responses from the Spring 2013 survey. i From those 12,750, we identified a subset whose circumstances were least complicated and who might have been considered, ex ante, as those most likely to switch and where comparison was relatively straightforward (for example only those with a single energy supplier). Our strategy was to take cases which give a naïve savings-based switching model its “best chance” and examine behavior among this “upper bound” group. To this end we applied two filters, retaining the respon dents: (a) who had a single energy supplier for gas and electricity at the time of the auction; and (b) who had opted to take part in the online Direct Debit14 auction. These criteria reduced the May 2012 sample from 119,125 to 86,904. For those who subsequently participated in the Spring 2013 survey, a further filter was applied to include only respondents who identified “to save money” as one of their motivations for taking part in TBS. This left us with 7,367 survey respondents who met all three criteria. 14. Direct Debit is the predominant payment method in Great Britain and involves monthly deductions from a bank account to spread the estimated cost of the energy evenly over the year, with an annual reconciliation from metered consump tion. Note that members of this group were not necessarily paying by Direct Debit before they entered the auction. 15. Table A3.1 in the Appendix shows that those who participated in the survey were older, more highly educated and more likely to own their own home (or have a mortgage) than the typical British household. 3. DATA The following summary statistics describe the characteristics of those used in the present analysis.ii The first column of the first row of Table 1 shows that only 27.1% of those participants who were offered a positive saving went on to switch, despite the fact that the median saving, reported in absolute and relative terms in the fifth and sixth rows, was over £100 in the first year, representing just over 10% of those participants’ pre-TBS energy bill. Given the ease of switching once the offer had been received, the relative sophistication of TBS survey participants15 and their prior action to investigate savings, this would seem to be a low take-up if energy savings are the main driver of switching. f The survey respondents (in the third and fourth columns of Table 1) were different from the larger sample in certain respects that are consistent with a higher likelihood of switching. The time and effort which they gave to respond to the survey might suggest that the relatively small amount of time and effort required to switch after receiving TBS offer was a less significant cost for them, despite the difference in the two activities. In addition, they were more likely to have referred to their actual bills (arguably a sign of greater financial awareness) and, once the filters were applied, specifically mentioned money-saving as part of their motivation for participating in TBS. Even so, almost 6 in 10 survey respondents did not switch. Table 2 provides a selection of summary statistics drawn from the survey responses which allow comparisons between respondents who did not switch and those who did.i The upper five rows of Table 2 show that in terms of household characteristics, switchers and non-switchers are reasonably similar, although switchers are more likely to be graduates and homeowners. The lower rows indicate that although switchers have a somewhat higher median in 14. Direct Debit is the predominant payment method in Great Britain and involves monthly deductions from a bank account to spread the estimated cost of the energy evenly over the year, with an annual reconciliation from metered consump tion. Note that members of this group were not necessarily paying by Direct Debit before they entered the auction. 15. 14. Direct Debit is the predominant payment method in Great Britain and involves monthly deductions from a bank account to spread the estimated cost of the energy evenly over the year, with an annual reconciliation from metered consump tion. Note that members of this group were not necessarily paying by Direct Debit before they entered the auction. 15. Table A3.1 in the Appendix shows that those who participated in the survey were older, more highly educated and more likely to own their own home (or have a mortgage) than the typical British household. 3. DATA As is to be expected if there is at least some sensitivity to price, the savings offered were higher both absolutely and relative to their bills for those who switched than for those who did not; and, unsurprisingly, exit fees were more prevalent among non-switchers. come, median bill sizes are much the same. As is to be expected if there is at least some sensitivity to price, the savings offered were higher both absolutely and relative to their bills for those who switched than for those who did not; and, unsurprisingly, exit fees were more prevalent among non-switchers. On the non-financial front, non-switchers were more likely to report other claims on their time during TBS period. In terms of the qualities of suppliers, those who switched were more likely to have a preference for the Co-op’s perceived ethical/environmental/tariff type profile; fewer switchers were happy with their pre-TBS supplier’s customer service. While this exploratory anal ysis identifies some of the potential drivers of the switching decision, we rely on a (reduced form) econometric analysis to identify more robustly the monetary and non-monetary factors associated with switching. 3. DATA fif * Indicates the statistic for analysed participants is significantly different at the 5% level from the statistic for all partici pants with complete data and offered a positive saving. fif p pf p g $ Indicates the statistic for survey respondents who were offered a positive saving is significantly different at the 5% level from the statistics for all participants with complete data and who were offered a positive saving.if ^ Indicates the statistic for the analysed survey respondents is significantly different at the 5% level from the statistic for analysed TBS participants.if ! Indicates the statistic for analysed survey respondents is significantly different at the 5% level from the statistic for all participants with complete data and who were offered a positive saving. 1 These are households who were paying by Direct Debit before TBS. 2 This percentage combines participants who entered a “Round Amount” for their bill, suggesting they may have estimated their bill, and participants who had their bill estimated by Which? on the basis of their dwelling’s characteristics. Other respondents are assumed to have used their actual bills. 2 This percentage combines participants who entered a “Round Amount” for their bill, suggesting they may have estimated their bill, and participants who had their bill estimated by Which? on the basis of their dwelling’s characteristics. Other respondents are assumed to have used their actual bills. p 3 Single supplier households either only had an electricity connection (around 12% of each group) or received both their electricity and gas from a single supplier before TBS. A “Dual Fuel” tariff refers to tariffs where consumers buy both their electricity and gas from a single supplier as part of a combined deal. 4 Tested using Mood’s median test 3 Single supplier households either only had an electricity connection (around 12% of each group) or received both their electricity and gas from a single supplier before TBS. A “Dual Fuel” tariff refers to tariffs where consumers buy both their electricity and gas from a single supplier as part of a combined deal. 4 Tested using Mood’s median test come, median bill sizes are much the same. 3. DATA Table A3.1 in the Appendix shows that those who participated in the survey were older, more highly educated and more likely to own their own home (or have a mortgage) than the typical British household. Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 101 Table 1: Summary statistics on energy bills and TBS savings Variable Participants Offered Saving at TBS with Complete Data from 2012 Participants with Complete Data from 2012—Filters Applied 2013 Survey Respondents Offered Saving at TBS 2013 Survey Respondents— Filters Applied % Switching supplier at TBS 28.1 27.1* 39.5$ 41.9^, ! Median bill size (actual and estimated) (£)5 1176 1168* 1161$ 1162! % Using estimated bill2 27.9 28.8* 21.2$ 20.2^, ! % Facing an exit fee 10.4 12.8* 13.6$ 16.5^, ! Median saving offered by best supplier (£)4 117.48 112.5* 111.48$ 106.66^, ! Median saving as % of existing bill4 10.6 10.3* 10.3$ 9.9^, ! % Shown two offers 46.1 50.7* 45.5 49.0^, ! % Paying for their energy by Direct Debit1 89.8 96.4* 90.9$ 97.3^, ! % of single supplier households on a Dual Fuel tariff3 87.1 87.9* 87.0 87.8 Total Number of Observations 119,126 86,904 12,750 7,367if Table 1: Summary statistics on energy bills and TBS savings Notes: The first column covers all TBS participants who provided complete data and were offered a positive saving at TBS, while the third column is the subset of this group who responded to the survey (sent to half of all participants). The second column is a subset of the first formed from TBS participants who were supplied by a single supplier before TBS and entered the direct debit auction. The fourth column is a subset of the second column where additionally participants responded to the survey and stated saving money as one of the top three reasons for participating in TBS. * Indicates the statistic for analysed participants is significantly different at the 5% level from the statistic for all partici responded to the survey and stated saving money as one of the top three reasons for participating in TBS. * Indicates the statistic for analysed participants is significantly different at the 5% level from the statistic for all partici pants with complete data and offered a positive saving. Open Access Article 4. ECONOMETRIC METHOD To analyse the switching decision, we used a Probit model of the likelihood to accept the offer received in TBS on the basis of both monetary considerations and non-price preferences. The Open Access Article Open Access Article 102 / The Energy Journal Table 2: Comparisons between non-switchers and switchers from 2013 survey Variable Non-switchers Switchers Household Characteristics Age group containing median age1 55–64 55–64 % Male 72.1 72.3 % Respondents with first degree or higher 59.8 64.0* % Respondents who fully or partly own home 93.2 94.4* % Households with at least one person working full/part-time 53.4 55.5 Financial and Non-financial Factors Category containing median income2 £30,000–34,999 £35,000–39,999 Median bill size (£) 1162 1161 Median saving (£)3 90.55 124.55* Median saving (% of existing bill)3 8.7 11.3* % With current exit fee 24.0 6.0* % Strongly Agree/Agree that “Timing of TBS was an especially busy period” 23.0 8.4* % Happy with pre-TBS supplier customer service 82.1 73.6* % Prefer offered supplier over existing supplier re: ethics/environment 23.7 55.9* % Prefer offered supplier over existing supplier re: tariff type 8.0 41.4* Total Number of Observations 4,279 3,088if * Indicates a significant difference at the 5% level between the mean statistic for Switchers and Non-Switchers. Medians have been tested using Mood’s median test. We were unable to test for a significant difference in income levels. 1 Based on the 4 666 observations for which age information was available * Indicates a significant difference at the 5% level between the mean statistic for Switchers and Non-Switchers. Medians have been tested using Mood’s median test. We were unable to test for a significant difference in income levels. * Indicates a significant difference at the 5% level between the mean statistic for Switchers and Non-Switchers have been tested using Mood’s median test. We were unable to test for a significant difference in income levels 1 Based on the 4,666 observations for which age information was available. 2 Based on the 7,064 observations for which income information was available. if 3 These figures relate solely to people offered a positive saving as part of TBS. dependent variable, iy , takes a value of 1 when an individual accepted the TBS offer and a value of 0 when an individual did not accept it. 16. No variables were excluded on grounds of multicollinearity, following a Variance Inflation Factor test which revealed no multicollinearity between them. Based on the 4,666 observations for which age information was available. g Based on the 7,064 observations for which income information was available. if These figures relate solely to people offered a positive saving as part of TBS. 17. Technical details of the econometric methodology discussed in this section are provided in Appendix 4, while details of the regression resulting in the IMR are given in table A3.2. 18. The respondents’ current energy bill and the alternative energy cost offered by the new TBS offer were initially in cluded separately in the regression, but a test on the restriction of equal coefficients for these two variables revealed it was possible to use their difference (which we label saving amount) directly in our Probit model. 4. ECONOMETRIC METHOD For each individual the probability, pi, of acceptance was modelled as: dependent variable, iy , takes a value of 1 when an individual accepted the TBS offer and a value of 0 when an individual did not accept it. For each individual the probability, pi, of acceptance was modelled as: ( ) ( ) 1\| i i p Prob y F = = = i i x x'β (1) ( ) ( ) 1\| i i p Prob y F = = = i i x x'β (1) Here pi is the probability that acceptance was observed conditional on the vector of explan atory values for individual i, ix . These include financial characteristics of the current and proposed supply contract, individual preferences for characteristics of the suppliers and features of their of fers, and individual characteristics of the survey respondents. For the Probit model, ( ) . F is the Normal cumulative distribution function. We assume that when deciding whether to accept the offer (for which we use the shorthand “switch’), individual i compared the utility of switching (UiS) to the utility of not switching (UiNS); the probability of observing a switch by individual i equalled the probability that, for individual i, the utility from switching exceeded the utility from not switching: ( ) ( ) 1 i iS iNS Prob Y Prob U U = = > ( ) ( ) 1 i iS iNS Prob Y Prob U U = = > We assume that the unobservable utility associated with the two options could be captured by the observable variables included in the vector of explanatory variables ix . We therefore mod elled the difference in utility derived from switching and not switching by a set of individual-specific characteristics (e.g. gender and education), choice-specific characteristics (e.g. the respondent’s view of the new supplier’s environmental and ethical credentials) and characteristics which vary across both individuals and choices. We estimated the likelihood of switching using a reduced form equation of the decision to switch. Our analysis was based on the extensive set of variables included in the 2013 survey.16 However, it is clear that those who responded to the survey were a self-selecting subsample of 16. No variables were excluded on grounds of multicollinearity, following a Variance Inflation Factor test which revealed no multicollinearity between them. 4. ECONOMETRIC METHOD Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 103 those who took part in TBS, leading to important potential differences between the main and survey samples. To correct for any self-selection bias associated with the decision to take part in our survey we adopted the Heckman 2-stage model where, in addition to estimating an equation for the prob ability of switching, we also estimated an equation for the probability of taking part in the survey. The exclusion restriction imposed on the “survey participation” equation implies that the decision to participate in the survey is significantly influenced by the method through which the respondents were recruited for TBS (e.g. advertising campaign) and the numbers of reminders they were sent during the TBS campaign (as a measure of their interest to engage in the energy market), but these factors do not affect the decision to switch supplier after receiving the TBS offer. Based on the latter equation we calculated the Inverse Mills Ratio (IMR) for the probability of taking part in our survey and included it in our main regression17. We found no evidence of a statistically significant distortion arising from self-selection; however, we recognise that any conclusions and policy recommenda tions driven by the results obtained from the responses of these potentially more engaged consumers might overestimate the likelihood of switching within the general population of consumers, both of TBS participants and more widely. A preliminary depiction of the relationship between the frequency of switching and the amount of savings offered is shown in Figure 1. Figure 1: Switching rates by size of annual saving (£) For both the main sample and for the survey sample, there is a clear correlation between the size of saving offered by TBS and the likelihood of switching. However, the rate of increase in the percentage of respondents switching slows at savings amounts higher than £100. For the main sample, moving from the category of £0–20 in savings to that of £100–120 increases the probability of switching by 24 percentage points, while moving from the category of £100–120 to that of £300– 320 increases the probability by only 12 percentage points. The corresponding figures for the survey sample are 36 and 10 percentage points respectively. Open Access Article 4. ECONOMETRIC METHOD This led us to adopt a quadratic specification for the (continuous) saving variable in our Probit model.18 The average marginal effects presented in Table 3 have been calculated taking into account the quadratic treatment of this variable. Figure 1: Switching rates by size of annual saving (£) Figure 1: Switching rates by size of annual saving (£) Figure 1: Switching rates by size of annual saving (£) For both the main sample and for the survey sample, there is a clear correlation between the size of saving offered by TBS and the likelihood of switching. However, the rate of increase in the percentage of respondents switching slows at savings amounts higher than £100. For the main sample, moving from the category of £0–20 in savings to that of £100–120 increases the probability of switching by 24 percentage points, while moving from the category of £100–120 to that of £300– 320 increases the probability by only 12 percentage points. The corresponding figures for the survey sample are 36 and 10 percentage points respectively. This led us to adopt a quadratic specification for the (continuous) saving variable in our Probit model.18 The average marginal effects presented in Table 3 have been calculated taking into account the quadratic treatment of this variable. Open Access Article 104 / The Energy Journal In addition to the savings offered as part of TBS process, we also included the minimum amount of savings which our respondents said they had required in order to switch. While including such information helped us to understand the cost-benefit evaluation undertaken by our respon dents, the inclusion of the minimum required saving variable created an endogeneity problem, as it is conceivable that unobserved factors which affected their decision to switch in 2012 might also affect the required savings to switch stated in 2013. These factors could, for instance, include their attitude towards the uncertainty associated with moving to a different supplier. Indeed, one might expect individuals who are more “cautious” to be both less likely to switch and require more money to be persuaded to switch. Due to the potential endogeneity of the minimum required saving variable, our Probit model was estimated using conditional maximum likelihood estimation,19 an instrumental variable method. This method involves the joint estimation of two equations, the first of which has the potentially endogenous variable as the dependent variable. 4. ECONOMETRIC METHOD The predicted values of the endogenous variable were included as regressors in the main Probit model (i.e. as part of ix in equation (1)). Following this procedure to correct for potential endogeneity, the magnitude and sign of the main estimated effects were not substantially affected, compared to the simple Probit model with no correction for endogeneity; nevertheless, we have included the correction in the regression reported in Table 3. f Lastly, it seemed possible that unobserved factors might affect both the probability of being presented with two (rather than just one) offers and the decision to switch. In order to account for this possibility, we used a recursive Probit model where in a first stage we modelled the probability of being presented with two offers and then we modelled the probability of switching conditional on the number of offers made to the participant, in addition to all the other factors which could have affected the switching decision.20f When considering the different explanatory factors included in our reduced form analysis we would expect that positive monetary incentives, such as higher savings, or a motivation to save money as part of the TBS process, would increase the probability to switch, while we would expect the opposite effect from disincentives such as having to pay an exit fee or losing other benefits as a result of switching. We would also expect that the likelihood of switching would be reduced by uncertainty about the actual size of the savings or about the switching process. A negative effect would also be expected as result of constraints on the respondents’ time. Another key factor in the decision to switch has been identified in the literature as the existence of heterogeneous preferences for different suppliers and their brands (see e.g. Dubé et al., 2010). We account for these effects by including in our analysis the respondents’ preferences for characteristics and offers of their current supplier and for those of the new “proposed” supplier. We would expect that a preference for the characteristics of the current supplier would reduce the probability of switching, while the opposite would hold if the characteristics of the new supplier were preferred, or if the respondents were dis satisfied with the service provided by their current supplier. 20. In Appendix 2, we report the results of separate regressions for those shown one or two offers at TBS; although a likelihood ratio test indicated that separate regressions should be run for those shown one offer and those shown two offers, the main results are not qualitatively different. Open Access Article 19. Technical details about this estimation approach are provided in Appendix 4. 19. Technical details about this estimation approach are provided in Appendix 4. 20. In Appendix 2, we report the results of separate regressions for those shown one or two offers at TBS; although a likelihood ratio test indicated that separate regressions should be run for those shown one offer and those shown two offers, the main results are not qualitatively different. 5. RESULTS The econometric approach described in the previous section was used to explore the role of a wide range of financial and non-financial variables in individuals’ switching decisions. Table 3 Switching Energy Suppliers: It’s Not All About the Money / 105 Table 3: Selected average marginal effects on the probability of switching energy supplier at TBS Switching Factor Variable Average Marginal Effects Filtered Survey Respondents Survey respondents with positive savings Excluded survey respondents with positive savings Monetary Savings 1. Saving amount of the best offer (£10 units) 0.016* 0.014* 0.013* 2. Has an Exit Fee –0.173* –0.158* –0.125* 3. No other penalty/loss of benefits if switch supplier 0.054* 0.063* 0.086* 4. Top 3 factor persuading to switch: Large enough saving –0.040* –0.020 –0.003 5. Stated minimum required saving to switch (Spring 2013, £1 units) –0.001* –0.001* –0.001* Non-Price Preferences 6. Electricity/energy supplier before TBS: Co-Operative Energy 0.098 0.154* 0.246* 7. Prefers existing supplier re: tariff type –0.149* –0.141* –0.131* 8. Prefers offered supplier on ethical/environmental grounds 0.115* 0.111* 0.104* 9. Prefers offered supplier re: tariff type 0.188* 0.178* 0.162* 10. Prefers offered supplier for “Other” reason 0.118* 0.101* 0.083*** 11. Top 3 factor to switch: ethical/environmental reasons 0.024* 0.005 –0.011 Uncertainty/ Preparedness 12. Confidence in accuracy of TBS saving (continuous scale 0 to 1) 0.031 0.040*** 0.040* 13. Energy bill estimated by Which –0.070* –0.070* –0.084* 14. Respondent states bill as ‘Round’ amount –0.048* –0.046* –0.048 15. Unsure if other penalty/loss of benefits if switch supplier –0.052* –0.052* –0.029 16. Top 3 factor to switch: confidence in getting best deal 0.023 0.031* 0.032* 17. Shown two offers –0.060* –0.051* –0.049* Concerns with Switching Process 18. Worried re: “Other” issues –0.104* –0.101* –0.090* 19. Additional help wanted: phone support –0.057* –0.030 0.000 20. Additional help wanted: simpler switching process –0.112* –0.109* –0.107* 21. Additional help wanted: something else –0.069* –0.068* –0.065*** 22. Top 3 factor to switch: Quick & easy switching process 0.025* 0.018* 0.009 Time Pressures 23. Worried switching would be time consuming 0.050* 0.055* 0.062* 24. TBS was a very busy period: Strongly Agree –0.228* –0.204* –0.173* 25. TBS was a very busy period: Agree –0.106* –0.104* –0.094* 26. TBS was a very busy period: Disagree 0.036* 0.034* 0.027 27. TBS was a very busy period: Strongly Disagree –0.014 0.001 0.024 Respondent Characteristics 28. Number of people in household: One 0.037* 0.040* 0.042* 29. 5. RESULTS Open Access Article 106 / The Energy Journal reports the average marginal effects of key explanatory variables on the probability of switching energy supplier, using information from the original TBS data and from our survey.21 The role of savings in incentivising switching is placed in the context of the perceived uncertainties and other non-financial considerations involved in the switching decision.f We present the variables in broad categories which might affect the likelihood of switching, but which sit largely within a rational choice framework: savings offered, non-price preferences, uncertainty on the part of the participant, concerns with the switching process, time pressures and characteristics of the participant themselves. While such subdivisions are inevitably somewhat ar bitrary, they enable a more manageable discussion of the results, whose nature and size are not affected by the categorisation. Most of the variables are relevant to the development of general policies towards consumer switching in energy, but the interpretation of some is specific to the TBS experience, in particular: variable 6, being a Co-op customer before TBS; variables 24 to 27 which refer specifically to the TBS period; and variable 17, being shown two offers. We take the specifics of the exercise into account in our discussion of each of these categories below. ii Table 3 summarises the key results for the respondents identified by the filters discussed in section 3 (filtered survey respondents in column 1), alongside the results for the whole group of survey respondents who were presented with the offer of positive savings (column 2), and for the respondents who were excluded from our analysis (column 3). For the majority of the variables considered in our analysis we notice that the estimated average marginal effects are significant (to a similar extent) across the 3 groups of survey respondents, but the magnitude of the average marginal effects is higher for the filtered participants, except for the impact of consumers who were already supplied by Coop energy. Also, the average marginal effect associated with the desire for confidence in getting the best deal is higher for the excluded respondents (and for the survey respondents over all), possibly reflecting the complexity of working out the “best” deal for consumers who are not driven primarily by financial considerations. 5. RESULTS Highest Educational Qualification: Masters/PhD 0.026 0.020** 0.007 Other 30. IMR for survey response 0.137 0.152 0.087 N 7,367 12,750 5,383 Notes: * indicates significance at the 10% level indicates significance at the 5% level and * indicates significance at the 1% level The Table 3: Selected average marginal effects on the probability of switching energy supplier at TBS A M i l Eff t : Selected average marginal effects on the probability of switching energy supplier at TBS Average Marginal Effects Average Marginal Effects Notes: * indicates significance at the 10% level, indicates significance at the 5% level and * indicates significance at the 1% level. The current table focuses on variables that were statistically significant. A wide range of additional variables were included in the regression, including: payment method before TBS, respondent gender, household tenure, employment status of household members, whether in receipt of a fuel related benefit and communication with existing supplier triggered by TBS. In particular, dummy variables for electricity supply regions and median household income in a respondent’s postcode area were included and generally found to be statistically insignificant. Additional dummies for extra options beyond those listed in the current table were included for: factors that would persuade a respondent to switch, preferences between previous and offered suppliers, worries about the switching process, additional help wanted, number of house hold members and highest educational qualification obtained. Details of the complete regression results are available on request. Default categories for reported dummy variables. 2: No Exit Fee; 3 and 14: Has a penalty/loss of benefits if switch; 4, 11, 16 and 22: List ed factor not in the top 3 factors that would persuade the respondent to switch in the future; 6: Electricity/energy supplier before TBS—All energy suppliers other than Co-Operative Energy and EBICo; 7 to 10: Indifferent between existing and offered supplier on stated dimension; 13 and 14: Respondent used actual bill and stated ‘Non-Round’ amount; 17: Shown one offer; 18 and 23: Not worried about stated issue; 19 to 21: The form of additional help stated was not required; 24 to 27: Neither agree nor disagree with the statement “TBS was a very busy period”; 28: Two people in household; 29: Highest educational qualification - first degree or equivalent Observations dropped by regressions: No observations were recorded for the postcode area income category £75,000–80,000. Open Access Article 21. The marginal effects of only those variables which are available for these respondents from the TBS exercise itself are reported in Table A1.1. 22. The reported marginal effect of the saving amount reflects the quadratic treatment of this term in the Probit model. 23. This is calculated as the ratio of the estimated coefficient (N.B. not the marginal effect reported in Table 3) associated with the dummy variable for the exit fee and the estimated coefficients associated with the level of savings (taking into ac count the quadratic treatment of this variable). 24. While some companies were charging up to £100 exit fees, £25-£30 was more typical, see: http://www.thisismoney. co.uk/money/bills/article-2934318/Don-t-let-exit-fee-switching-energy-deals-50-levy-potential-savings-eye-popping.html . 21. The marginal effects of only those variables which are available for these respondents from the TBS exercise itself are reported in Table A1.1. Open Access Article 5. RESULTS Having considered in general terms the results for all survey respondents and for those who did not meet our criteria for a high propensity to switch, we now discuss in more detail the results for the group of respondents that we consider as most likely to switch given their interest in financial gains and the relative simplicity of their switching process.l Dealing with each category in turn, monetary savings continue to exert an important influ ence on the probability of switching: an increase of £10 in the saving offered at TBS is associated with a 1.6 percentage point increase in the probability of switching.22 However the results illustrate why an offer of monetary savings alone is insufficient to guarantee switching. The presence of an exit fee from a consumer’s existing energy supplier is associated with an average reduction in the probability of switching of 17.3 percentage points. In monetary terms this corresponds to a required increase in savings of about £12023 in order to renege on the existing contract and having to pay an exit fee, which was on average about £5024 at the time of TBS. From our results it therefore appears Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 107 that the existence of an exit fee has a disproportionate deterrent effect on switching, perhaps reflect ing “loss aversion”.25 that the existence of an exit fee has a disproportionate deterrent effect on switching, perhaps reflect ing “loss aversion”.25 Variable 4 relates to a question asking about factors which would induce switching in the future. A respondent who reported that one of these factors was a “large enough saving” may have been indicating that they required a particularly large saving to switch supplier, and/or that the saving presented at TBS was insufficient. So we see that headline monetary savings themselves may have been moderated by other considerations, including exit fees and other penalties and the respondent’s own evaluation of financial rewards. i A variety of non-financial preferences and characteristics are captured by variables 6 to 11. Although in comparison with other consumer goods energy may appear to be homogenous, it is clear that respondents had preferences over suppliers beyond the price charged. 25. Loss aversion is the idea that losses weigh more heavily in consumer decisions than corresponding gains, and numer ous studies have suggested a ratio of between 1.5 and 2.5 to 1, similar to our results (see Kahneman, 2011, p.284) 26. Of the 22 filtered survey respondents who were already with Co-Op before TBS and switched through TBS, 4 changed to other suppliers (equivalent to “external switching” as used by the Council of European Energy Regulators, 2018) and 18 remained with Co-Op after TBS, equivalent to “internal switching”. At least 3 of these internal switchers chose Co-Op even though a larger saving was available from an alternative supplier. An existing Co-Op customer might have chosen to make such an “internal switch” through TBS because they were offered a special deal in the auction. 27. We thank an anonymous referee for this suggestion. 25. Loss aversion is the idea that losses weigh more heavily in consumer decisions than corresponding gains, and numer ous studies have suggested a ratio of between 1.5 and 2.5 to 1, similar to our results (see Kahneman, 2011, p.284)i 5. RESULTS For example, preferring the ethical/environmental stance of an offered supplier to that of the respondent’s existing supplier is associated with an increase in switching probability of 11.5 percentage points (compared to the base case of indifference between suppliers regarding this factor). Since the Co-op had a positive ethical and environmental reputation at the time of TBS, it is not surprising that those who were influenced by ethical and environmental reasons (variables 8 and 11) were more likely to ac cept the offer (although it may be hard to separate this entirely from the fact that consumers already with the Co- op were effectively only switching to a different tariff and faced reduced uncertainty about the service they would receive).26 Consumers who were uncertain about the size of saving were less likely to switch for a given “expected” gain, and this is reflected in variables 12 through 17. Greater confidence in the accuracy of the offered savings increases the chances of switching, while not knowing the exact amount of the bill or whether exit penalties exist may be interpreted as lower confidence in the accuracy of any saving offered and may thereby have negative effect on the probability of switching. ff We have also included under uncertainty the negative effect of being shown two offers in TBS rather than one (variable 17). One possible interpretation is that being shown two offers may have prompted some participants to wonder whether there might be other (possibly better) deals in the wider market, either now or in the near future, encouraging postponement of a decision. Another is that being presented with two offers may indicate that there was another pre-existing cheap offer on the market, which the participant had already “ignored’, suggesting greater inertia.27 However, the unique circumstances of TBS, where participants expected only one offer and received two, may mean that any interpretations do not necessarily generalise to other switching situations. There may have been uncertainties not just about the gains to be realised, but also about the switching process itself, and these are captured by variables 18 to 23. This group of variables suggest that higher “anxiety” and/or effort costs are associated with a lower probability of switching at TBS, while the desire for a quick and easy switching process (possibly facilitated by the support and auctioning system put in place by Which?) increases the probability of switching. 27. We thank an anonymous referee for this suggestion. 5. RESULTS The size Open Access Article 108 / The Energy Journal of these positive effects is generally smaller than the negative impact of other concerns about the switching process. f Another possible reason for not switching, even when offered substantial savings and sent several reminders, is pressure on consumers’ time, such that even the small amount of time and attention needed to accept the offer felt excessive at that moment. The effects of variables 24 to 27, taken together, are as might be expected in a rational choice framework: individuals who reported greater time pressure around TBS were less likely to switch. For example, strongly agreeing that TBS came during a busy period is associated with a 23-percentage point reduction in switching probability. The sign of the average marginal effect for variable 27 is unexpected, although not statistically significant. We therefore conclude that time pressure contributes to understanding unre sponsiveness to money saving offers. i Once the specific characteristics and contexts of a respondent’s choice decision are con trolled for, few socio-economic characteristics are associated with the probability of switching.28 Gender, housing tenure, receipt of fuel related benefits, regional location and median income in the respondents” postcode area all have statistically insignificant relationships with the probability of switching. Two exceptions were education and household size. Those with postgraduate quali fications were more likely to switch, as were single person households. The 3.7 percentage point increase in the probability of switching associated with being a single person household might re flect the greater simplicity of decision making when there is no need to reach agreement with other household members. Moreover, a single individual can more easily identify the benefits that would accrue to them personally from switching and balance these against the (individually incurred) efforts involved. We summarise the story told by these results as follows. When all we have are data about monetary savings and penalties, even when supplemented by a limited set of variables that proxy uncertainty or lack of confidence, households appear to leave a considerable amount of money on the table in the British retail energy market. However, a number of other factors, such as non-price preferences, time pressures and concerns about the switching process itself, appear to be affecting consumers’ behavior. 5. RESULTS The “enhanced” respondent model is not perfect and influences from some variables are possibly still not identified—including, perhaps, some heuristics or biases that might be conventionally regarded as “irrational”. Nevertheless, the model derived from our survey con tributes to explaining why financial rewards alone may fail to induce switching, even among people who are well-educated and more engaged than most within the retail energy market. 28. However recall that our sample is far from representative of the population as a whole where variations in socio-eco nomic characteristics are likely to be larger and hence may have effects on the probability of switching which we do not see in our self-selected sample. Open Access Article 6. CONCLUSION TBS provided a unique opportunity to observe the detailed decisions and circumstances of a large group of energy consumers faced with a real choice of providers in the residential energy market. These consumers were generally more pro-active in this market than the average house holder, as demonstrated by their participation in TBS itself, and within this group we have focused on those who joined the exercise to save money and responded to a follow-up survey. Consequently, our findings could be viewed as an upper bound on engagement in the UK energy market. The sam ple is self-selected, as individuals took several active steps to participate in the auction and respond 28. However recall that our sample is far from representative of the population as a whole where variations in socio-eco nomic characteristics are likely to be larger and hence may have effects on the probability of switching which we do not see in our self-selected sample. Switching Energy Suppliers: It’s Not All About the Money / 109 109 to the survey, and they possess underlying traits associated with more activity/engagement than the general population. if While we find that switching is positively correlated with the savings offered to partici pants, the raw data clearly demonstrate that the prospect of substantial savings is not by itself suffi cient to induce a majority of participants to switch, despite the small additional effort required and several reminders from Which?. A range of non-price factors—various sources of uncertainty, the non-monetary characteristics of different offers, concerns about the switching process and time pres sures when TBS occurred—are all associated with the switching decision. Some other features, such as the seemingly disproportionate weight attached to exit fees and the negative impact of seeing two offers rather than one, may have elements of behavioral bias; but most of the factors we identify are consistent with consumers making a largely “rational” decision when declining to switch, even if this results in substantial monetary savings apparently being left on the table. Moreover the relation ship between switching and savings levels off, so that increased savings have little additional effect on the probability of switching beyond one hundred pounds per year.i Our findings have some important implications for policy makers and managers. The sig nificant role of non-financial factors means that switching cannot be relied on to put all consumers on the cheapest deal for them. 6. CONCLUSION Indeed, our results suggest that some consumers consciously choose to remain with more expensive suppliers due to non-price preferences. These non-price preferences confirm that consumers do not regard energy as a homogeneous product, despite the view of many analysts. So our second policy conclusion is that actions which automatically move consumers to a cheaper supplier may reduce utility for at least some consumers, since they do not regard suppliers as completely interchangeable. Management which can differentiate its supply/service offering may be able to create considerable value—at least for some consumers—even though the gas or electric ity itself is homogeneous. In terms of process, the restrictions on switching fees introduced by European Commis sion’s Directive on Clean Energy (2019), Article 12, are supported by the disproportionate effect of exit fees, our third conclusion. Our fourth conclusion is that opt-in collective switching processes, such as TBS, do not constitute a panacea in attracting a wide variety of consumers to switch to cheap energy deals. These collective switches rely on a different kind of consumer engagement, both to “opt in” to the process and to accept the auction offer. Those who opted into this auction displayed characteristics which are typically associated with higher engagement (see e.g. Competition and Markets Authority, 2016), but they still “left money on the table” by not switching after the offer had been made.ii However, the results confirm that financial gains are positively associated with switching, so policies which restrict potential savings, including price caps, are likely to reduce switching rates. Indeed, there is a danger that such policies may foster disengagement if consumers suppose that the regulator is looking after their interests so that they need not concern themselves. On the other hand, the proportion of TBS participants still not switching suggests that relying substantially on consum ers to drive margins down to competitive levels is likely to prove disappointing to policymakers. Open Access Article ACKNOWLEDGMENTS We are very grateful to Which? for making available the data for this study and working with us on the analysis, and to the Economic and Social Research Council for funding the initial stages of this study through its support for the Centre for Competition Policy (CCP) at the Univer sity of East Anglia (grant RES-578-28-0002); David Deller and Catherine Waddams also received Open Access Article 110 / The Energy Journal support from the UK Energy Research Centre under Phase 3 (grant EP/L024756/1); Monica Gi ulietti received financial support from the EPSRC (grants EP/N001745/1, EPR062258/1 and EP/ K002228) and from UKERC (grant FF3/3); and Graham Loomes received support from the Eco nomic and Social Research Council’s funding of the Network for Integrated Behavioural Science (grants ES/K002201/1 and ES/P008976/1). support from the UK Energy Research Centre under Phase 3 (grant EP/L024756/1); Monica Gi ulietti received financial support from the EPSRC (grants EP/N001745/1, EPR062258/1 and EP/ K002228) and from UKERC (grant FF3/3); and Graham Loomes received support from the Eco nomic and Social Research Council’s funding of the Network for Integrated Behavioural Science (grants ES/K002201/1 and ES/P008976/1). 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APPENDICES APPENDIX 1: SWITCHING PROBABILITIES Table A1.1: Selected average marginal effects on the probability of switching energy supplier at TBS using TBS data only Average Marginal Effects Switching Factor Variable (1) All Participants with Positive Savings (2) Filtered Participants (3) All Survey Respondents with Positive Savings (4) Filtered Survey Respondents Monetary Savings 1. Saving amount of the best offer (£10 units) 0.012* 0.012* 0.013* 0.015* 2. Has an Exit Fee –0.205* –0.206* –0.320* –0.317* Uncertainty or Preparedness 3. Energy bill estimated by Which –0.179* –0.172* –0.125* –0.124* 4. Respondent states bill as ‘Round’ amount –0.082* –0.085* –0.051* –0.076* Other 5. Shown two offers –0.057* –0.074* –0.104* –0.141* N 119,125 86,888 12,748 7,363 Notes: * indicates significance at the 10% level, indicates significance at the 5% level and * indicates significance at the 1% level. Dummy variables for electricity supply regions, electricity/energy supplier before TBS, other payment types and purchasing from the incumbent supplier were also included as controls in the regressions but are not reported for brevity. By column, the number of the electricity supply region dummies (null region: London) significant at the 5% level is: column 1–12, column 2–11, column 3–3, and column 4–2. By column, the number of the dummy variables for electricity/energy suppliers (null supplier: British Gas) significant at the 5% level is: column 1–11, column 2–9, column 3–7, and column 4–3. Details of the complete results are available on request. Sample Selection: The regressions in columns 2-4 were all found to be subject to a significant sample selection effect compared to the ‘All participants’ sample. This sample selection effect was indicated by, and controlled for, including the relevant Inverse Mills Ratio (IMR) in each regression. In column 2 the IMR was negative and significant at the 1% level, while in columns 3 and 4 the IMR was positive and significant at the 1% level. Null categories for reported dummy variables: 2. No Exit Fee; 3. Energy bill not estimated by Which; 4. “Non-round” energy bill figure entered by participants; and 5. Shown one offer Observations dropped by regressions: Column 1: 1 observation dropped for Utilita perfectly predicting non-switching. Column 2: No observations were recorded for Utilita; 16 observations were dropped for Green Energy UK and Spark Energy perfectly predicting non-switching. Column 3: No observations were recorded for Utilita or Spark Energy; 2 observations were dropped for Sainsbury’s Energy perfectly predicting non-switching. REFERENCES and M. 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Open Access Article 112 / The Energy Journal APPENDICES Column 4: No observations were recorded for Utilita, Spark Energy or Sainsbury’s Energy; 3 observations were dropped for Green Energy UK and Good Energy perfectly predicting non-switching; 1 observation was dropped for National Trust perfectly predicting switching. Table A1.1: Selected average marginal effects on the probability of switching energy supplier at TBS using TBS data only Average Marginal Effects Switching Factor Variable (1) All Participants with Positive Savings (2) Filtered Participants (3) All Survey Respondents with Positive Savings (4) Filtered Survey Respondents Monetary Savings 1. Saving amount of the best offer (£10 units) 0.012* 0.012* 0.013* 0.015* 2. Has an Exit Fee –0.205* –0.206* –0.320* –0.317* Uncertainty or Preparedness 3. Energy bill estimated by Which –0.179* –0.172* –0.125* –0.124* 4. Respondent states bill as ‘Round’ amount –0.082* –0.085* –0.051* –0.076* Other 5. Shown two offers –0.057* –0.074* –0.104* –0.141* N 119,125 86,888 12,748 7,363iii 1: Selected average marginal effects on the probability of switching energy supplier at TBS using TBS data only Notes: * indicates significance at the 10% level, indicates significance at the 5% level and * indicates significance at the 1% level. Dummy variables for electricity supply regions, electricity/energy supplier before TBS, other payment types and purchasing from the incumbent supplier were also included as controls in the regressions but are not reported for brevity. By column, the number of the electricity supply region dummies (null region: London) significant at the 5% level is: column 1–12, column 2–11, column 3–3, and column 4–2. By column, the number of the dummy variables for electricity/energy suppliers (null supplier: British Gas) significant at the 5% level is: column 1–11, column 2–9, column 3–7, and column 4–3. Details of the complete results are available on request.if Sample Selection: The regressions in columns 2-4 were all found to be subject to a significant sample selection effect compared to the ‘All participants’ sample. This sample selection effect was indicated by, and controlled for, including the relevant Inverse Mills Ratio (IMR) in each regression. In column 2 the IMR was negative and significant at the 1% level, while in columns 3 and 4 the IMR was positive and significant at the 1% level. Sample Selection: The regressions in columns 2-4 were all found to be subject to a significant sample selection effect compared to the ‘All participants’ sample. APPENDIX 2: ONE VS TWO OFFERS In this appendix we present some additional analysis used to explore the finding that being shown two offers is associated with a lower probability of switching. In table A2.1 the main demographic and socio-economic descriptive statistics are split by those who received one offer and those who received two offers. Table A2.1 reveals that the differences in respondent characteristics between the one and two offer groups are generally small in magnitude. Table A2.1: Demographic and socio-economic characteristics of those receiving one and two offers Statistic One Offer Two Offers Age group containing median age1 55–64 55–64 % Male 72.4 71.9 % Respondents with first degree or higher 61.5 61.7 % Respondents who fully or partly own their home 93.8 93.6 % Households containing at least one person who is employed (part-time or full-time) 54.2 54.4 % Respondents receiving a disability benefit 7.9 7.6 % Respondents receiving an energy related benefit (excluding Winter Fuel Payments) 8.4 8.5 Income category containing median income2 £30,000–34,999 £35,000–39,999 Total Number of Observations 3,754 3,613 1 Based on the 4,666 observations for which age information is available. 2 Based on the 7,064 observations for which income information is available. None of the percentages were significantly different at the 5% level. Table A2.1: Demographic and socio-economic characteristics of those receiving one and two offers Table A2.2 shows that those receiving two offers had a higher median bill and were offered larger savings in both absolute and percentage terms. Table A2.2: Financial information for those receiving one and two offers Table A2.2: Financial information for those receiving one and two offers Statistic One Offer Two Offers Financial Factors Median size of bill (£) 1131 1209* Median size of saving (£)1 103.82 110.07* Median saving as percentage of existing bill1 9.8 10.2* % Existing energy deal includes an exit fee 16.2 16.7 Total Number of Observations 3,754 3,613 * Indicates the median for the two offers group is different to the median for the one offer group at the 5% significance level. 1 The median saving was calculated based only on participants who were offered a positive saving as part of TBS. Average marginal effects for separate one and two offer regressions are reported in Table A2.3 below. While most variables remain highly significant in both the one and two offer regres sions, there are some notable exceptions. 29. Due to the reduced sample sizes of the separate one and two offer regressions compared to the combined regression it is difficult to know if the loss of significance is due to a more accurate model of respondents’ choice decisions from using two separate regressions rather than just a loss of statistical power. APPENDICES This sample selection effect was indicated by, and controlled for, including the relevant Inverse Mills Ratio (IMR) in each regression. In column 2 the IMR was negative and significant at the 1% level, while in columns 3 and 4 the IMR was positive and significant at the 1% level. Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 113 Open Access Article APPENDIX 2: ONE VS TWO OFFERS For example, Co-Op as a respondent’s existing energy supplier loses statistical significance in the one offer model and is only significant at the 10% level in the two offer model.29 29. Due to the reduced sample sizes of the separate one and two offer regressions compared to the combined regression it is difficult to know if the loss of significance is due to a more accurate model of respondents’ choice decisions from using two separate regressions rather than just a loss of statistical power. Open Access Article 114 / The Energy Journal Using the two models in Table A2.3 it is possible to estimate the predicted probability of switching for those shown one offer and those shown two offers. The mean predicted probability of switching for those shown two offers is 12.4 percentage points lower than for those shown one offer (35.3% vs 47.9%). However, this does not control for the fact that those shown two offers have different characteristics from those shown one offer. This issue can be overcome by calculating the mean predicted probability of switching for all survey respondents using the one offer model and comparing this against the mean predicted probability of switching for all survey respondents using the two offer model. This latter approach still yields a lower average predicted probability of switch ing associated with two rather than one offer; however, the magnitude of the effect is reduced to only 2.8 percentage points (39.6% vs 42.4%).30 While this result is interesting in the context of TBS, it is unclear how far it is generalizable. Table A2.3: Selected average marginal effects on the probability of switching (separate 1 vs 2 offer regressions) Switching Factor Variable Average Marginal Effect—One Offer Average Marginal Effect—Two Offers Monetary Savings 1. Saving amount of the best offer (£10 units) 0.018* 0.015* 2. Has an Exit Fee –0.167* –0.162* 3. No other penalty/loss of benefits if switch supplier 0.061* 0.040 4. Top 3 factor persuading to switch: Large enough saving –0.032 –0.039 5. Stated minimum required saving to switch (Spring 2013, £1 units) –0.002* –0.001* Non-Price Preferences 6. Electricity/energy supplier before TBS: Co-Operative Energy 0.081 0.111 7. Prefers existing supplier re: tariff type –0.135* –0.161* 8. Prefers offered supplier on ethical/ environmental grounds 0.118* 0.100* 9. Prefers offered supplier re: tariff type 0.217* 0.136* 10. (continued) Open Access Article APPENDIX 2: ONE VS TWO OFFERS Prefers offered supplier re: payment method –0.015 0.096* 11.Prefers offered supplier for ‘Other’ reason 0.170* 0.068 Uncertainty/ Preparedness 12. Confidence in accuracy of TBS saving (0 to 1) 0.061 –0.005 13. Energy bill estimated by Which –0.084* –0.064* 14. Respondent states bill as ‘Round’ amount –0.037 –0.062 15. Unsure if other penalty/loss of benefits if switch supplier –0.024 –0.094* Concerns with Switching Process 16. Worried re: ‘Other’ issues –0.111* –0.104* 17. Additional help wanted: phone support –0.046 –0.052 18. Add’l help wanted: simpler switching –0.106* –0.099* 19. Additional help wanted: something else –0.075* –0.061* Time Pressures 20. Worried switching time consuming 0.005 0.085* 21. TBS was a very busy period: Strongly Agree –0.209* –0.245* 22. TBS was a very busy period: Agree –0.094* –0.128* 23. TBS was a very busy period: Disagree 0.028 0.040* 24. TBS was a very busy period: Strongly Disagree –0.022 –0.009 Selected average marginal effects on the probability of switching (separate 1 vs 2 offer regressions) 3: Selected average marginal effects on the probability of switching (separate 1 vs 2 offer regressions) 30. Both of the reported differences in predicted switching probability are significant at the 1% level. The statistically significant drop in the predicted probability of switching when shown two offers is robust to removing variable 35 in Table A2.3 (the difference between offers) from the two-offer regression. Switching Energy Suppliers: It’s Not All About the Money / 115 Table A2.3: Selected average marginal effects on the probability of switching (separate 1 vs 2 offer regressions) (continued) Switching Factor Variable Average Marginal Effect—One Offer Average Marginal Effect—Two Offers TBS Specific Factors 25. Top 3 factor persuading to switch: Confidence in getting best possible deal 0.010 0.039** 26. Top 3 factor persuading to switch: Ethical/ environmental reasons 0.035* 0.019 27. Top 3 factor persuading to switch: Quick and easy switching process 0.025 0.020 Respondent Characteristics 28. Number of people in household: One 0.044* 0.026 29. Highest Educational Qualification: Masters/PhD 0.015 0.036 30. Gender: Male 0.029 -0.012 Other 31. Saving of best offer less saving of the Co-Op -0.001* 32. APPENDIX 2: ONE VS TWO OFFERS IMR for survey response -0.025 0.243 N 3,754 3,613 * i di i ifi h 10% l l i di i ifi h % l l d * i di i ifi ed average marginal effects on the probability of switching (separate 1 vs 2 egressions) (continued) Notes: * indicates significance at the 10% level, indicates significance at the 5% level and * indicates significance at the 1% level. The table focuses on statistically significant variables. A wide range of additional variables were includ ed in the regression, but are not reported for brevity. These variables include: payment method before TBS, household tenure, employment status of household members, whether in receipt of a fuel related benefit and interactions with existing supplier triggered by TBS. In particular, dummy variables for electricity supply regions and median household income in a respondent’s postcode area were included and generally found to be statistically insignificant. Additionally, dummies for extra options beyond those listed in the current table were included for: factors that would persuade a respondent to switch, preferences between previous and offered suppliers, worries about the switching process, additional help wanted, number of household members and highest educational qualification obtained. Details of the complete regression results are avail able on request.i Null categories for reported dummy variables: 2. No Exit Fee; 3. and 15. Has a penalty/loss of benefits if switch; 4. and 25. to 27. Listed factor not in the top 3 factors that would persuade the respondent to switch in the future; 6. Electricity/en ergy supplier before TBS - Not Co-operative Energy or EBICo; 7. to 11. Indifferent between existing and offered supplier on stated dimension; 13. and 14. Respondent used actual bill and stated ‘Non-Round’ amount; 16. and 20. Not worried about stated issue; 17. to 19. The form of additional help stated was not required; 21. to 24. Neither agree nor disagree with the statement ‘TBS was a very busy period’; 28. 2 people in household; 29. Highest educational qualification - first degree or equivalent; 30. Female. Open Access Article APPENDIX 2: ONE VS TWO OFFERS Open Access Article Open Access Article 116 / The Energy Journal APPENDIX 3: SUPPORTING MATERIALS Table A3.1: Comparison of Analysed group with all survey respondents and with average UK household characteristics Characteristic 2013 Survey Respondents Offered Saving at TBS 2013 Survey Respondents— Filters Applied Equivalent figure for GB3 Age group containing median age1 55–64 55–64 35–44 % male 70.6* 72.2,^ 48.3 % with first degree or higher 60.0 61.6,^ 23.0 % who rent their home 6.6 6.2* 35.5 % of households receiving disability benefit 7.5* 7.7* 9.84 Category containing median household income2 £30,000–34,999 £35,000–39,999 £35,000–39,999 Total number of observations 12,750 7,367 —i 1: Comparison of Analysed group with all survey respondents and with average UK household characteristics Notes: The first column represents all those who responded to the survey (it was sent to half of all TBS participants) and who were offered a positive saving at TBS. The second column is a subset of the first column involving those respondents who were supplied by a single supplier before TBS, entered the direct debit auction and who stated saving money as one of the top three reasons for participating in TBS. p p p g icates the statistic is significantly different from the figure for GB as a whole at the 5% level.ifi g y y pi y p 2 No specific question about income was asked in the survey. These figures are based on the median income of inhabitants of the six digit post code area where the respondent lived. Such income information was available for only 12,202 survey respondents (7,064 respondents among the filtered group). 3 3 These statistics are based on tables available in Ofgem (2014). 4 This is the percentage of respondents having responsibility for members of the immediate family with long-standing illness, physical or mental health problems or disability. Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 117 Table A3.2: Average marginal effects for statistically significant variables predicting the selection from all TBS participants to filtered survey respondents Variables Average Marginal Effect 1. Saving amount of the best offer (£10 units) 0.002* 2. Energy expenditure before TBS –0.000* 3. Energy bill estimated by Which 0.026* 4. Has an Exit Fee 0.016* 5. Doesn’t know if has an Exit Fee –0.006* 6. Before TBS received electricity and gas from a single supplier 0.012* 7. APPENDIX 2: ONE VS TWO OFFERS Open Access Article 118 / The Energy Journal Table A3.3: Coefficients for potential instrumental variables from the regression estimating the minimum saving required to switch Potential Instrumental Variable Regression Coefficient 1. Before TBS: On a Dual Fuel Tariff 12.171* 2. Respondent reminded of saving they were offered in TBS 7.347* 3. Respondent states saving needed using a slider covering the range £0 to £1,000 29.134* 4. Respondent states saving needed using a slider covering the range £0 to £500 15.225* 5. Household member receives disability benefit 4.815 6. With the incumbent supplier(s) for electricity (and gas where applicable) –2.833 7. Both gas and electricity from one supplier: either British Gas or the incumbent electricity supplier 1.354 8. Top 3 factor persuading to switch: Confidence that the switching process will be problem free –0.662 9. Top 3 factor persuading to switch: Having spare time to devote to switching supplier –9.138 10. Top 3 factor persuading to switch: Confidence that deal will remain relatively good for more than a year 4.032** N 7,367 Notes: * indicates significance at the 10% level indicates significance at the 5% level and * Table A3.3: Coefficients for potential instrumental variables from the regression estimating the minimum saving required to switch Potential Instrumental Variable Regression Coefficient 1. Before TBS: On a Dual Fuel Tariff 12.171* 2. Respondent reminded of saving they were offered in TBS 7.347* 3. Respondent states saving needed using a slider covering the range £0 to £1,000 29.134* 4. Respondent states saving needed using a slider covering the range £0 to £500 15.225* 5. Household member receives disability benefit 4.815 6. With the incumbent supplier(s) for electricity (and gas where applicable) –2.833 7. Both gas and electricity from one supplier: either British Gas or the incumbent electricity supplier 1.354 8. Top 3 factor persuading to switch: Confidence that the switching process will be problem free –0.662 9. Top 3 factor persuading to switch: Having spare time to devote to switching supplier –9.138 10. Top 3 factor persuading to switch: Confidence that deal will remain relatively good for more than a year 4.032** N 7,367 Notes: * indicates significance at the 10% level, indicates significance at the 5% level and * indicates significance at the 1% level. The variables listed were treated as potential instruments as they were only included in the regression modelling the minimum saving needed to switch. APPENDIX 2: ONE VS TWO OFFERS The dependent variable for the regression is “Smallest amount of saving per year I would have needed to persuade me to switch (£)”. Details of the complete regression results are available on request. Null categories for reported dummy variables: 1. Not on a Dual Fuel tariff; 2. Respondent not reminded of saving offered in TBS; 3. and 4. Respondent types ‘saving needed’ into a free text box with no upper limit; 5. No household member receives disability benefit; 6. At least one of gas or electricity is with a non-incumbent supplier; 7. Does not receive both gas and electricity from a sin gle supplier which is either British Gas or the incumbent electricity supplier; 8. to 10. Listed factor is not in the top three factors that would persuade a consumer to switch energy supplier in the future. Table A3.3: Coefficients for potential instrumental variables from the regression estimating the minimum saving required to switch APPENDIX 2: ONE VS TWO OFFERS Before TBS with incumbent supplier(s) for electricity and/or gas –0.029* 8. Before TBS received both gas and electricity from either British Gas or the incumbent regional electricity supplier –0.006* 9. Bill before TBS paid by Cash –0.058* 10. Bill before TBS paid by variable Direct Debit –0.015* 11. Shown two offers 0.007* 12. Method/venue where participant joined TBS known 0.006* 13. Reminder email sent as part of TBS phase II –0.012* 14. Reminder email sent as part of TBS phase III 0.005 15. Electricity supply region: Scottish Hydro 0.011 16. Electricity supply region: Seeboard –0.010* 17. Electricity supply region: Southern Electric 0.005* 18. Electricity supply region: Yorkshire –0.007 19. Electricity/energy supplier before TBS: npower –0.006 20. Electricity/energy supplier before TBS: Good Energy –0.099* 21. Electricity/energy supplier before TBS: Southern Electric –0.016* 22. Electricity/energy supplier before TBS: Ecotricity –0.049* 23. Electricity/energy supplier before TBS: EDF Energy –0.011* 24. Electricity/energy supplier before TBS: The Utility Warehouse –0.034* 25. Electricity/energy supplier before TBS: first:utility 0.018* 26. Electricity/energy supplier before TBS: OVO Energy 0.009* 27. Electricity/energy supplier before TBS: M&S Energy –0.015 28. Electricity/energy supplier before TBS: Co-Operative Energy 0.025* N 139,594ii Notes: * indicates significance at the 10% level, indicates significance at the 5% level and * indicates significance at the 1% level. Dummy variables for other electricity supply regions, other electricity/energy suppliers before TBS, other payment types were also included as controls in the regressions but are not reported. Details of the complete regression results are available on request. Null categories for reported dummy variables: 3. “Non-round” energy bill stated by respondent; 4. and 5. No Exit Fee; 6. Before TBS respondent received electricity and gas from separate suppliers or consumed only one fuel; 7. Receives gas or electricity from a non-incumbent supplier; 8. Does not receive both electricity and gas from either British Gas or the incumbent supplier; 9. and 10. Bill before TBS paid by Fixed Direct Debit; 11. Shown one offer; 12. Method/venue where participant joined TBS not known; 13. Reminder email not sent as part of TBS phase II; 14. Reminder email not sent as part of TBS phase III; 15. to 18. Electricity supply region: London; 19. to 28. Electricity/ energy supplier: British Gas. Observations dropped by regressions: 50 observations for Utilita, Spark Energy and Sainsbury’s Energy were dropped for perfectly predicting non-inclusion in the final analysis. (i) Heckman 2-stage We acknowledge that the sample we used for the empirical analysis could be subject to sample selection issues and for this reason we modelled the self-selection mechanism as: zi* = wi′γ + ui, with zi =1 if zi* > 0 and 0 otherwise. Where zi* represent a latent variable measuring the pro pensity to participate to our survey while wi represents a matrix of explanatory variables affecting the propensity to participate and γ represents the vector of associated coefficients. zi is a 0/1 variable reflecting actual participation in the survey. i Based on this set up we can calculate the probability of taking part in the survey by first estimating by maximum likelihood a Probit model for the probability of participating (stage 1 of the Heckman 2-stage approach): ( ) ( ) 1\| i i p Prob z ′ = = = i i w w Φ γ (A.2) ( ) ( ) 1\| i i p Prob z ′ = = = i i w w Φ γ (A.2) Where φ is the Normal probability distribution function and Φ the cumulative distribution function. From this equation we can construct the Inverse Mills Ratio (IMR) as: Where φ is the Normal probability distribution function and Φ the cumulative distribution function. From this equation we can construct the Inverse Mills Ratio (IMR) as:  ( ) ( ) ˆ / ˆ i i i w w λ ϕ γ γ ′ ′ = Φ (A.3)  ( ) ( ) ˆ / ˆ i i i w w λ ϕ γ γ ′ ′ = Φ (A.3) Stage 2 of the Heckman 2-stage approach involves including ˆλ in equation (A.1) as an additional explanatory variable in xi to correct for the potential non-random nature of our sample, which includes only individuals who chose to participate in the survey. APPENDIX 4: ECONOMETRIC METHODOLOGY Our analysis models the probability of switching supplier based on an unobservable latent variable yi * which measures utility consumers derive from switching, with * i i y ε ′ = + ix β where xi is a set of observable exogenous variables and εi a Normally distributed error term. The econometric model used in the analysis is a Probit model where the dependent variable, iy , takes a value of 1 when an individual switches energy supplier and a value of 0 when an individual does not do so. For each individual “i” it is possible to model the probability, pi, of a switch occurring as: 1 0 1 i i i with probability p y with probability p    =   −    1 0 1 i i i with probability p y with probability p    =   −    We expect to observe yi = 1 if yi * > 0. Formally, the probability of an individual switching, pi, can be modelled as: ( ) ( ) 1\| i i p Prob y F ′ = = = i i x x β (A.1) (A.1) where pi is the probability that switching is observed given the values of the explanatory variables, ix . where pi is the probability that switching is observed given the values of the explanatory variables, ix . Open Access Article When modelling the decision to switch supplier we encountered two main modelling chal lenges: (i) sample selection, and (ii) endogeneity issues. In this technical appendix we discuss the Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 119 methods used to address these issues, namely the Heckman 2-stage approach and the conditional maximum likelihood (instrumental variable approach). The main sources for the material discussed below are Cameron and Trivedi (2005) and Greene (2011). methods used to address these issues, namely the Heckman 2-stage approach and the conditional maximum likelihood (instrumental variable approach). The main sources for the material discussed below are Cameron and Trivedi (2005) and Greene (2011). 31. By endogeneity we mean that there could be common unobserved factors determining both the probability of switch ing and the minimum saving required to induce switching. Failing to properly account for this endogeneity could lead to biased and inconsistent estimates in the regressions. 32. Respondents were randomly allocated to six different treatments. Firstly, half the sample was reminded of the saving they were offered at TBS and half did not receive this reminder. Also, there were three variations in the way respondents were asked to record the saving they required: (i) on a grid with assigned values, (ii) using a slider with a maximum value of £500 and (iii) using a slider with a maximum value of £1,000. 31. By endogeneity we mean that there could be common unobserved factors determining both the probability of switch ing and the minimum saving required to induce switching. Failing to properly account for this endogeneity could lead to biased and inconsistent estimates in the regressions. 32 Respondents were randomly allocated to six different treatments Firstly half the sample was reminded of the saving Open Access Article biased and inconsistent estimates in the regressions. 32. Respondents were randomly allocated to six different treatments. Firstly, half the sample was reminded of the saving they were offered at TBS and half did not receive this reminder. Also, there were three variations in the way respondents were asked to record the saving they required: (i) on a grid with assigned values, (ii) using a slider with a maximum value of £500 and (iii) using a slider with a maximum value of £1,000. (ii) Conditional Maximum Likelihood Estimation Our Probit model for the probability of switching has been estimated using the conditional maximum likelihood estimation, an instrumental variable method, which deals with the potential endogeneity31 of the minimum required saving. This process involves the joint estimation of two equations, the first of which has the potential endogenous variable as a dependent variable. In order to account for the endogeneity bias, we chose a set of “instrumental variables” to be included as regressors in the second equation. The “instrumental variables” need to be correlated with the en dogenous variable but independent of the decision to switch supplier. The main instrument we used in our analysis is the method we used for asking the respondents to report the minimum required saving to switch.32 While this variable is a highly significant predictor of the minimum required saving the random assignment of individuals between different methods means that it cannot be a predictor of switching, also recalling that our survey was conducted about one year after TBS, when the switching decision took place. Open Access Article 120 / The Energy Journal Suppose there are two endogenous variables, y* 1i and y2i (in other words, they are deter mined by the same underlying process). Assume that each endogenous variable can be represented by a linear equation. Also, assume each equation involves only two explanatory variables: * 1 1 1 2 1 i i i y y α ε ′ = + + i x β (A.4) 2 2 i i y ε ′ = + ix π (A.5) * 1 1 1 2 1 i i i y y α ε ′ = + + i x β (A.4) 2 2 i i y ε ′ = + ix π (A.5) (A.4) * 1 1 1 2 1 i i i y y α ε ′ = + + i x β (A.5) 2 2 i i y ε ′ = + ix π where x1i and xi are exogenous variables, with xi representing the instrumental (exogenous) variable which influences y2i but not y1i. β and π are vectors of coefficients associated with the explanatory variable whilst ε1 and ε2 are the error terms, assumed to be jointly Normally distributed. A Wald test for the independence of the error terms in the two equations, ε1i and ε2i, led us to reject the null hypothesis of y2 being exogenous. Open Access Article (ii) Conditional Maximum Likelihood Estimation Equation (A.4) explaining y1i is the structural equation of interest used to estimate a Probit model for the probability of switching, while equation (A.5) explains the endogenous regressor y2i. As in (i) we expect to observe y1i = 1 when y1i * > 0. We therefore express the probability of switching as: where x1i and xi are exogenous variables, with xi representing the instrumental (exogenous) variable which influences y2i but not y1i. β and π are vectors of coefficients associated with the explanatory variable whilst ε1 and ε2 are the error terms, assumed to be jointly Normally distributed. A Wald test for the independence of the error terms in the two equations, ε1i and ε2i, led us to reject the null hypothesis of y2 being exogenous. Equation (A.4) explaining y1i is the structural equation of interest used to estimate a Probit model for the probability of switching, while equation (A.5) explains the endogenous regressor y2i. As in (i) we expect to observe y1i = 1 when y1i * > 0. We therefore express the probability of switching as: ( ) ( ) 1 1 1 2 1 1 2 1\| , i i i p Prob y F y α ′ = = = + i i i x y x β (A.6) (A.6) Based on the relationships described above we estimated the probability of switching sup plier by conditional maximum likelihood, by maximising the following likelihood function: ( ) ( ) ( ) ( ) { } 1 1 1 1 2 1 1 1 2 1 1 ( , ) 1 ln 1 N i i i i y lnF y y F y α β α α = ′ ′ = + + − − + ∑ i i x x β β (A.7) ( ) ( ) ( ) ( ) { } 1 1 1 1 2 1 1 1 2 1 1 ( , ) 1 ln 1 N i i i i L y lnF y y F y α β α α = ′ ′ = + + − − + ∑ i i x x β β (A.7) Open Access Article
https://openalex.org/W4320481930	https://hal.science/hal-03999495/file/journal.pone.0280071.pdf	English	null	Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes	PloS one	2,023	cc-by	8,455	To cite this version: Sardor Israilov, Li Fu, Jesús Sánchez-Rodríguez, Franco Fusco, Guillaume Allibert, et al.. Rein- forcement learning approach to control an inverted pendulum: A general framework for educational purposes. PLoS ONE, 2023, 18 (2), pp.e0280071. 10.1371/journal.pone.0280071. hal-03999495 HAL Id: hal-03999495 https://hal.science/hal-03999495v1 Submitted on 21 Feb 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. 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PLOS ONE RESEARCH ARTICLE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes Sardor Israilov1,2☯, Li Fu1☯, Jesu´s Sa´nchez-Rodrı´guezID1,3☯, Franco Fusco2☯, Guillaume Allibert2☯, Christophe RaufasteID1,4☯, Me´de´ric ArgentinaID1☯* 1 Universite´ Coˆte d’Azur, CNRS, INPHYNI, Valbonnes, France, 2 Universite´ Coˆte d’Azur, CNRS, I3S, Sophia Antipolis, France, 3 Laboratory of Fluid Mechanics and Instabilities, E´ cole Polytechnique Fe´de´rale de Lausanne, Lausanne, Switzerland, 4 Institut Universitaire de France (IUF), Paris, France Sardor Israilov1,2☯, Li Fu1☯, Jesu´s Sa´nchez-Rodrı´guezID1,3☯, Franco Fusco2☯, Guillaume Allibert2☯, Christophe RaufasteID1,4☯, Me´de´ric ArgentinaID1☯* Sardor Israilov1,2☯, Li Fu1☯, Jesu´s Sa´nchez-Rodrı´guezID1,3☯, Franco Fusco2☯, Guillaume Allibert2☯, Christophe RaufasteID1,4☯, Me´de´ric ArgentinaID1☯* 1 Universite´ Coˆte d’Azur, CNRS, INPHYNI, Valbonnes, France, 2 Universite´ Coˆte d’Azur, CNRS, I3S, Sophia Antipolis, France, 3 Laboratory of Fluid Mechanics and Instabilities, E´ cole Polytechnique Fe´de´rale de Lausanne, Lausanne, Switzerland, 4 Institut Universitaire de France (IUF), Paris, France 1 Universite´ Coˆte d’Azur, CNRS, INPHYNI, Valbonnes, France, 2 Universite´ Coˆte d’Azur, CNRS, I3S, Sophia Antipolis, France, 3 Laboratory of Fluid Mechanics and Instabilities, E´ cole Polytechnique Fe´de´rale de Lausanne, Lausanne, Switzerland, 4 Institut Universitaire de France (IUF), Paris, France a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. * mederic.argentina@univ-cotedazur.fr OPEN ACCESS Citation: Israilov S, Fu L, Sa´nchez-Rodrı´guez J, Fusco F, Allibert G, Raufaste C, et al. (2023) Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes. PLoS ONE 18(2): e0280071. https://doi.org/10.1371/journal.pone.0280071 Editor: Ning Sun, Nankai University, CHINA Received: June 30, 2022 Accepted: December 20, 2022 Published: February 13, 2023 Copyright: © 2023 Israilov 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: Ning Sun, Nankai University, CHINA Received: June 30, 2022 Accepted: December 20, 2022 Published: February 13, 2023 Editor: Ning Sun, Nankai University, CHINA Received: June 30, 2022 Accepted: December 20, 2022 Published: February 13, 2023 Copyright: © 2023 Israilov et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: We provide all our codes in github.com/francofusco/pendule_pi. Data Availability Statement: We provide all our codes in github.com/francofusco/pendule_pi. Abstract Machine learning is often cited as a new paradigm in control theory, but is also often viewed as empirical and less intuitive for students than classical model-based methods. This is par- ticularly the case for reinforcement learning, an approach that does not require any mathe- matical model to drive a system inside an unknown environment. This lack of intuition can be an obstacle to design experiments and implement this approach. Reversely there is a need to gain experience and intuition from experiments. In this article, we propose a general framework to reproduce successful experiments and simulations based on the inverted pen- dulum, a classic problem often used as a benchmark to evaluate control strategies. Two algorithms (basic Q-Learning and Deep Q-Networks (DQN)) are introduced, both in experi- ments and in simulation with a virtual environment, to give a comprehensive understanding of the approach and discuss its implementation on real systems. In experiments, we show that learning over a few hours is enough to control the pendulum with high accuracy. Simula- tions provide insights about the effect of each physical parameter and tests the feasibility and robustness of the approach. Introduction Inverted pendulums—also known as “cart-pole” apparatuses—belong to simple type of sys- tem that have a long history in the field of mechanics and dynamical systems [1, 2]. Their dynamics is described by a set of mathematical equations that are simple to derive, while still featuring interesting properties such as nonlinearity and under-actuation. This makes an inverted pendulum a perfect candidate to benchmark and showcase new control algorithms before deploying them on more complex systems such as quadrotors or humanoid robots [3]. In addition, given the simplicity required to build an experimental prototype, cart-pole systems are very well-suited for teaching a wide variety of topics, ranging from Lagrangian mechanics to control theory. Indeed, the literature includes numerous examples of low-cost Funding: This work was supported by the French government through the UCA Joint Excellent Dynamic Initiative (JEDI) and UCA Digital Systems for Humans (UCA DS4H) Investments in the Future Programs, managed by the National Research Agency (ANR), in the form of funds to MA, CR and GA [reference no. ANR-15-IDEX-0001] and GA [reference no. ANR-17-EURE-0004]. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 1 / 15 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes pendulums designed and built with the purpose of teaching one or more subjects to under- graduates [4–6]. In this article, we aim at controlling an inverted pendulum in its unstable position, by rein- forcement learning (RL). This machine learning method has shown great interest in many applications such as playing games [7, 8] and system controlling [9–11], and focuses on how agents perform actions in an environment so as to maximize some notion of cumulative reward [12]. The advantage of RL is that it avoids modeling the dynamics involved, unlike in model-based approaches [13, 14]. Many numerical studies have implemented an inverted pendulum virtual environment as a benchmark to test RL algorithms [15–22], but to our knowledge, there is no study that pro- vides successful RL implementations in experiments. First, except for a few studies that have discussed non ideal systems [16, 17], most of these numerical implementations discard the effects associated to realistic (and thus more complex) control methods: in experiments, the control of the cart is subject to delay, hysteresis, biases and noise that can significantly alter the learning process. Introduction Second, most of the existing virtual environments consider only motion of the pendulum in a small angle range around the upward and unstable position and do not treat the whole control from the downward and stable position as expected in experiments. This ambition makes the control task significantly more difficult. The goal of the article is twofold. First, we expose the basic ingredients to build an intuition about RL approaches. We focus here on Q-learning and Deep Q-Network approaches to give insights about the implementation and the conditions of successful controls. Simulations with a virtual environment are provided to test the feasibility of the two approaches as well as to probe the effect of physical parameters that can not be easily tuned in experiments. Second, we provide all the material to perform experiments. This paper is accompanied by an open-source code repository which allows to replicate all the approaches presented here [23]. It includes detailed instructions to build the prototype used in this work, configure its software interface and implement several controllers. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 Modelling the inverted pendulum and the controller We assume a mass m located at the end of a massless rigid rod of length ℓand subjected to gravity g. Its other extremity is free to rotate on a motorized cart located at abscissa x(t). The angle θ(t) separating the rod to the downward vertical direction, as shown in Fig 1, follows the dynamics of a damped oscillator in the absence of cart motion. If the cart is actuated, the dynamics is driven by the equation: €y þ kv _y þ o2 sin y þ €x ‘ cos y ¼ 0; ð1Þ ð1Þ with o ¼ ffiffiffiffiffiffiffi g=‘ p the natural frequency of the pendulum and kv a viscous friction coefficient [1]. fififififififi The purpose is to stabilize the pendulum in its unstable equilibrium position θ = π by con- trolling the motion of the cart only, which is itself driven by a target velocity provided by a con- troller. Unlike ideal systems implemented in virtual environments, experimental systems need to account for delay, hysteresis and biases between the target and measured values. For the present setup, the cart velocity _x and the control velocity _xc are linked through the equation: €x ¼ 1 t _xc _x ð Þ fc sign ð_xÞ fd: ð2Þ €x ¼ 1 t _xc _x ð Þ fc sign ð_xÞ fd: ð2Þ 2 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes Fig 1. Sketch of the inverted pendulum. https://doi.org/10.1371/journal.pone.0280071.g001 Fig 1. Sketch of the inverted pendulum. https://doi.org/10.1371/journal.pone.0280071.g001 https://doi.org/10.1371/journal.pone.0280071.g001 The first term on the right-hand side models the motorized cart with τ, a relaxation time scale to account for the linear dynamics. fc and fd are two coefficients to account for the asym- metric dry friction acting on the motorized base. In experiments, the cart target velocity _xc is proportional to the applied voltage U: _xc ¼ kUU; ð3Þ ð3Þ _xc ¼ kUU; where kU is a constant. The cart is constrained to move on a track of length 2xmax. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 Controlling the pendulum using reinforcement learning RL exploits the framework of Markov Decision Process (MDP), which is an extension of the Markov process. There are four components in MDP: a set of states, a set of actions, a reward and a policy. We refer to the internal decision maker who uses a RL algorithm as an agent, and the whole physical system as the environment. During the learning process, the agent evolves in an environment and tries to maximize its cumulative reward. At each time step, the state of the agent is assessed and an action is performed. After the actuation, the environment provides a new state and a reward. The choice of the action follows the policy π(a\|s) which is the proba- bility of taking action a while in state s. The objective in RL is to determine the best policy π(a\|s) for the agent, that maximizes the cumulative reward. For the cart-pole problem, at each time step ti = iΔt, the state si is given by the pendulum’s orientation θ(ti) and its angular velocity _yðtiÞ, as well as the cart’s position x(ti) and velocity _xðtiÞ, i.e.: si ¼ ðyðtiÞ; _yðtiÞ; xðtiÞ; _xðtiÞÞ: ð4Þ ð4Þ Here i is the time step number and Δt is the time interval between two successive state observations (or between two successive actions). According to the policy π(a\|s), the agent chooses and executes an action ai which controls the cart movement for a given state si. This action changes the agent’s state si to si+1, and the environment provides a reward ri+1 related to the proximity of the pendulum to its unstable position. This process is then iterated at stage i+1: the loop is depicted in Fig 2. In order to construct the policy π(a\|s), it is essential to PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 3 / 15 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes Fig 2. RL learning process. The state of environment s is measured and given to the agent. The agent updates it policy and accordingly choose the action a for the next step among −U, 0 or U. After sampling time, the state s evolves and the cycle continues. In our case, s ¼ ðy; _y; x; _xÞ. The policy is updated by updating the action-value function for Q-Learning and DQN, using a so-called Q-table and artificial neural networks, respectively. https://doi.org/10.1371/journal.pone.0280071.g002 Fig 2. RL learning process. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 Controlling the pendulum using reinforcement learning The state of environment s is measured and given to the agent. The agent updates it policy and accordingly choose the action a for the next step among −U, 0 or U. After sampling time, the state s evolves and the cycle continues. In our case, s ¼ ðy; _y; x; _xÞ. The policy is updated by updating the action-value function for Q-Learning and DQN, using a so-called Q-table and artificial neural networks, respectively. https://doi.org/10.1371/journal.pone.0280071.g002 estimate a return function Ri, designed as the discounted cumulative reward: Ri ¼ ri þ griþ1 þ g2riþ2 þ g3riþ3 þ :::; ð5Þ ð5Þ Ri ¼ ri þ griþ1 þ g2riþ2 þ g3riþ3 þ :::; where 0 < γ < 1 is the discount factor which measures the importance of the future unitary reward in computing the expected cumulative reward. Since the cumulative reward depends on the states si, si+1, si+2, . . . and the actions ai, ai+1, an+2, . . ., one can define an action-value function Q(si, ai) (Q refers to Quality) which computes the expected cumulative reward at the state si when performing the action ai: Qðsi; aiÞ ¼ E½Rijðsi; aiÞ: ð6Þ ð6Þ This function could be the basis for constructing an optimal policy. For example a greedy policy will always select the best action a = argmaxaQ(si, a) for an agent in state si. The learning process consists in visiting a large number of states and taking various actions, and to compute the reward expectation (6). However, it is usually time consuming and very difficult, if not impossible, to travel through all the states and actions to accurately determine the action-value function Q(s, a), as it is necessary to sample the state and the action spaces to accumulate statistics for the rewards. In addition, a control task could be infinitely long so it is not practical to wait to the end of the experiment and measure the cumulative reward, and update the function Q. In the MDP framework, one can rewrite Eq 6 as [12]: te the function Q. Controlling the pendulum using reinforcement learning In the MDP framework, one can rewrite Eq 6 as [12]: Qðsi; aiÞ ri þ gQðsiþ1; aiþ1Þ: ð7Þ ð7Þ Qðsi; aiÞ ri þ gQðsiþ1; aiþ1Þ: 4 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes Here we use the reward ri after a sampled action ai to represent the expected immediate reward, and γQ(si+1, ai+1) to represent the cumulative discounted future reward. In order to determine the action-value function, the agent interacts constantly with the environment dur- ing the learning phase and update its Q function. This function can be updated through an iterative procedure: Qðsi; aiÞ Qðsi; aiÞ þ aDQ; ð8Þ ð8Þ which is similar to the Euler scheme for numerically integrating differential equation _Q ¼ DQ, where α plays the role of a time step. This is the idea of what is called the temporal differencing (TD) approach [12]. By defining ΔQ = Q −Q, we know that the differential equation will drive Q to the target Q. The idea of the Q-Learning algorithm is to hypothesize that: Q ri þ g max a0 Qðsiþ1; a0Þ; ð9Þ ð9Þ with a0 being the accessible actions at state si+1, which is consistent to the definition (7). It models that an approximation of the cumulative expected reward is the reward ri plus the dis- counted cumulative reward at step i + 1 by taking the best action a iþ1 ¼ argmaxðQðsiþ1; a0ÞÞ. To summarize, the Q-learning iterative procedure writes [24, 25]: Qðsi; aiÞ Qðsi; aiÞ þ aðri þ g max a0 Qðsiþ1; a0Þ Qðsi; aiÞÞ; ð10Þ ð10Þ where the parameter α measures the learning rate. The effect of the discount factor γ becomes even clearer: as it tends to zero, the learning agent only takes into account the immediate reward, while as γ is nonzero, the agent integrates future rewards in the learning phase. With this iterative approach, the agent learns while it evolves in the environment. Q-learning employs an -greedy policy during the learning process. It chooses the best action most of the time, and slightly explore the consequences of randomly taken actions: at each time step, a random number NR is drawn, if NR < < 1, a random action will be chosen, otherwise the greedy policy will be applied. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 RL environment Our objective is to maintain the pendulum at the target position θ = π while centering the cart (x = 0) at the same time. We perform both experiments and simulations. The system state s has been defined with Eq (4). To avoid an angle discontinuity, sin(θ) and cos(θ) are given to the learning agent instead of only θ. The inverted pendulum system is driven by a motor on the cart and it has direct control on the meant cart’s velocity _x via an applied voltage on the motor. Three actions are offered to the agent at each time step, i.e., ai = (−U, 0, + U), with U 2 (0, 12V) a fixed voltage. At each time step, the cart can translate in both directions or to keep its current position, according to its dynamics. Another crucial component for RL is the reward function. The reward is maximum as the objective is reached, i.e., the pendulum in its unstable position (θ = π). In addition, we add the requirement for the cart to be centered around the middle of the track (x = 0). For this pur- pose, there are many options to design the reward function [12], and for simplicity, we have chosen: ð11Þ rðy; xÞ ¼ ð1=2Þð1 cos ðyÞÞ ðx=x0Þ 2; ð11Þ where x0 < xmax. This additional discard ———constraint does not prevent the agent to reach the control objective on the angle. The maximum of this function is equal to one, as θ = π and x = 0. The normalized return of an episode is computed as the cumulative reward of the entire episode divided by the maximum episode length, i.e. 800. Such a definition gives an evaluation of the policy: the closer to 1 the normalized return, the better the episode. An episode is inter- rupted when the state si meets at least one of the following conditions: 1. the dimensionless cart’s position exceeds the physical boundaries, i.e., \|x\|>xmax; In this case, the agent is strongly penalized and the cumulative reward of the episode is reduced by -400. 2. the angular speed exceed 14 rad/s, since in practice, we would like to avoid the pendulum spinning too rapidly. This value has been chosen according to the mechanical limit of our experimental system. 3. the maximum duration Tep = 800Δt is attained, where Δt = 0.05 s. Controlling the pendulum using reinforcement learning To store the expectation of the cumulative reward, the Q-Learning algorithm uses a Q-table that covers the whole state space and action space. This object takes the form of a huge matrix of dimension Ns × Na, where Ns is the number of discretized states, and Na is the number of possible actions (Na = 3 here). This representation already underlines the limitation of this approach, because of the finite size of memory of modern computers. To overcome this obstacle, it appears necessary to exploit a more efficient function approxi- mator. Deep Q-Network (DQN) [7] is a reinforcement learning algorithm based on the Q- learning approach that takes advantage of neural networks in place of the matrix “Q-table” to approximate the true “action-value” function. Neural networks provide an effective way to approximate Q(s, a), because they can incorporate non-linearity and aggregate among the states due to the interconnection between the neighboring layers of the neural net. This leads to a more efficient action-value approximations. In addition, to stabilize the learning process and obtain more reliable results, DQN also employs a number of additional techniques that we summarize in S1 File. In the next section, we perform the control task on a real system and we demonstrate the capacity of both Q-learning and DQN algorithms for the full control including swing-up and stabilization of the inverted pendulum. We first present in details our RL environment; then we discuss the limitations of the basic Q-Learning for this system; the more advanced DQN approach is then exploited and we show that it successfully maintains the pendulum at the PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 5 / 15 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes target position in both experiments and simulations. Finally, we explore in the virtual environ- ment the influence of different system’s physical parameters on the control quality. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 RL environment This choice has been set to diversify the experience and avoid being stuck in local minimums, which corresponds to roughly 2 or 3 times optimal swing-up time. These values are indeed adapted for an accept- able control quality. In the real experiment, one episode takes approximately 40 s. At the beginning of every episode, we initialize the system with the cart and the pendulum at rest, i.e., θ = 0 and x = 0. Between two episodes, the system waits 120 s to ensure that the con- dition y ¼ _y ¼ 0 is satisfied. The learning process consists in accumulating statistics during successive episodes. Plotting the normalized return as a function of the episode number can be noisy and we smooth the data by performing a moving average in Q-learning and DQN over 300 and 30 episodes, respectively, as the former is less stable. Finally, we prefer to represent the learning curve by plotting the normalized return as a function of the total number of time steps to give insights about the true time of the learning process, because some episodes might not run to the end. 6 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes Fig 3. The experimental setup. https://doi.org/10.1371/journal.pone.0280071.g003 Fig 3. The experimental setup. https://doi.org/10.1371/journal.pone.0280071.g003 https://doi.org/10.1371/journal.pone.0280071.g003 Experimental setup and methods. The experimental realization of the pendulum is shown in Fig 3. It features a DC motor (model: MFA 970D 12V) which can apply a horizontal force to the sliding base thanks to a transmission belt. An incremental encoder measures the position x of the base on a linear track, assuming that x = 0 m corresponds to the centered position. The finite length of the track gives the constraint \|x\|<xmax, with xmax = 0.35 m. A sec- ond encoder mounted on the moving base assesses the angle θ. Both are incremental encoders (model: LD3806–600BM-G5–24C) with two phases in quadrature, for a total of 2400 steps per revolution. A Raspberry Pi 4 is used to handle the electronic devices and control the system. It runs a C++ executable, namely the low-level interface (LLI), which is responsible of handling the different hardware components and expose the current state of the pendulum to client control applications (see S1 File). RL environment The algorithm running on the Raspberry Pi then commands the motor, within the three possible actions. All the code to control the pendulum is open source and available, as well as a reference manual [23]. The exact procedures to measure the physical parameters that appear in Eqs (1) to (3) are described in S2. Their values are summarized in S1 File. Simulations. In the experimental setup, the state information is gathered directly from the physical world, and the agent interacts with the environment via the LLI. In the virtual setup, the agent’s state is updated through Eqs (1) to (3). Details can be found in our code [23]. The effects of the voltage U, the dry friction acting on the motorized base fc and the viscous friction kv of the pendulum were investigated systematically in simulation. The same holds for the influence of the noise amplitude σθ and s_y on the control quality: we have introduced a Gaussian noise to the measurement of the pendulum angle θ, i.e., at each instant t = iΔt, yi N ðym i ; s2 yÞ, where N refers to the normal distribution, ym i ¼ yi1 þ _yDt is updated from the previous state. Naturally, a noise of amplitude s_y ¼ sy=Dt was then introduced to the _y measurement. Q-learning In Q-learning, the observation space ðsinðyÞ; cosðyÞ; _y; x; _xÞ is discretized into different num- ber of bins, whose sizes is matter of compromise. A Q-table with low resolution results in relatively fast simulations and limits the use of computer memory. On the other hand, the res- olution needs to be high enough to ensure the success of the learning process. As an example we start with a sparse and homogeneous discretization with nBins = (10, 10, 10, 10, 10). In this case we expect the Q-table to contain 3 105 elements, given that there are three possible actions. 7 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes The Q-table size gives a minimal estimate of the total number of time steps to learn assum- ing that the agent needs to visit each element of the table. This number is 10–100 times higher in practice given that the basic Q-learning algorithm usually suffers a low sample-efficiency [12]: some elements are never evaluated while some others can be updated regularly. We have tested the Q-learning approach in simulation with different total number of epi- sodes NT from 104 to 107. We recall that one episode contains 800 time steps at maximum; the average number of time steps per episode is lower in practice due to numerous interrupted epi- sodes at the beginning of the learning process. The technical details such as the value of the hyperparameters are found in S4. Given the expression of the reward function and of the pen- alty, the cumulative reward spans from -0.5 (the cart goes quickly out of the track) to 1 (suc- cessful learning). Below 106 time steps, the normalized return remains close to its minimum. The system requires at least 107 time steps (105 episodes) to observe an increase of the normal- ized return above 0 (Fig 4a). Even in this case, the cumulative reward remains low, around 0.3, and reaches 0.55 at most as the number of time steps is increased to 1010. For such an episode, the pendulum can be maintained at its vertical position only in a short amount of time, other- wise the pendulum oscillates (Fig 4b). Transposed to experiments with a physical time interval Δt = 0.05s, 107 time steps correspond to 6 days of experiments! https://doi.org/10.1371/journal.pone.0280071.g004 Q-learning Between two iterations the angle varies within an increment Δθ and we write θ(t) = θ(0) + Δθ, Δθ 1: cos yðtÞ ¼ cosðyð0ÞÞ Dy sin yð0Þ þ oðDyÞ 2 ð12Þ ð12Þ cos yðtÞ ¼ cosðyð0ÞÞ Dy sin yð0Þ þ oðDyÞ 2 _y ¼ Dy Dt ; ð13Þ _y ¼ Dy Dt ; ð13Þ such that we deduce that: ny ¼ p o2Dt2 1 sin yð0Þ : ð14Þ ð14Þ This gives the order of magnitude nθ 50. The presence of a divergence near the unstable equilibrium shows that the discretization must be refined at least near cos(θ) = −1. Consequently, we tested a finer resolution nBins = (50, 50, 50, 10, 10) with sin(θ), cos(θ) and _y discretized into 50 bins. The computation memory increases exponentially with the size of the Q-table and any finer resolution would be unpractical. As observed in Fig 4c, it takes at least 108 time steps to see a normalized return above 0. After about 5.6 × 109 time steps (7 × 106 episodes), the system has finally learned reasonably well and obtain a normalized return of 0.8: the pendulum can stay in the goal position for a finite period, but quickly falls over to be quickly swung back up again (Fig 4d). The inefficiency of the learning is rationalized by the fact that the matrix representation of Q-Table is not adapted to solve the swing-up problem. To update the action-value function more efficiently, a better function approximation is needed. In that regard, artificial neural net- works show very promising capabilities and is data efficient [10]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 Q-learning We can nevertheless discuss the effect of the discretization of the Q-table, which is too low in the former example to reach a cumulative reward close to 1 even after a very large number of time steps. In the following, we estimate the typical value nθ for the bin in θ: the Fig 4. Learning results using the basic tabular Q-learning implementation. Left: Normalized return as a function of the number of time steps for different total number of episodes NT. Right: Temporal evolution of cos θ in the best episode of the longest learning process (NT = 107). The observation space ðsinðyÞ; cosðyÞ; _y; x; _xÞ is discretized homogeneously into different number of bins: a) and b) nBins = (10, 10, 10, 10, 10), c) and d) nBins = (50, 50, 50, 10, 10). https://doi org/10 1371/journal pone 0280071 g004 Fig 4. Learning results using the basic tabular Q-learning implementation. Left: Normalized return as a function of the number of time steps for different total number of episodes NT. Right: Temporal evolution of cos θ in the best episode of the longest learning process (NT = 107). The observation space ðsinðyÞ; cosðyÞ; _y; x; _xÞ is discretized homogeneously into different number of bins: a) and b) nBins = (10, 10, 10, 10, 10), c) and d) nBins = (50, 50, 50, 10, 10). https://doi.org/10.1371/journal.pone.0280071.g004 https://doi.org/10.1371/journal.pone.0280071.g004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 8 / 15 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes discretization interval is Δθ = 2π/nθ. In order to ensure the learning objective, the time interval separating two actions must not be too large with respect to this discretization. We expect that Δt should be smaller than the typical time the agent lasts in one interval: we can assess this duration in the limit of small damping. By assuming that the pendulum is weekly damped, we approximate the Eq (1) with €y þ o2 sin y ¼ 0. Consequently we write the energy conservation 1 2 _y2ðtÞ ¼ o2 cos y tð Þ cos y 0 ð Þ ð Þ, where _yð0Þ ¼ 0. Deep Q learning In this section, we implement the Deep Q Learning technique. In this approach, the Q-Table for approximating the Q-function is replaced by an Artificial Neural Network (ANN), which is named Deep Q Network (DQN). We have shortly introduced ANN in S3. For our purposes, we use for the ANN a dense neural network architecture with 2 hidden lay- ers having 256 nodes each. Five nodes receive the values of the state s, and three terminal nodes give the Q-value for each of the 3 actions. Similar to any other deep learning algorithm, the training of DQN depends on the hyperparameters, which determine the network policy structure, the learning strategy and the learning speed. We offer a set of fixed hyperparameters (see S1 File), which is robust for our system. In parallel to experiments, we performed simulations of the model. For both approaches, the features and quality of the learning process are evaluated. Note that the maximal number of time steps (150000) for the complete training is chosen so that the steady state average value is reached in both real and virtual experiments. We evaluate the policy performance every 5000 time steps with an inference. It consists in testing a greedy policy during one complete episode, with the initial condition We evaluate the policy performance every 5000 time steps with an inference. It consists in testing a greedy policy during one complete episode, with the initial condition ðy; _y; x; _xÞ ¼ ð0; 0; q0; 0Þ. This protocol is applied directly in experiments, while in PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 9 / 15 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes simulations, the inference curve consists in computing the evolution of the average normalized return of 10 episodes (instead of only one in experiments) with equidistant initial conditions: (θ0 2 (−10˚, 10˚) and ð_y; x; _xÞ ¼ ð0; 0; 0Þ). This allows to test the robustness of the policy in simulation, i.e., the capacity to generalize and achieve a high normalized return from different initial states, other than the particular initial state of the learning process. This protocol how- ever is not viable in experiments since in practice it is difficult to control precisely the initial angle of the pendulum other than its equilibrium position. Deep Q learning Finally, the best learned policy in the sequel corresponds to the DQN model that obtained the highest normalized return among all inferences. Experimental results. We first discuss the results of the outlined DQN algorithm obtained with the experimental setup. The only control parameter is the applied voltage U, which is directly proportional to the target cart’s velocity value _xc Eq (3). Fig 5 displays the temporal evolutions (a) of the cart’s position and (b) of the pendulum’s angle during a single episode for the best learned policies. Two distinct voltages were tested: U = 2.4 V and U = 7.1 V: we illustrate the corresponding learning processes in a movie (See S1 Video). The voltage U = 2.4 V is not sufficient to swing up the pendulum, and the best policy yields an oscillation of the pendulum around 0. This means that the energy provided with this voltage is not high enough to swing up the pendulum or that the total duration of one episode, 800 time steps, is not large enough to increase the maximal angle, period after period. Given that the maximum angle is reached after 300 times steps already, the first assumption is probably the good one. For the other voltage U = 7.1 V, the cart initially oscillates with a large amplitude and the pendulum swings up after about the equivalent of almost 3 periods. As soon as the unstable equilibrium is reached, the cart turns into a vibration regime with smaller amplitude to main- tain the pendulum balanced upward around θ = π. The learning and the inference curves (see Fig 6, thick solid lines) reveal exactly the same results that for U = 7.1 V, the normalized return in both learning and inference reaches a high plateau value of 0.8 −0.9, indicating a success- ful control, while for U = 2.4 V, the normalized return stays very low around 0.1. Simulation results. In this section, we perform simulations and test different important physical parameters which could influence the control quality. All the parameters are kept con- stant and defined with the Table 1 and 2 of the SI except the one investigated. The voltage is set to U = 12 V and the noise sy ¼ s_y ¼ 0 if not specified. Effect of action amplitude—Applied voltage on the DC motor. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 Deep Q learning We have shown with our experimental setup that the action amplitude plays a crucial role in the task: a low voltage applied on the DC motor results in a failure of control. Here we test a range of U from 2.4 to 12 V in the virtual environment and the results are presented in Fig 6. First, we note that the simulation results are consistent with those found in experiments (thick curves), i.e., both nor- mal and thick curves of (U = 7.1 V) as well as (U = 2.4 V) show similar trend. Fig 6a displays the learning curves. The normalized return increases and then reaches a plateau for all the applied voltages. However, up to U = 4.7 V, the plateau value is smaller than 0.4, close to that observed using Q-learning algorithm, referring to an oscillation around the stable position. Above 4.7 V, DQN algorithm gives satisfying performance during the learning process. To assess the performance of the optimal policy obtained for each applied voltage, we plot the inference results in Fig 6b. Because there is no exploration and the optimal action is chosen at each time step, the plateau value of each inference curve is expected to be greater than the corresponding learning curve. Nevertheless, some inferences exhibit negative peaks associated to the fact that among the 10 episodes that are averaged to measure the normalized return of an inference, some of them are terminated by the cart reaching xmax and are strongly penalized PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 10 / 15 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes Fig 5. Experimental results with the best policies in inference for two different applied voltages U = 2.4 V (blue) and 7.1 V (green): a) Temporal evolution of the cart’s position x during one episode b) trajectory of the cart in the ðx; _xÞ space c) Temporal evolution of the pendulum’s angle θ during one episode d) Trajectory of the pendulum in the ðy; _yÞ space e) Temporal evolution of the applied voltage during the first 200 time steps. https://doi org/10 1371/journal pone 0280071 g005 Fig 5. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 Deep Q learning Experimental results with the best policies in inference for two different applied voltages U = 2.4 V (blue) and 7.1 V (green): a) Temporal evolution of the cart’s position x during one episode b) trajectory of the cart in the ðx; _xÞ space c) Temporal evolution of the pendulum’s angle θ during one episode d) Trajectory of the pendulum in the ðy; _yÞ space e) Temporal evolution of the applied voltage during the first 200 time steps. https://doi.org/10.1371/journal.pone.0280071.g005 https://doi.org/10.1371/journal.pone.0280071.g005 consequently. These negative peaks disappear as the number of time steps increases and the learning process continues. A normalized return between 0.8–0.9 is a good value as it is calcu- lated from the averaging on one episode, and this includes the initial stage before swing up. This can be seen in Fig 6c where the learning process is probed by plotting the time evolution of the reward for an episode initiated at θ0 = 0 following the best learned policy obtained after the 150000 time steps. From U = 5.9 V, the plateau of the reward is around 1 and the system reaches the objective. This figure also reveals that the higher the applied voltage is, the quicker the swing-up is achieved. To probe the robustness of the best learned policy for each applied voltage, we have measured the average of the plateau reward for 10 episodes initiated with PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 11 / 15 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes Fig 6. Influence of the applied voltage on the learning process. Thin curves correspond to different simulations, while thick curves refer to the experimental observations. a) Learning curve. b) Inference curve built from inferences performed every 5000 time steps. c) Temporal evolution of the reward for the episode initiated at θ0 = 0 following the best learned policy. d) Statistics over 10 episodes initiated with θ0 between −10˚ and 10˚ of the plateau reward following the best learned policy. Fig 6. Influence of the applied voltage on the learning process. Thin curves correspond to different simulations, while thick curves refer to the experimental observations. a) Learning curve. b) Inference curve built from inferences performed every 5000 time steps. c) Temporal evolution of the reward for the episode initiated at θ0 = 0 following the best learned policy. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 Deep Q learning Influence of the physical parameters on the control: Inference curves of a) static friction, b) viscous friction, c) measurement noise and d) action noise. Fig 7. Influence of the physical parameters on the control: Inference curves of a) static friction, b) viscous friction, c) m action noise. Fig 7. Influence of the physical parameters on the control: Inference curves of a) static friction, b) viscous friction, c) measurement noise and d) action noise. https://doi.org/10.1371/journal.pone.0280071.g007 https://doi.org/10.1371/journal.pone.0280071.g007 https://doi.org/10.1371/journal.pone.0280071.g007 mrad, result in a perfect control quality as observed with high plateau values of the inference curves. A noise amplitude of 17.5 mrad is still acceptable. Beyond this value, the pendulum can’t be driven to its unstable position. Finally, we examine the effect of an associated degree of uncertainty on the command sent to the motor, thus a Gaussian noise of standard deviation σU is added to the voltage U in simu- lation. We show in Fig 7d that, up to a noise level of σU/U ’ 0.1, a good control is achieved. This condition is not restrictive and is easily obtained with classical systems. A moderate noise does not seem to impact the quality of the learning process. Deep Q learning d) Statistics over 10 episodes initiated with θ0 between −10˚ and 10˚ of the plateau reward following the best learned policy. https://doi.org/10.1371/journal.pone.0280071.g006 https://doi.org/10.1371/journal.pone.0280071.g006 equidistant initial values of θ0 between −10˚ and 10˚. Statistics over these 10 episodes are rep- resented by a box-plot of the reward as a function of U (Fig 6d). It shows that the pendulum can operate and maintain a swing up for some values of θ0 even for U = 4.7V, but that this behavior becomes robust only for U 5.9V. Effect of the physical parameters. In what follows, we numerically investigate the robust- ness of the learning process with respect to the two frictions coefficients and to the two sources of noise. In Fig 7a, the static friction is varied from 0 to 11.7 N.kg−1, keeping the other parameters constant. We observe that the value 1.17 N.kg−1 measured with the real system does not per- turb the learning process in comparison to a system without friction. However, increasing ten- fold this parameter value prevents the system from learning correctly. In Fig 7b, the viscous friction is varied from 0 to 0.70 N.kg−1. Again the experimental value 0.07 N.s.rad−1 exhibits a good learning performance but multiplying this value by 10 would prevent the agent to drive the pendulum to the target. As mentioned in the experimental setup description, the real system has uncertainties asso- ciated to the measurement of the angle θ. In the virtual environment, this is accounted for by Gaussian noises of standard deviations σθ and σθ/Δt for the measurements of θ and _y respec- tively. From the real system, we have evaluated σθ 2.6 mrad. Here we probe values ranging between 0 and 175 mrad in simulation (Fig 7c). Clearly, low measurement noises, i.e., σθ < 8.7 PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 12 / 15 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes Fig 7. Influence of the physical parameters on the control: Inference curves of a) static friction, b) viscous friction, c) measurement noise and d) action noise. https://doi.org/10.1371/journal.pone.0280071.g007 Fig 7. Influence of the physical parameters on the control: Inference curves of a) static friction, b) viscous friction, c) measurement noise and d) action noise Fig 7. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 Author Contributions Formal analysis: Guillaume Allibert, Christophe Raufaste, Me´de´ric Argentina. Investigation: Sardor Israilov, Li Fu, Jesu´s Sa´nchez-Rodrı´guez, Franco Fusco, Guillaume Alli- bert, Christophe Raufaste, Me´de´ric Argentina. Investigation: Sardor Israilov, Li Fu, Jesu´s Sa´nchez-Rodrı´guez, Franco Fusco, Guillaume Alli- bert, Christophe Raufaste, Me´de´ric Argentina. Software: Franco Fusco. Software: Franco Fusco. Writing – original draft: Sardor Israilov, Li Fu, Jesu´s Sa´nchez-Rodrı´guez, Franco Fusco, Guil- laume Allibert, Christophe Raufaste, Me´de´ric Argentina. Writing – original draft: Sardor Israilov, Li Fu, Jesu´s Sa´nchez-Rodrı´guez, Franco Fusco, Guil- laume Allibert, Christophe Raufaste, Me´de´ric Argentina. Writing – review & editing: Sardor Israilov, Li Fu, Jesu´s Sa´nchez-Rodrı´guez, Franco Fusco, Guillaume Allibert, Christophe Raufaste, Me´de´ric Argentina. Conclusion In this article, we have revisited in a pedagogical context, the stabilization of an inverted pen- dulum, a classical problem in dynamics and control theory. We first recalled the physical model of such a system and the control objective. Two model-free Reinforcement Learning algorithms were investigated both in experiments and in simulations, which offers an accurate description of real experiments. In terms of the control quality, the basic Q-Learning method is found not efficient while the more advanced algorithm DQN successfully accomplishes the stabilization of the pendulum in its unstable position, independently of the initial condition. Finally, we studied the influence of some extensive physical parameters on the control quality in simulation with the virtual environment. The robustness of the DQN approach has been therefore validated, both in terms of parameter range, but also in terms of initial conditions: the RL always drives the pendulum in its unstable position, independently of the initial state. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 13 / 15 PLOS ONE Reinforcement learning approach to control an inverted pendulum: A general framework for educational purposes An admissible range of physical parameters were determined, which can be used to guide the elaboration of experimental setups. Meanwhile, we deliberately chose to use discrete actions for simplicity, but there exists many other RL algorithms which can work with continuous action spaces, for instance the Soft Actor-Critic (SAC) algorithm [26]. Using continuous action space unquestionably enables a finer control, but it would take more resources and time to train the RL model due to addi- tional complexity, and is less suitable for the scope of this article. For public outreach, we provide all the details in an open-source code repository. S1 File. Supplementary material to the manuscript. (PDF) S1 Video. The learning process and the quality of the control for the pendulum. (MP4) S1 Video. The learning process and the quality of the control for the pendulum. (MP4) Supporting information S1 File. Supplementary material to the manuscript. (PDF) PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 References 1. Lundberg KH, Barton TW. History of Inverted-Pendulum Systems. IFAC Proceedings Volumes. 2010 Jan; 42(24):131–135. https://doi.org/10.3182/20091021-3-JP-2009.00025 2. Boubaker O. The inverted pendulum benchmark in nonlinear control theory: a survey. 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Kim H, Jordan M, Sastry S, Ng A. Autonomous Helicopter Flight via Reinforcement Learning. In: Thrun S, Saul L, Scho¨lkopf B, editors. Advances in Neural Information Processing Systems. vol. 16. MIT Press; 2004. 12. Sutton RS, Barto AG. Reinforcement learning: An Introduction ( Second edition). The MIT Press, Sec- ond edition; 2012. 13. Huang J, Ding F, Fukuda T, Matsuno T. Modeling and velocity control for a novel narrow vehicle based on mobile wheeled inverted pendulum. IEEE Transactions on Control Systems Technology. 2012; 21 (5):1607–1617. https://doi.org/10.1109/TCST.2012.2214439 14. Sun W, Su SF, Xia J, Wu Y. PLOS ONE \| https://doi.org/10.1371/journal.pone.0280071 February 13, 2023 References Adaptive tracking control of wheeled inverted pendulums with periodic dis- turbances. IEEE Transactions on Cybernetics. 2018; 50(5):1867–1876. https://doi.org/10.1109/TCYB. 2018.2884707 PMID: 30582561 15. OpenAI Gym;. https://gym.openai.com/. 16. 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https://openalex.org/W2995134587	https://www.intechopen.com/citation-pdf-url/68597	English	null	Rare Disease Advocacy Groups and Their Significance in Diagnosis, Management, Treatment, and Prevention of Rare Diseases	IntechOpen eBooks	2,020	cc-by	3,429	Rare Disease Advocacy Groups and Their Significance in Diagnosis, Management, Treatment, and Prevention of Rare Diseases Yashodhara Bhattacharya, Gayatri Iyer, Aruna Priya Kamireddy, Subhadra Poornima, Keerthi Konda Juturu and Qurratulain Hasan Abstract Rare diseases are those diseases that are not seen frequently in a population. There are about 7000 rare diseases that have been identified worldwide, and 80% of them are caused by genetic changes. Since a small number of individuals are affected with rare diseases, most clinicians are not aware of such diseases, and thus, they remain undiag- nosed and untreated. Awareness regarding such diseases is essential to train clinicians to diagnose individuals affected with these disorders and to develop National/International Registries, which will serve to give information about the disease prevalence, its natural course, treatment, and management options available, to the medical fraternity. Patient advocacy groups play a remarkable and unique role in forming the collective voice of individuals living with rare diseases. They help in the identification, diagnosis, manage- ment, treatment, and prevention of such diseases. Advocacy Groups form collaborative partnerships with scientists studying such rare diseases, clinicians managing these diseases, pharmaceutical companies developing drugs, and Government officials over- seeing and policy makers implementing medical regulatory processes. Thus, advocacy groups play a key role in helping patients and families with rare diseases. Keywords: rare disease, patient advocacy group, rare disease registry, diagnosis, management, treatment • Non-availability of treatment for the disease in that country The USA defines a rare disease as a condition that affects less than 200,000 people. The definition of rare diseases as is defined in the USA was coined in the Congress during the Orphan Drug Act of 1983. Such diseases also came to be known as Orphan Diseases as drug companies were indifferent to adopting the research and manufacture of novel drugs for their treatment [2, 3]. The World Health Organization defines a rare disease as a disease with a frequency of less than 6.5–10 per 10,000 people. In Europe, it is defined as a disease seen in less than 5 of every 10,000 people, while in Australia, it is taken as one in 10,000 individuals, and in India, it is taken as one in 5000 individuals [4, 5]. A rare disease in isolation may affect a small population because of which clinicians are not aware of the disease and their symptoms, hence such individuals remain undiagnosed and untreated [1, 6, 7]. Although individu- ally these disorders are rare, when taken together the people affected with rare diseases constitute a large population of the country. Such diseases are debilitat- ing and without a proper diagnosis may cause gross morbidity and mortality, thereby posing a challenge to the healthcare system of the country [8]. A rare disease has an adverse impact on the everyday lives of the whole family and their care givers [9–12]. The cost of treatment/management is high and causes considerable financial burden to the individuals and their families [8, 11, 12], as there is a lack of Government policies regarding this aspect, hindering their treatment. 1. Introduction A rare disease is so called because its frequency in any given population is very low [1]. There are about 7000 rare diseases that have been discovered, and more are being described in medical literature. Rare diseases have different causes, and about 80% of them have a genetic basis that could be chromosomal or genomic [2]. Rare diseases also include certain rare infections, cancers, and even autoimmune disor- ders. A rare disease is defined differently in individual countries and is based on the following parameters: 1 Rare Diseases Rare Diseases • The total number of people affected by the disease in that country • Prevalence of the disease in that country • Non-availability of treatment for the disease in that country 2. Advocacy groups Since several rare diseases are being diagnosed and brought into light, it is required that more time and effort should go into research for understanding and preventing such diseases. A remarkable and unique aspect of rare disease treatment and management is the evolving role of advocacy groups and their collaborative partnerships with scientists studying such diseases, pharmaceuti- cal companies developing drugs, and Government officials and policy makers overseeing medical research and health care [3, 6]. Rare disease advocacy groups have played a vital role over the years in the adoption of public policies, relocation of available research funding, and other factors affecting the research for rare diseases [1, 7]. In most settings, the rare disease advocacy groups are created by the family members of the affected individuals. They are the ones who look into the formation of public policies, help fast-track treatment approvals by regulatory bodies, and facilitate the welfare of individuals and their care givers. The National Organization for Rare Disorders (NORD) in the USA was one of the first advocacy groups to be formed, followed by Rare Diseases International, which is a global alliance of patients with rare diseases across various nationali- ties and is dedicated for supporting treatment and formulating policies for rare disorders. Apart from these, patient advocacy groups have been formed all over the world, which individually or in alliance help to alleviate the various problems faced by individuals with rare diseases and to pressurize companies and countries to provide life-saving drugs and at a reasonable cost. 2 Rare Disease Advocacy Groups and Their Significance in Diagnosis, Management, Treatment… DOI: http://dx.doi.org/10.5772/intechopen.88630 Rare Disease Advocacy Groups and Their Significance in Diagnosis, Management, Treatment… DOI: http://dx.doi.org/10.5772/intechopen.88630 3. Patient advocacy groups in India There are many patient advocacy groups currently active in India. The Pompe Foundation started by Mr. Prasanna Shirol in 2008 and caters to individuals and families affected with Lysosomal Storage Disorders. The Metabolic Errors and Rare Diseases Organization of India (MERD), founded by Mr. Vikas Bhat, promotes awareness regarding Inborn Errors of Metabolism and newborn screen- ing. Both these organizations have been founded by parents having an affected child. There are a few other advocacy groups for Spinal Muscular Atrophy (SMA), Spino-Cerebellar Ataxia (SCA), Duchenne Muscular Dystrophy (DMD), and Osteogenesis Imperfecta. Twenty-five such organizations together joined hands to form the Organization for Rare Diseases India (ORDI), which is actively involved in helping patients and their families through the involvement of NGOs. These advocacy groups, however, need to be better organized, so that they can obtain and disseminate information about diseases, diagnostics, and treatment avenues to the affected families. India has a huge diversity in the kind of rare diseases seen in different states, which can be attributed to certain cultural practices such as consanguinity in South India and endogamy in the North [13]. Based on the data from these organizations, a Rare Disease Registry has been initiated. This has helped in re-classifying rare diseases based on their prevalence in different states. Diseases like β-thalassemia are more prevalent in Punjab, Gujarat, West Bengal, Odisha, and Andhra Pradesh but are rare in other states [14, 15]; hence, they cannot be classified under rare diseases in these states. Similarly, house-to-house survey carried out by Molecular Diagnostics, Counseling, Care and Research Center (MDCRC) Coimbatore esti- mates that the prevalence of Duchenne Muscular Dystrophy in Tamil Nadu is high and cannot come under a rare disorder in that state [16]. Gradually, such data need to be combined, so that advocacy groups can focus their efforts on rare diseases and would help in developing a comprehensive and factual National registry, which would further aid in framing the National Policy for Rare Diseases [17, 18]. 4. Multi-specialty hospital-based advocacy group In this chapter, we would like to highlight our study that was to evaluate the feasibility of initiating an advocacy group for rare diseases in a multi-specialty hospital setting with the support of the Department of Genetics and Molecular Medicine. The genetic counselors were instrumental in liaising between different departments such as pediatrics, nephrology, neurology, orthopedics, and oncology for the identification of patients with suspected rare disease. Around 200 such patients were identified during the period of April 2016 to April 2019. The patient families were encouraged to register with the advocacy group, which would support and follow-up the patient and their families and provide the necessary management and treatment options as required. p q Patients evaluated were identified and categorized based on age into the pedi- atric and the adult age group. About 63% of the patients were in the pediatric age group, and the remaining 37% were in the adult age group. 4.1.1 MAITRI (bond of friendship) – Kamineni Hospital’s rare disease advocacy group The Department of Genetics and Molecular Medicine along with its team of genetic counselors created Maitri to collectively address the various problems faced by individuals suffering from rare diseases. y g Objectives of Maitri: y g Objectives of Maitri: • To raise awareness about rare diseases among healthcare professionals, general public, and policy makers • To evaluate and diagnose an individual with a suspected rare disease • To identify individuals with rare diseases at Kamineni Hospital and include them in the Kamineni Rare Disease Registry • To counsel the patient, parents, and family about the disease and its prognosis, management, and treatment options (if any) • To identify individuals in the extended family at risk of having the disease • To create self-help groups of individuals with similar symptoms/problems for discussion and possible management options • To help cope with psychosocial issues • To conduct parental/family group sessions about the different rare diseases and on the various psychosocial problems faced by them • To involve teachers from the pre-school level, school, special schools, and college level in the advocacy group. To create awareness among them and other teaching staff about dealing with an individual affected with rare diseases to integrate individuals into the mainstream schools/colleges and allow them to lead a normal life. 4.1 Rare disease advocacy group at Kamineni Hospital To cater to patients affected with such diverse diseases, the first hospital-based advocacy group was created at Kamineni Hospital, a multi-specialty hospital located in the cosmopolitan city of Hyderabad in South India. It was named Maitri, 3 Rare Diseases which originates from the Sanskrit word meaning “friendship.” Maitri looks into the collective interests of individuals with rare diseases. The rare disease community is often denied the most basic of rights. Society is ill-equipped to understand the cause and gravity of the diseases. This often leads to a number of psychological problems. A diagnosis is important to understand a disease, its progression, symp- toms, possible treatment options, and also for its prevention in future generations. Most individuals can lead a normal life. However, due to the lack of awareness, such individuals are not allowed to do so. People in general lack the sensitivity to accept and work alongside individuals suffering from such diseases. Maitri aims to change this scenario by raising awareness among clinicians, the general public, in schools, and colleges. It also looks into extended family screening and counseling for making informed reproductive decisions. a. Pediatric age group: Age 0–5 years: Parental discussions on symptom management, addressing vari- ous needs of the child. Refer for clinical follow-up and psychological evaluation of the child prior to school admission. p Age 6–15 years: Addressing psychological needs of the patient. Group discussion programs with teachers, parents, and other healthcare professionals. Address issues faced at school and mitigate it. 4.2 General workflow of Maitri Every patient visiting the Department of Genetics and Molecular Medicine was evaluated in two or more sessions as a part of primary evaluation (Figure 1). 4 Rare Disease Advocacy Groups and Their Significance in Diagnosis, Management, Treatment… DOI: http://dx.doi.org/10.5772/intechopen.88630 Rare Disease Advocacy Groups and Their Significance in Diagnosis, Management, Treatment… DOI: http://dx.doi.org/10.5772/intechopen.88630 Figure 1. Flowchart for the working of Maitri. Figure 1. Flowchart for the working of Maitri. g Flowchart for the working of Maitri. g Flowchart for the working of Maitri. g Flowchart for the working of Maitri. Patients were segregated based on the following age groups and advised accordingly • Individual patients requiring vocational, behavioral, or occupational therapy are referred for the same 5. Conclusion Hospital-based Rare Disease Advocacy Groups like Maitri are crucial in a number of ways they help in establishing prevalence of rare disorders through the hospital-based registries. They promote awareness, so that every affected individual may be tested for a diagnosis. Such advocacy groups help bring together families with same, similar, or even different rare diseases, so that they can help and sup- port each other. Groups like Maitri also perform a very important role in extended family screening, wherein they identify individuals at risk of developing the disorder and are counseled regarding appropriate testing and preventive measures. It also encourages reproductive genetic counseling for the families with an affected individual, which would help prevent the disorder in the future generations. p p g Advocacy groups along with policy makers are instrumental in creating public awareness about such diseases. Increase in awareness would make their prevention a public health concern, thus making it mandatory to have definitive screening and preventive strategies in the country. Not only advocacy groups are important for the support of individual families, but they also play a role in mass awareness and prevention of rare diseases. b. Adult age group: Age 16–22 years: Patient and parental psychological consultation to address vari- ous psychosocial issues. One-on-one patient meetings with geneticist and psycholo- gist to address puberty-related problems. Reproductive counseling is also given. g p y p p g g Age 23–40 years: Pre-marital, pre-pregnancy genetic counseling, group sessions with other patients and their family to address common issues. Refer for clinical follow-up if needed. Age > 40 years: Group sessions for patient and families for discussion of symp- toms, their management, possible treatment options, and psychosocial problems faced due to the condition. Pre-symptomatic genetic testing and subsequent advice for children of individuals affected with rare diseases. • Individual patients requiring vocational, behavioral, or occupational therapy are referred for the same • Individual patients requiring vocational, behavioral, or occupational therapy are referred for the same 5 Rare Diseases The importance of a rare disease advocacy group at the National and International level has been established by many esteemed clinicians, geneticists, and social workers. However, such groups at a hospital setting are important in a country like India, where there are limited electronic medical records, and there is huge literary and financial disparity in the population, such advocacy groups may contribute to maintaining crucial information for providing better healthcare and support to patient families. Acknowledgements We would like to thank Kamineni Hospitals Ltd. for the support they have extended to us for this project. We would also like to thank all the clinicians and other healthcare providers who have referred patients to the Department of Genetics and Molecular Medicine. They have been instrumental in the identifica- tion and diagnosis. We would also like to thank our patients for the support and cooperation. Conflict of Interest There was no conflict of interest for the given project. 6 Rare Disease Advocacy Groups and Their Significance in Diagnosis, Management, Treatment… DOI: http://dx.doi.org/10.5772/intechopen.88630 Rare Disease Advocacy Groups and Their Significance in Diagnosis, Management, Treatment… DOI: http://dx.doi.org/10.5772/intechopen.88630 References [8] Valdez R, Ouyang L, Bolen J. Public health and rare diseases: Oxymoron no more. Preventing Chronic Disease. 2016;13:150491. DOI: 10.5888/ pcd13.150491 [1] Choudhury MC, Saberwal G. The role of patient organizations in the rare disease ecosystem in India: An interview based study. 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Rare Disease Advocacy Groups and Their Significance in Diagnosis, Management, Treatment… DOI: http://dx.doi.org/10.5772/intechopen.88630 Author details Yashodhara Bhattacharya, Gayatri Iyer, Aruna Priya Kamireddy, Subhadra Poornima, Keerthi Konda Juturu and Qurratulain Hasan* Department of Genetics and Molecular Medicine, Kamineni Hospitals, Hyderabad, India Address all correspondence to: qhasan2000@yahoo.com © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ Address all correspondence to: qhasan2000@yahoo.com © 2019 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. 7 Rare Diseases [14] Mohanty D, Colah RB, Gorakshakar AC, Patel RZ, Master DC, Mahanta J, et al. Prevalence of β-thalassemia and other haemoglobinopathies in six cities in India: A multicentre study. Journal of Community Genetics. 2013;4:33-42. DOI: 10.1007/s12687-012-0114-0 [13] Tamang R, Singh L, Thangaraj K. Complex genetic origin of Indian populations and its implications. Journal of Biosciences. 2012;37:911-919 References DOI: 10.1007/978-90-481-9485-8_28 [13] Tamang R, Singh L, Thangaraj K. Complex genetic origin of Indian populations and its implications. Journal of Biosciences. 2012;37:911-919 [13] Tamang R, Singh L, Thangaraj K. Complex genetic origin of Indian populations and its implications. 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https://openalex.org/W4240472546	https://www.researchsquare.com/article/rs-3893/latest.pdf	English	null	Activating the interleukin-6-Gp130-STAT3 pathway ameliorates ventricular electrical stability in myocardial infarction rats by modulating neurotransmitters in the paraventricular nucleus	Research Square (Research Square)	2,020	cc-by	6,048	Activating the interleukin-6-Gp130-STAT3 pathway ameliorates ventricular electrical stability in myocardial infarction rats by modulating neurotransmitters in the paraventricular nucleus Meng Gao First Affiliated Hospital of Harbin Medical University Dechun Yin First Affiliated Hospital of Harbin Medical University Jugang Chen First Affiliated Hospital of Harbin University Xiufen Qu (  xfqu_hmu@163.com ) First Affiliated Hospital of Harbin Medical University https://orcid.org/0000-0002-3237-546X Research article Keywords: hypothalamic paraventricular nucleus; interleukin-6; glycoprotein 130; STAT3; sympathetic activity; cardiac electrophysiological activity. Posted Date: January 27th, 2020 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 BMC Cardiovascular Disorders on February 5th, 2020. See the published version at https://doi.org/10.1186/s12872-020-01363-x. Page 1/16 Abstract Background: Malignant ventricular arrhythmia (VA) is the most common cause of death associated with acute myocardial infarction (MI). Recent studies have revealed direct involvement of the paraventricular nucleus (PVN) in the occurrence of VA. However, the underlying mechanisms remain incompletely understood. In this study, we investigated changes in the interleukin-6 (IL-6)-glycoprotein 130-signal transducer and activator of transcription 3 (STAT3) pathway in the PVN during acute MI and the effects of this pathway on ventricular stability. Methods: Rats were divided into a control group, a MI group, a PVN-injected anti-IL-6 antibody group and a PVN-injected SC144 group to observe how IL-6 and its downstream glycoprotein 130-STAT3 pathway in the PVN affect ventricular stability. The left anterior descending coronary artery was ligated to induce MI. After that, an anti-IL-6 antibody and SC144 were injected into the PVNs of rats. All data are expressed as the mean ± SE and were analysed by ANOVA with a post hoc LSD test. p<0.05 was considered to indicate statistical significance. Results: After MI, the concentration of the inflammatory factor IL-6 increased, and its downstream glycoprotein 130-STAT3 pathway was activated in the PVN. After injection of MI rat PVNs with the anti-IL-6 antibody or glycoprotein 130 inhibitor (SC144), glutamate levels increased and γ-aminobutyric acid (GABA) levels decreased in the PVN. Plasma norepinephrine concentrations also increased after treatment, which increased the vulnerability to VA. Conclusions: In summary, IL-6 in the PVN exerts a protective effect in MI rats, and the glycoprotein 130-STAT3 pathway plays a key role in this process. We anticipate that our findings will provide new ideas for the prevention and treatment of arrhythmia after MI. Background Acute myocardial infarction (MI) is a condition of myocardial necrosis caused by acute, persistent ischaemia and hypoxia in the coronary arteries [1]. There are some complications of MI, including heart failure, arrhythmia, heart rupture, pericarditis, papillary muscle rupture and others. Arrhythmia occurs in most MI patients and most commonly occurs within 24 hours [2]. Furthermore, lethal ventricular arrhythmia (VA) is the most common cause of death among patients with acute MI. It is well known that autonomic imbalance, especially excessive activation of sympathetic nerves (called a sympathetic storm), plays the most important role in promoting the occurrence of arrhythmia. In recent years, there have been many reports on the mechanisms by which peripheral autonomic nerves, such as local cardiac nerves, renal sympathetic nerves, and star ganglions, regulate arrhythmia [3, 4]. However, the mechanism by which the central nervous system (CNS) affects VA remains unclear. Lampert et al. have demonstrated that ventricular tachycardia and ventricular fibrillation (VF) can be induced by psychological stress, sudden changes in mental state, brain trauma, and elevated intracranial pressure [5]. Davis et al. have demonstrated that brain tissue regions and nuclei from the medulla to the cerebral cortex play important roles in the development of arrhythmia and revealed that there are complex and variable interconnections among these areas [6]. Stimulation of different brain regions and nerve nuclei can lead to different types of arrhythmia. Among these regions, the paraventricular nucleus (PVN) is the main area of sympathetic preganglionic neuron accumulation and innervates other autonomic Page 2/16 nuclei, including the midbrain periaqueductal grey region, the parabrachial region, the rostral ventrolateral medulla, the solitary tract nucleus, the dorsal vagal nucleus and the nucleus ambiguus. Moreover, the PVN is an important integrative site within the brain composed of magnocellular and parvocellular neurons. Parvocellular neurons project to other sites within the CNS, including regions that are important for autonomic control [7, 8]. However, the exact mechanism by which the PVN affects arrhythmia remains unclear and needs further investigation. Changes in neurochemical factors, such as reactive oxygen species and inflammatory cytokines, in the hypothalamic PVN during MI may be important factors in the increase in sympathetic nerve sensitivity that occurs during MI. Kang et al have shown that microinjection of pro-inflammatory cytokine inhibitors into the CNS can alleviate the symptoms of MI and that the effects of central administration are significantly better than those of peripheral administration [9, 10]. Background Neurotransmitters play important roles in this process. For example, glutamate is enhanced and γ- aminobutyric acid (GABA) declines in the PVN during MI, thereby affecting sympathetic overactivation and further affecting heart function [11]. Glutamate, one of the most important excitatory amino acids in the CNS, regulates sympathetic nerve activity and cardiovascular function through N-methyl-D-aspartic acid (NMDA) receptors. Stimulation of NMDA receptors in the PVN can increase sympathetic discharge. GABA is the main inhibitory neurotransmitter in the PVN of the hypothalamus. Injecting GABA into the PVN of the hypothalamus can reduce heart rate and attenuate arrhythmia. GAD67 is a rate-limiting enzyme of GABA and a marker for GABAergic neurons, and its distribution is parallel to that of GABA. In contrast to other inflammatory factors, interleukin-6 (IL-6) is a pleiotropic regulator that has multiple functions, not only exerting pro-inflammatory effects but also affecting tissue regeneration, metabolism and other processes. IL-6 is upregulated significantly during acute injury and plays key roles in mediating the acute-phase response. IL-6 has two kinds of receptors, a membrane-bound receptor and a soluble receptor, both of which can bind to Glycoprotein 130 (Gp130). After dimerization, intracellular signalling occurs through IL-6 classic signalling and trans-signalling pathways. Interestingly, these two pathways strongly differ in their biological influences. While classic signalling is primarily associated with protection, promoting tissue regeneration and maintaining physiological homeostasis, trans-signalling has deleterious effects [12]. With regard to the CNS, Suzuki et al. demonstrated that IL-6 plays a protective role in the early stage of brain injury. Intracerebroventricular injection of rhIL-6 dramatically reduces ischaemic brain damage measured 24 hours after middle cerebral artery occlusion [13, 14, 15]. Gp130 is a receptor of IL-6 and is the main signalling molecule for intracellular signal transduction. Currently, three signalling pathways are known to be associated with Gp130: the JAK-signal transducer and activator of transcription (STAT) pathway, the EKA pathway, and the PI3K/Akt pathway. The most prominent proteins recruited to Gp130 are the STAT family transcription factors STAT3 and (to a certain extent) STAT1. Furthermore, it is currently well accepted that STAT3 and (to a much lesser extent) STAT1 are activated by IL-6. Binding to IL-6 causes phosphorylation of Gp130 and then activates the cytoplasmic region [16]. Gp130 phosphorylation exposes a STAT3 binding site to induce STAT3 phosphorylation and then enters the nucleus to initiate transcription. Habecker et al. Cardiac electrophysiological studies After the surgery, we recorded the arrhythmia occurrence in rats within 24 hours using a single-lead dynamic electrocardiogram (Good Friend, Shenzhen, China). The rats were anaesthetized by i.p. injection of Ulatan (150 mg/kg), and a second thoracotomy was carried out to perform an open-chest electrophysiological study for assessment of endpoints including the VF threshold (VFT) and VF inducibility. A 1.9 F electrophysiological catheter (Scisense, Canada) was placed on the left ventricle, and eight poles recorded electrocardiograms with an Electrophysiology Lab Amplifier (GY-6000, Huanan Medical Science and Technology, Henan, China). To determine the VFT, the minimum voltage to induce sustained VF, 60 ms S1-S1 stimuli were repeatedly applied to the left ventricular apex, and the stimulus intensity was increased by 0.5 V each time until VF was induced. Ten bursts of ventricle pacing (25 Hz) lasting for 10 s each were used to assess the inducibility of VF. VF was defined as >1000 ms of irregular VA. Coronary ligation and paraventricular nucleus injection (PNI) [20, 21] The rats underwent sterile surgery under anaesthesia (Ulatan，concentration：20%, 150 mg/kg, intraperitoneal [i.p.]) for induction of MI by ligation of the left anterior descending coronary artery (MI group) or the same surgery without ligation of the vessel (sham group). The PVN in each rat was injected with artificial cerebrospinal fluid (ACSF; given to sham rats and MI rats), an anti-IL-6 antibody or a Gp130 antagonist (SC144) according to the rat stereotaxic atlas coordinates, and each group included 12 rats (n=12) [22, 23, 24]. Animals Adult male Sprague-Dawley rats (200-250 g) were purchased from the Animal Experimental Center of the Second Affiliated Hospital of Harbin Medical University. The rats were housed at a density of 8 rats per cage with 12 hours of light and freely available food and water at a temperature 23±2°C and a relative humidity of 40%-50%. Background confirmed that Gp130 mediates the conversion of peripheral sympathetic neurons to cholinergic neurons after MI [17]. The sympathetic co-release of acetylcholine (Ach) and norepinephrine (NE) impairs adaptation to high Page 3/16 Page 3/16 heart rates and increases arrhythmia susceptibility. In the CNS, the Gp130 pathway promotes the differentiation and growth of nerves [18, 19]. However, the effect of Gp130 on neurotransmitter conversion in the PVN has not been studied. heart rates and increases arrhythmia susceptibility. In the CNS, the Gp130 pathway promotes the differentiation and growth of nerves [18, 19]. However, the effect of Gp130 on neurotransmitter conversion in the PVN has not been studied. The aim of this study is to investigate whether IL-6 in the hypothalamic PVN exerts a protective effect against the incidence of VA after MI and whether the Gp130-STAT3 pathway plays a key role in this process. Western blot analysis The tissue was homogenized in RIPA buffer containing a protease inhibitor cocktail (Beyotime Biotechnology). A BCA protein assay (Beyotime Biotechnology) was used to determine the protein concentrations. Equal amounts of protein were separated by SDS-PAGE and then transferred electrophoretically to polyvinylidene fluoride membranes (Bio-Rad) [29, 30]. The membranes were incubated with the following primary antibodies for 2 hours at room temperature: IL-6 (1:1000, Abcam, England), Gp130 (1:1000, Santa, America), pSTAT3 (1:1000, Bioss, China), and NMDA receptors (1:1000, Bioss, China). The membranes were then incubated with GAPDH (1:1000, Solarbio, China), goat anti- mouse IgG (Bioss, China, Gp130, 1:1000 and GAD67, 1:1000) or goat anti-rabbit IgG (Bioss, China, pSTAT3, 1:1000, NMDA receptors, 1:3000 and IL-6, 1:1000) secondary antibodies for 2 hours at room temperature. Finally, the membranes were placed in a gel imaging analysis system for exposure and analysis (AlphaView FluorChem FC3). Immunohistochemistry After the brain tissue was embedded in paraffin, the part between the optic chiasm and mammillary body was resected in the rostro-caudal direction. The tissue was serially sectioned on a paraffin slicer, and sections that were approximately 1.50 mm from the bregma were obtained [25, 26]. After dewaxing the slices, 3% H2O2 was used to block endogenous peroxidase activity, and 0.01 M citric acid was used to retrieve the antigens prior to antibody incubation. Then, the slices were incubated with primary antibodies overnight at 4°C [27, 28]. The sections were immunohistochemically labelled to identify IL-6 (Bioss, China, 1:100), Gp130 (Santa Cruz, America, 1:20), pSTAT3 (Bioss, China, 1:50), NMDA receptors (Bioss, China, 1:50), and GAD67 (Abcam, England, 1:2000) and then incubated with secondary antibodies (anti-mouse for Gp130 and GAD67; anti-rabbit for NMDA receptors, pSTAT3 and IL-6) for 20 minutes at room temperature. For each rat, the positive neurons within the bilateral borders of the PVN were manually counted in three consecutive sections, and the average value is reported. Methods of animal euthanasia and tissue collection Page 4/16 Page 4/16 The animals were sacrificed by rapid excision of the heart to confirm permanent cessation of the circulation under anaesthesia (Ulatan, concentration：20%, 150 mg/kg i.p.). Then the rats were decapitated to get the whole brain. For western blotting, brain tissue was quickly extracted in a low- temperature environment, and Palkovits's microdissection procedure was used to isolate the PVN. For immunohistochemistry, 4% paraformaldehyde was inserted into the left ventricle and the ascending aorta to fix the brain tissue, and then the rats were decapitated to obtain the brains. Measurement of glutamate and GABA in PVN tissues Brain tissue was separated as previously described. Perchloric acid (0.1 mol/L, Sigma) was added to the brain tissue. Then, the tissue was dissolved on an ice pack or crushed ice, fully crushed and homogenized, and sonicated for 5 minutes. Finally, the samples were centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was aspirated, diluted and filtered through a filtration membrane. The Page 5/16 Page 5/16 concentrations of glutamate and GABA were measured using a liquid chromatograph mass spectrometer (Singapore, Xevo). Measurement of circulating catecholamine levels Arterial blood was drawn from the left heart chamber and centrifuged at 3,000 rpm for 15 minutes at 4 °C. The supernatant was obtained and stored in a freezer at -80 °C. An ELISA kit purchased from Bioss was used to measure NE levels. The standards were diluted and loaded for a total well volume of 50 μl. The standard concentrations were 120 ng/L, 80 ng/L, 40 ng/L, 20 ng/L, and 10 ng/L. The samples to be tested on the enzyme-labelled plate were first diluted; 40 μl and then 10 μl of each sample was added. Fifty microliters of enzyme labelling reagent were added per well. After sealing it with a sealing film, the plate was incubated at 37 °C for 30 minutes. The plate was zeroed with blank wells, and the absorbance of each well was measured in sequence at 450 nm and 630 nm wavelengths. The concentration in each sample was calculated based on the absorbance. Statistical analysis All analyses were carried out with Statistical Product and Service Solutions 17.0 (SPSS Inc, Chicago, II, USA). Normality testing was performed to evaluate whether the data conforms the normal distribution prior to the analysis. All data were expressed as the mean ± SEM, data which fit the normal distribution were analysed by ANOVA followed by a post-hoc LSD test. Statistical significance was accepted at p < 0.05 for all analyses. NMDA and GAD67 expression in the PVN Fig. 3A demonstrates the immunohistochemical staining for NMDA receptors in the four groups, and Fig. 3C shows the immunohistochemical staining for GAD67 expression in the four groups. Fig. 3B and D display the results of densitometric analysis for NMDA receptors and GAD67, respectively. Fig. 3E shows representative immunoblot image of NMDA receptors and GAD67 levels, which demonstrate that MI rats had higher NMDA receptors levels and lower GAD67 levels in the PVN than sham rats (p<0.01). PVN injections of the anti-IL-6 antibody and SC144 promoted an increase of NMDA receptors levels and a decrease of GAD67 levels within the PVNs of MI rats (p<0.05). Cardiac electrophysiological study Fig. 1A shows a typical graph of VF induced by an electrophysiological catheter. Fig. 1B displays a ventricular premature beat recorded by dynamic electrocardiography. As shown in Fig. 1C-E, MI rats showed a 4-fold greater incidence of spontaneous VA (5.5±0.8 vs 1.3±0.6) (p<0.01) compared with sham rats. The incidence of spontaneous VA (54.67±5.59 and 200.8 ±19.12) in anti-IL-6 antibody-treated and SC144-treated PNI rats was significantly higher than that in sham rats and MI rats (p<0.0001). To further study the changes in cardiac electrophysiology, we measured the catheter-induced VF and VFT in each group of rats. The induction rates of VF in MI rats were over 50% higher than those in sham rats (31.83±3.43% vs 13.33±1.41%) (p<0.01), while anti-IL-6 antibody-treated rats and SC144-treated PNI rats had about 2-fold and 3-fold induction rates of VF (58.17±5.74% and 81.33±6.01%, respectively) when compared with MI rats (p<0.0001). The VFT of MI rats (7.25±0.63 V), in comparison with sham rats (10.75±1.20 V), showed a 25% decrease (p<0.0001), and the VFT of PNI anti-IL-6 antibody-treated PNI rats and SC144-treated PNI rats (6.16±0.60V and 3.66±0.45V) lessened 15% and 40% than in MI rats (p<0.05). IL-6, Gp130 and pSTAT3 expression in the PVN IL-6, Gp130 and pSTAT3 expression in the PVN Page 6/16 Page 6/16 The immune system was activated when the left anterior descending coronary artery was ligated. The immunohistochemical images in Fig. 2A show the expression of IL-6 in the four groups and those in Fig. 2C illustrate the expression of Gp130 in the four groups. The expression of pSTAT3 in the four groups are displayed in Fig. 2E. Fig. 2B, D and F show the densitometric analysis results for IL-6, Gp130 and pSTAT3, respectively. Fig. 2G shows a representative immunoblot image of IL-6, Gp130 and pSTAT3 levels. From these images, we observed that compared to sham rats PVNs, MI rat PVNs had significantly higher IL-6 concentrations (p<0.0001). In addition, the elevations in IL-6 activated the Gp130 receptor (p<0.0001) and its downstream mediator pSTAT3 (p<0.01). With the reduction in IL-6 content upon injection of the anti-IL- 6 antibody, Gp130 and pSTAT3 activation was blunted (p<0.05). Gp130 and STAT3 activation was also blunted conspicuously by SC144, which could bind to Gp130 and eventually abrogate STAT3 phosphorylation and nuclear translocation. Fig. 2H-J show the densitometric analysis results for the protein expression of IL-6, Gp130 and pSTAT3. Plasma humoral factors To determine the sympathoexcitatory effects of MI and PVN infusion, we measured plasma NE levels in blood using ELISA. Fig. 4C shows that, as expected, MI rats showed 1.5-fold higher plasma NE levels than sham rats (412.7±16.5 pg/ml vs 630.5±21.2 pg/ml) (p<0.0001). Additionally, anti-IL-6 antibody-treated PNI rats and SC144-treated PNI rats had about 1.3-fold and 1.5-fold higher levels of plasma NE (850.5±23.2 pg/ml and 1002.0±29.9 pg/ml, respectively) than MI rats did (p<0.0001) (Fig. 4). Neurotransmitters in the PVN As shown in Fig. 4A and B, we observed significant differences in the levels of excitatory and inhibitory neurotransmitters in the PVNs of MI and PNI rats compared to those of sham rats. In comparison with sham rats, MI rats had 6-fold higher levels of glutamate (0.64 ±0.08 vs 0.90±0.09 μg/mg, p<0.05) and 1.3-fold lower levels of GABA in the PVN (304.0±12.0 vs 232.3±9.1 ng/mg, p<0.0001). Furthermore, anti- IL-6 antibody-treated PNI rats and SC144-treated PNI rats had 2-fold and 4-fold higher levels of glutamate (1.28±0.09 μg/mg and 2.57±1.13 μg/mg, respectively) (p<0.0001), and 1.4-fold and 1.7-fold lower levels of GABA (165.9±8.2 ng/mg and 135.2±8.1 ng/mg, respectively) (p<0.0001) in the PVN than MI rats did. Discussion Page 7/16 Page 7/16 The novel finding of the present study is that changes in IL-6 and its downstream molecules Gp130 and STAT3 induce an imbalance between excitatory and inhibitory neurotransmitters and their rate-limiting enzymes in the PVN in MI rats, which contributes to sympathoexcitation and the incidence of VA. The novel finding of the present study is that changes in IL-6 and its downstream molecules Gp130 and STAT3 induce an imbalance between excitatory and inhibitory neurotransmitters and their rate-limiting enzymes in the PVN in MI rats, which contributes to sympathoexcitation and the incidence of VA. CNS diseases can induce multiple types of arrhythmia, including ventricular tachycardia and VF. Exploring CNS-related ventricular premature contractions is of great significance for clinical work. Thus, the PVN may be a potential target for the prevention and treatment of VA in patients with acute MI. Neuroanatomical studies have shown that the PVN sends direct projections to spinal preganglionic neurons of sympathetic ganglia. Stimulation of cell bodies in the PVN increases blood pressure, heart rate and circulating NE concentrations [31]. Injecting glutamate or a GABA antagonist into the PVN increases renal nerve activity and circulating NE concentrations, suggesting that a sympathoadrenal component to cardiovascular responses is associated with PVN stimulation [32]. Recent research has demonstrated that pathophysiological changes in the PVN are undoubtedly critical to the elevated sympathetic nerve activity in MI. In response to MI, microglia in the PVN become activated and secrete cytokines. In this study, we observed increased expression of IL-6 and activation of Gp130 and STAT3 in PVN neurons of MI rats. Interestingly, while previous studies have demonstrated that other cytokines in the PVN, such as TNF-α and IL-1, play devastating roles, our study indicated that IL-6 in the PVN plays a protective role. When MI rats were treated with an IL-6 antagonist, the sympathetic outflow increased. The role of IL-6 is complicated during inflammation, which contributes to both injury and repair processes. However, the peak in IL-6 expression at 24 hours is associated with neuroprotection [12]. Many studies have shown that IL-6 dose-dependently protects neurons against NMDA toxicity. Activation of NMDA receptors can increase sympathetic discharge. In our study, we observed that blocking IL-6 increased glutamate concentrations and elevated NMDA receptor expression in the PVN, whereas it decreased GABA concentrations and reduced GAD67 expression in the PVN. Moreover, the Gp130-STAT3 pathway plays a key role in this process. Discussion Treatment of MI rats with a Gp130 antagonist (SC144) gave rise to the same changes in neurotransmitters in the PVN as treatment with the anti-IL-6 antibody. This effect led to an increase in sympathetic outflow with increased incidence of VA. Gp130, a common signal-transducing receptor subunit, acts in association with ligand-specific receptors of IL-6. The drugs currently used in the clinic to antagonize IL-6 mostly target IL-6 and IL-6R. However, detrimental side effects, such as bacterial infections, can occur. In this trial, we chose SC144, a novel specific small-molecule inhibitor of Gp130, to block the signal of IL-6, as Gp130 is a new target for IL-6 signalling inhibition [33]. The intracellular signal transduction induced by IL-6 involves the activation of JAK tyrosine kinase family members, leading to the activation of transcription factors of the STAT family. STAT3 is an important element in the JAK-STAT pathway. The phosphorylation of STAT3 at Tyr705 in response to Gp130-stimulating cytokines leads to the formation of STAT3 dimers followed by the translocation of these dimers to the nucleus, where they regulate the transcription of target genes [16]. Increases in cholinergic genes within the stellate ganglion and widespread coexpression of the ChAT protein in TH+ neurons have been detected in MI rats. The acquisition of cholinergic function requires the Page 8/16 expression of the Gp130 cytokine receptor in sympathetic neurons. Removal of Gp130 from sympathetic neurons also prevents local noradrenergic transmission in the left ventricle after acute MI. In this study, Gp130 played the same role in the PVN by transforming glutamate into GABA and inducing an imbalance between excitatory and inhibitory neurotransmitters, thereby further affecting the outflow of sympathetic activity. expression of the Gp130 cytokine receptor in sympathetic neurons. Removal of Gp130 from sympathetic neurons also prevents local noradrenergic transmission in the left ventricle after acute MI. In this study, Gp130 played the same role in the PVN by transforming glutamate into GABA and inducing an imbalance between excitatory and inhibitory neurotransmitters, thereby further affecting the outflow of sympathetic activity. Conclusions In summary, the present study demonstrates that MI rats have higher concentrations of IL-6, Gp130 and STAT3 in the PVN than sham rats and that the elevations in these molecules contribute to sympathetic nerve inhibition and increased ventricular electrical stability. Our findings provide new insights into the potential treatment of VA in MI rats. Preservation of the IL-6-Gp130-STAT3 pathway in the PVN can reduce the occurrence of VA in the acute phase of MI. Limitations We must consider the effect of the depth of anaesthesia on the autonomic nervous system (ANS). Although we administered anaesthetic according to the weight of each rat, there were individual differences in efficacy. Another limitation of the study is that we elected to diagnose VA from a single- lead electrocardiogram during 24 hours of recording. Although the diagnostic accuracy is good in humans, more experiments are needed to confirm the diagnostic accuracy of this method in rats. Abbreviations MI: Myocardial infarction VA: Ventricular arrhythmia CNS: Central nervous system VF: Ventricular fibrillation PVN: Paraventricular nucleus GABA: γ-aminobutyric acid NMDA: N-methyl-D-aspartic acid IL-6: Interleukin-6 Gp130: Glycoprotein 130 JAK: Janus Kinase MI: Myocardial infarction VA: Ventricular arrhythmia CNS: Central nervous system VF: Ventricular fibrillation PVN: Paraventricular nucleus GABA: γ-aminobutyric acid NMDA: N-methyl-D-aspartic acid IL-6: Interleukin-6 Gp130: Glycoprotein 130 JAK: Janus Kinase MI: Myocardial infarction Page 9/16 Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Consent to publish Not applicable Ethics approval and consent to participate All experimental protocols were approved by the local animal care and use committee (Animal Experimental Ethics Association of the First Affiliated Hospital of Harbin Medical University). The methods were carried out in accordance with the revised Animals (Scientific Procedures) Act 1986. Competing interests The authors declare that they have no competing interests. Page 10/16 Page 10/16 All authors have read and approved the manuscript. The design of the study; the collection, analysis and interpretation of the data; and the writing and editing were supported by the Science Foundation of the First Affiliated Hospital of Harbin Medical University (2018L001), the Heilongjiang Postdoctoral Science Foundation (LBH-Z18213), and the Chinese Postdoctoral Science Foundation (2018M631959). Authors' contributions All authors have read and approved the manuscript. Conceptualization, MG, DCY and XFQ; data curation, MG; formal analysis, MG; funding acquisition, DCY and XFQ; investigation, MG; methodology, MG and JGC; project administration, XFQ; resources, XFQ; software, JGC; supervision, DCY and XFQ; writing- original draft, MG; writing-review and editing, DCY, and XFQ. 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Immunity, 2019, 50(4): 1007-1023. [33] Kang S, Tanaka T, Narazaki M, et al. Targeting interleukin-6 signaling in clinic[J]. Immunity, 2019, 50(4): 1007-1023. Figures Figures Figure 1 Effects of PVN infusion of an anti-IL-6-antibody or SC144 on ventricular electrophysiological activity in MI rats. (A) Recordings of typical ventricular arrhythmia induced by programmed electrical stimulation. (B) Recording of ventricular premature beats within 24 hours by small-animal dynamic electrocardiography. (C) Quantitative analysis of induced ventricular fibrillation. (D) Quantitative analysis of the ventricular fibrillation threshold. (E) Quantitative analysis of premature ventricular beats over 24 hours. The values are the means±SEs. &p<0.05 vs sham rats, #p<0.05 vs MI rats. Abbreviations: PVN, paraventricular nucleus; IL-6, interleukin-6; MI, myocardial infarction. g Figure 1 Effects of PVN infusion of an anti-IL-6-antibody or SC144 on ventricular electrophysiological activity in MI rats. (A) Recordings of typical ventricular arrhythmia induced by programmed electrical stimulation. (B) Recording of ventricular premature beats within 24 hours by small-animal dynamic electrocardiography. (C) Quantitative analysis of induced ventricular fibrillation. (D) Quantitative analysis of the ventricular fibrillation threshold. (E) Quantitative analysis of premature ventricular beats over 24 hours. The values are the means±SEs. &p<0.05 vs sham rats, #p<0.05 vs MI rats. Abbreviations: PVN, paraventricular nucleus; IL-6, interleukin-6; MI, myocardial infarction. Page 14/16 Page 14/16 Figure 2 Effects of PVN infusion of an anti-IL-6 antibody or SC144 on IL-6, Gp130 and pSTAT3 levels within the MI rats. (A) Representative image of IL-6 immunohistochemical staining. (B) Densitometric analysis of IL-6 staining. (C) Representative image of Gp130 immunohistochemical staining. (D) Densitometric analysis of Gp130 staining. (E) Representative image of pSTAT3 immunohistochemical staining. (F) Densitometric analysis of pSTAT3 staining. (G) Representative immunoblot image of IL-6, Gp130 and pSTAT3 levels. (H-J) Densitometric analysis of the protein expression of IL-6, Gp130 and pSTAT3 (n=4). The values are the means ± SEs. &p<0.05 vs sham rats, #p<0.05 vs MI rats. Abbreviations: PVN, paraventricular nucleus; IL-6, interleukin-6; MI, myocardial infarction; Gp130, glycoprotein 130. Figure 2 Figure 2 Figure 4 Effects of PVN infusion of an anti-IL-6 antibody or SC144 on PVN glutamate and GABA concentrations in MI rats. (A) Glutamate. (B) GABA. The values are the means ± SEs. (C) Quantitative analysis of plasma NE levels. &p<0.05 vs sham rats, #p<0.05 vs MI rats. Abbreviations: PVN, paraventricular nucleus; IL-6, interleukin-6; MI, myocardial infarction; Gp130: glycoprotein 130; GABA, γ-aminobutyric acid. Figure 2 Effects of PVN infusion of an anti-IL-6 antibody or SC144 on IL-6, Gp130 and pSTAT3 levels within the MI rats. (A) Representative image of IL-6 immunohistochemical staining. (B) Densitometric analysis of IL-6 staining. (C) Representative image of Gp130 immunohistochemical staining. (D) Densitometric analysis of Gp130 staining. (E) Representative image of pSTAT3 immunohistochemical staining. (F) Densitometric analysis of pSTAT3 staining. (G) Representative immunoblot image of IL-6, Gp130 and pSTAT3 levels. (H-J) Densitometric analysis of the protein expression of IL-6, Gp130 and pSTAT3 (n=4). The values are the means ± SEs. &p<0.05 vs sham rats, #p<0.05 vs MI rats. Abbreviations: PVN, paraventricular nucleus; IL-6, interleukin-6; MI, myocardial infarction; Gp130, glycoprotein 130. Page 15/16 Figure 3 Page 15/16 Figure 3 Figure 3 Page 15/16 Effects of PVN infusion of an anti-IL-6 antibody or SC144 on NMDA and GAD67 levels within the PVN in MI rats. (A) Representative image of NMDA immunohistochemical staining. (B) Densitometric analysis of NMDA staining. (C) Representative image of GAD67 immunohistochemical staining. (D) Densitometric analysis of GAD67 staining. (E) Representative immunoblot image of NMDA and GAD67 levels. (F) Densitometric analysis of the protein expression of NMDA and GAD67 (n=4). The values are the means ± SEs. &p<0.05 vs sham rats, #p<0.05 vs MI rats. Abbreviations: PVN, paraventricular nucleus; IL-6, interleukin-6; MI, myocardial infarction. Figure 4 NC3RsARRIVEGuidelinesChecklistfillable.pdf Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 16/16 Page 16/16
https://openalex.org/W4280649627	https://jrssem.publikasiindonesia.id/index.php/jrssem/article/download/103/531	English	null	Bilateral Trade Analysis of ASEAN and China Countries in ACFTA Cooperation (Gr avity Model Approach)	Journal Research of Social Science, Economics, and Management/Journal Research of Social Science, Economics and Management	2,022	cc-by-sa	4,530	JRSSEM 2022, Vol. 01, No. 7, 842 – 852 E-ISSN: 2807 - 6311, P-ISSN: 2807 - 6494 BILATERAL TRADE ANALYSIS OF ASEAN AND CHINA COUNTRIES IN ACFTA COOPERATION (GRAVITY MODEL APPROACH) JRSSEM 2022, Vol. 01, No. 7, 842 – 852 E-ISSN: 2807 - 6311, P-ISSN: 2807 - 6494 JRSSEM 2022, Vol. 01, No. 7, 842 – 852 E-ISSN: 2807 - 6311, P-ISSN: 2807 - 6494 BILATERAL TRADE ANALYSIS OF ASEAN AND CHINA COUNTRIES IN ACFTA COOPERATION (GRAVITY MODEL APPROACH) Devi Tri Wulandari1* Lilis Yuliati2 Siti Komariyah3 1,2,3University of Jember e-mail: devitwulandari@gmail.com1, lilisyuliati.feb@unej.ac.id2, Sitikomariyah.feb@unej.ac.id3 Correspondence: devitwulandari@gmail.com Devi Tri Wulandari1 Lilis Yuliati2 Siti Komariyah3 1,2,3University of Jember e-mail: devitwulandari@gmail.com1, lilisyuliati.feb@unej.ac.id2, Sitikomariyah.feb@unej.ac.id3 Correspondence: devitwulandari@gmail.com Devi Tri Wulandari1 Lilis Yuliati2 Siti Komariyah3 1,2,3University of Jember e-mail: devitwulandari@gmail.com1, lilisyuliati.feb@unej.ac.id2, Sitikomariyah.feb@unej.ac.id3 *Correspondence: devitwulandari@gmail.com Submitted: 27 January 2022, Revised: 07 February 2022, Accepted: 18 February 2022 Abstract. At the end of 2001 ASEAN and China agreed on free trade in Bandar Sri Begawan, Brunei Darussalam, known as the ASEAN-China Free Trade Agreement (ACFTA). Periodically, ASEAN and China make agreements, one of the goals of which is to eliminate or cut barriers to trade in goods, both tariffs and non-tariffs. Under ACFTA, tariff reduction began in July 2005 and aims to cut import duties to zero by 2010 on about four thousand types of goods for the relatively developed ASEAN countries namely Thailand, Malaysia, Singapore, Indonesia, the Philippines and Brunei. The Gravity Model predicts trade based on distances and interactions between countries in terms of their economic size. The Gravity Model in economics imitates Newton's law of gravity which also takes into account the physical distance and size between two objects. The application of this model to explain economic phenomena regarding the interaction between the two countries has been widely carried out by economists. The study uses panel data from China and ASEAN6 in the 2010- 2020 research period with ASEAN6 exports to China as the dependent variable, and the independent variables include the GDP of the destination country and the country of origin, economic distance proxied in the form of transportation tariffs, exchange rates and economic openness. Panel data regression analysis was used to see the effect of the independent variable on the dependent variable by determining the best model (common effect, fixed effect, random effect) and the classical assumption test performed was the multicollinearity test and the heteroscedasticity test. The results showed that the GDP of destination and origin countries, distance, and exchange rates significantly affected the export value of ASEAN6 to China. Meanwhile, economic openness has no significant effect on the value of ASEAN6 exports to China. Keywords: gravity model; international trade; ASEAN; ACFTA; panel data regression. Submitted: 27 January 2022, Keywords: gravity model; international trade; ASEAN; ACFTA; panel data regression eywords: gravity model; international trade; ASEAN; ACFTA; panel data regression. DOI : 10.36418/jrssem.v1i7.103 Devi Tri Wulandari, Lilis Yuliati, Siti Komariyah \| 843 INTRODUCTION The economic growth of a country cannot be separated from the role of international trade which is one of the factors that can be used as a driving force for economic growth or an increase in the value of GDP. To improve trade relations with countries in ASEAN, at the end of 2001 ASEAN agreed on free trade with China within the framework of ACFTA (ASEAN- China Free Trade Agreement) in Bandar Sri Begawan, Brunei Darussalam and fully implemented in 2010. Exports of ASEAN countries China, based on data from ASEAN Statistics, from 2010 to 2019 was in the top rank compared to other countries. This can be seen in Figure 1, where China occupies the top chart and shows a positive trend since 2010. In addition to China's exports which have been ranked the highest in the last ten years, the value of China's imports to ASEAN also ranks at the top (Zhang, Yang, Wang, Zhan, & Bian, 2020). This can be seen in Figure 2 which shows a positive trend of Chinese imports into the ASEAN market. Figure 1 shows that the exports of ASEAN countries had the highest number of exports to China during 2010 to 2019. Likewise with the number of imports to the ASEAN market (Webb, Strutt, Gibson, & Walmsley, 2020), China was in the top rank compared to other countries during 2010 to 2019. Of course the increase in export value and Chinese imports in the ASEAN market occur in line with the policy of implementing ACFTA cooperation (Chen & Lombaerde, 2019), one of which is that there is no tariff for imports. In addition, the ease of trade between China and ASEAN countries is also influenced by the distance between countries which is not far when compared to the distance between non- ASEAN countries. Since January 1, 2010, China and ASEAN-5 plus Brunei have removed tariffs on 7000 product categories covering 90% of traded goods (Li et al., 2016). Figure 1. Exports of goods from ASEAN countries to countries in 2010-2019 (Source: ASEAN Statistical Yearbook 2020) Figure 1. Exports of goods from ASEAN countries to countries in 2010-2019 (Source: ASEAN Statistical Yearbook 2020) DOI : 10.36418/jrssem.v1i7.103 844 \| Bilateral Trade Analysis of ASEAN and China Countries in ACFTA Cooperation (Gravity Model Approach) 844 \| Bilateral Trade Analysis of ASEAN and China Countries in ACFTA Cooperation (Gravity Model Approach) Figure 2. INTRODUCTION In addition, research also conducted by (Abbas & Waheed, 2015) in Pakistan found that distance has a negative relationship to Pakistan's exports. However, (Naudé, Bosker, & Matthee, 2010) found that the economic distance variable had a positive and significant effect on new coal exports. The type of data used in this research is annual data from 2010 to 2020. The selection of the research period is based on the full implementation of the ACFTA cooperation agreement. Data sources are taken from IMF, World Bank, ASEAN Statistics, comtrade.org and distanceworld.com. With the number of countries studied are 6 countries and a time period of 11 years using annual data, then the amount of data used in this study is 66 data. Research research Model specification adopts research that has been done previously by (Agung, Ishak, Asngari, & Bashir, 2019); (Irshad, Xin, Shahriar, & Arshad, 2017). The variables used as indicators in this research are Export, GDP, Distance, Exchange Rate, and Economic Openness. The econometric model that will be used in this study is written in equation 6 as follows. Referring to the exposure of empirical studies that have been carried out previously, this study wants to further examine the role of ACFTA cooperation, China's trade cooperation with ASEAN6 member countries (Brunei Darussalam, Indonesia, Malaysia, Philippines, Singapore, and Thailand) with the Gravity Model. 𝑙𝑛𝑋𝑖𝑗𝑡= 𝛼0 + 𝑎1𝑙𝑛(𝐺𝐷𝑃𝑖𝑡.𝐺𝐷𝑃𝑗𝑡) + 𝑎2𝑙𝑛𝐷𝐼𝑆𝑇𝑖𝑗𝑡+ 𝑎3𝑙𝑛𝐸𝑋𝐶𝑗𝑡 + 𝑎4𝑂𝑃𝐸𝑁𝑗𝑡+ 𝑒𝑖,𝑡 Whe e: 𝑙𝑛𝑋𝑖𝑗𝑡= 𝛼0 + 𝑎1𝑙𝑛(𝐺𝐷𝑃𝑖𝑡.𝐺𝐷𝑃𝑗𝑡) + 𝑎2𝑙𝑛𝐷𝐼𝑆𝑇𝑖𝑗𝑡+ 𝑎3𝑙𝑛𝐸𝑋𝐶𝑗𝑡 + 𝑎4𝑂𝑃𝐸𝑁𝑗𝑡+ 𝑒𝑖,𝑡 Wh INTRODUCTION Imports of goods from countries to ASEAN markets in 2010-2019 (Source: ASEAN Statistical Yearbook 2020) Figure 2. Imports of goods from countries to ASEAN markets in 2010-2019 (Source: ASEAN Statistical Yearbook 2020) International trade is created because There are differences in production from one country to another. Smith argued that trade between two countries was based on absolute advantage. Smith in his theory believes that all countries will benefit from free trade which causes the world's resources to be used efficiently and maximize welfare. But in Smith's view there is a paradox that most countries impose many restrictions on the free flow of international trade. While in reality, trade restrictions are only recommended by some industries and trade unions who feel threatened by imported products (Howse & Langille, 2012) Classical international trade theory has received criticism from modern theory because classical theory cannot explain why there are differences in the production function between two countries. Modern trade theory from the Hecker-Ohlin (HO) model explains that countries export what they are most efficient and produce the most (Espinoza, 2020). Economic integration in general is the removal (removal of) economic barriers between two or more economies (countries). Operationally, discrimination is defined as deprivation and political unity (policy) such as norms, rules, procedures. These instruments include import duties, taxes, currencies, laws, institutions, standardization, and economic policies. There are two approaches that are particularly useful as literature in international trade policy, namely the Gravity Model which predicts trade based on the distance between countries and the interaction between countries in terms of their economic size, and the Computable General Equilibrium Models (Babatunde, Begum, & Said, 2017). The Gravity Model in economics imitates Newton's law of gravity which also takes into account the physical distance and size between two objects. The application of this model to explain economic phenomena regarding the interaction between the two countries has been widely carried out by economists. In practice, export activities are economic Devi Tri Wulandari, Lilis Yuliati, Siti Komariyah \| 845 activities that are directly related to other countries. (Mulyadi, Zhang, Dutzer, Liu, & Deng, 2017) have conducted a similar study, which in his research found that the GDP of export destination countries had a significant effect and had a positive sign. In line with the theory described by the Gravity Model where the GDP of the destination country increases, exports to that country will increase. Operational Definition of Variables Several variables used in this study have various units and their respective operational definitions. Some of the variables used also consist of various different sources. The operational definitions of the variables used in this study consist of the following: 846 \| Bilateral Trade Analysis of ASEAN and China Countries in ACFTA Cooperation (Gravity Model Approach) 846 \| Bilateral Trade Analysis of ASEAN and China Countries in ACFTA Cooperation (Gravity Model Approach) logarithm (ln) OPEN : economic openness of ASEAN6 countries i : Indonesia, Singapore, Brunei, Malaysia, Philippines, Thailand j : China t : 2010 – 2020 if the best model chosen later is the Fixed Effect, then the autocorrelation test does not need to be carried out because the Fixed Effect does not require free equations from autocorrelation (Nachrowi, 2006; 334). logarithm (ln) OPEN : economic openness of ASEAN6 countries i : Indonesia, Singapore, Brunei, Malaysia, Philippines, Thailand j : China t : 2010 – 2020 Where: Objects and Types of Research Data The Objects in this study are ASEAN6 member countries (Brunei Darussalam, Malaysia, Philippines, Singapore, Thailand, Indonesia) and China. The selection of research objects is based on the ACFTA agreement which is fully valid only with 6 ASEAN6 countries in 2010. The data used in this study is panel data which includes export data of each ASEAN6 country to China, China's GDP and GDP of ASEAN6 countries, distance economy, exchange rates and economic openness. lnX : exports of each ASEAN6 country to China in t converted into natural logarithm (ln) lnX : exports of each ASEAN6 country to China in t converted into natural logarithm (ln) lnGDP : product of China's GDP with GDP of ASEAN6 countries in year t converted into natural logarithm (ln) lnDIST : transportation costs from each ASEAN6 country to China in year t converted into natural logarithm (ln) lnDIST : transportation costs from each ASEAN6 country to China in year t converted into natural logarithm (ln) lnEXC : exchange rate of national currencies of ASEAN6 countries per US$, average per period converted into natural Data Analysis Method The EXCj is the average domestic currency exchange rate (domestic currency) per period in US$ units in ASEAN countries6. The exchange rate used is the real exchange rate. Exchange rate data is taken from the IFS website. the Philippines and Brunei, and to 5 % in 2015 for other ASEAN members, namely Vietnam, Laos, Cambodia and Myanmar (Yang & Martinez-Zarzoso, 2014). d. EXCj is the average domestic currency exchange rate (domestic currency) per period in US$ units in ASEAN countries6. The exchange rate used is the real exchange rate. Exchange rate data is taken from the IFS website. The development of bilateral trade between China and ASEAN6 since the implementation of the ACFTA policy which cut import duties to zero since 2010 can be seen in Figure 3 China's net exports in Figure 3 show a positive trend during the period of zero import duty. Although at the beginning of 2010 – 2011 China's net exports to ASEAN6 countries decreased, but in the following year (2011 to 2015) the development of China's trade with ASEAN6 countries showed an increase. The development of this positive trend does not seem to be going well because net exports declined again in 2015 to 2017 for Brunei, Thailand, Malaysia and Singapore, while Indonesia and the Philippines still showed a positive trend. In the final year of the study period (2019-2020), Indonesia, Singapore and Brunei trade with China showed a decline. Based on ASEAN Key Figures 2020, the Covid-19 pandemic and the movement restrictions it causes have a significant impact on trade and supply chains around the world, including ASEAN. This ultimately resulted in weakening international trade (ASEAN Secretariat, 2020). In its latest forecast, the World Trade Organization (WTO) projects a 9.2% decline in trade volumes by the end of 2020 (WTO, 2020). e. OPENj is the percentage of economic openness of each ASEAN country6 expressed in percent. Data obtained from the World Bank. Data Analysis Method The Method used in explaining the analysis of ASEAN 6 bilateral trade with China is the Least Square Panel (PLS). The Panel Least Square (PLS) method will provide an explanation related to the formulation of the problem in this study (Hair, Sarstedt, Ringle, & Mena, 2012). PLS is an estimation method that uses panel data, which is a combination of time series and cross section data so that more data will be observed than time series or cross section. In addition, the use of panel data will make the regression results tend to be better than regressions that only use time series or cross section. In using the Least Square Panel (PLS), there are several approaches used to estimate the model parameters, namely the Common Effect, Fixed Effect and Random Effect approaches. a. Xij is the export of each ASEAN6 country to China with units of million US$. Net export data obtained from comtrade.org. b. GDPij is the product of the GDP of China and each ASEAN country6 (Irshad et al, 2018). GDP is a proxy for the size of the economy in the gravity model. The GDP used is real GDP based on the base year 2015. The unit used is US$ and the data is taken from the World Bank. c. DISTij is a proxy for transportation costs in conducting international trade. The value of the economic distance of the country of origin to the country of destination is obtained from the calculation of the geographical distance of the capital cities of the two countries multiplied by the nominal GDP of the destination country (China) in the last research period divided by the total nominal GDP of the destination country (China) in year t (Liu et al., 2021). Country distance data is taken from distanceworld.com and GDP data is taken from the World Bank. The next test after getting the best model results in this study will be a classical assumption test which generally consists of autocorrelation, multicollinearity, and heteroscedasticity tests. However, in this study, the classical assumption test that was only used was the Multicollinearity and Heteroscedasticity test. This is because in panel data which is a combination of time series and cross section there will be no autocorrelation because autocorrelation only occurs in time series data. In addition, Devi Tri Wulandari, Lilis Yuliati, Siti Komariyah \| 847 d. Devi Tri Wulandari, Lilis Yuliati, Siti Komariyah \| 848 Figure. 3 Net Exports of China – ASEAN6 2010 – 2020 (Source: comtrade.org) Figure. 3 Net Exports of China – ASEAN6 2010 – 2020 (Source: comtrade.org) The positive trend of China's bilateral trade with ASEAN6 countries cannot be separated from the country's economic conditions. This can be seen from the development of the country's GDP which in this study can be seen in Figure 4. Based on data obtained from the World Bank, the GDP of ASEAN6 countries showed a positive trend from 2009 to 2019. Similar to trade, the Covid-19 pandemic 19 also affects the GDP of ASEAN countries6. Based on a report from ASEAN Key Figures 2021, the continuous increase in GDP per capita from 2000-2019 has decreased due to the COVID-19 outbreak in 2020 (Tailor, 2020). Figure. 4 GDP of ASEAN Countries6 2009 – 2020 (Source: World Bank) Figure. 4 GDP of ASEAN Countries6 2009 – 2020 (Source: World Bank) RESULTS AND DISCUSSION China's trade growth rate has increased rapidly since 2001, when the country joined the WTO and held two initial meetings to discuss the establishment of the ASEAN– China Free Trade Area (ACFTA). More specifically, the average annual growth rate in bilateral trade from 2001 to 2008 was about 30%. In 2011, ASEAN became China's third largest trading partner behind the United States and the European Union. China and ASEAN consider the period between 2002 and 2009 to be a transition period before the completion of ACFTA. During this period, the tariffs imposed on goods traded between China and ASEAN will be lowered gradually. Under the goods trade agreement, the reduction in tariffs began in July 2005 and aims to cut import duties to zero by 2010 on about four thousand types of goods for six relatively developed ASEAN countries namely Thailand, Malaysia, Singapore, Indonesia, Devi Tri Wulandari, Lilis Yuliati, Siti Komariyah Table 2 Results of the Hausman Test Summary Chi-Sq.statistics Chi-Sqdf Prob. Cross-section random 11.042514 4 0.0261 Source: author's preparation regression estimation using the Random Effects show the results where there are 3 independent variables having a probability value of less than 0.05, which means that these three variables significantly affect the exports of each ASEAN6 country to China with an alpha of 5%, while one independent variable is economic openness does not have a significant effect on exports of each ASEAN6 country6 to China. The results of the panel data regression estimation with the Random Effect can be seen in Table 3 as follows. From the model testing that has been carried out using the Chow test and Hausman test, the best model for research using panel data regression is Random Effect (REM) which will be discussed in the next subsection. Panel Regression Model Selection Test by comparing the Common Effect (CEM), Fixed Effect (FEM) and Random Effect (REM). The significance test of the model is carried out using the Chow test for the first, Before entering the Panel Regression estimation, a series of panel regression model selection tests will be carried out based on the significance test of the model DOI : 10.36418/jrssem.v1i7.103 Devi Tri Wulandari, Lilis Yuliati, Siti Komariyah \| 849 where this test will see the best model from the comparison of the CEM model and FEM. Furthermore, Hausman test will be carried out to see the best model from the comparison of the FEM model and REM. The first model selection test was conducted, namely the Chow test. The results of the Chow test in Table 1 show a cross-section F 0.0232 which means less than 0.05, so it can be determined that the best model between CEM and FEM is FEM. Furthermore, in testing the model selection between FEM and REM which was carried out with the Hausman test. The results of the Hausman test can be seen in Table 2 where the table shows that the probability value of a random cross section is 0.0435, which means less than 0.05. From the Hausman test, it can be determined that the best model between FEM and REM is FEM. Table 1. Test Results Chow Effects Test Statistics df Prob. Cross-section F 5.487768 (5.56) 0.0004 Cross-section Chi-square 26.318308 5 0.0001 Source: author's preparation Table 2 Results of the Hausman Test Summary Chi-Sq.statistics Chi-Sqdf Prob. Cross-section random 11.042514 4 0.0261 Source: author's preparation i i i i h Table 1. Test Results Chow Effects Test Statistics df Prob. Cross-section F 5.487768 (5.56) 0.0004 Cross-section Chi-square 26.318308 5 0.0001 Source: author's preparation Table 2 Results of the Hausman Test Summary Chi-Sq.statistics Chi-Sqdf Prob. Cross-section random 11.042514 4 0.0261 Source: author's preparation OPENESS -0.002 OPENESS -0.002 each ASEAN6 country to China by 0.288815, which means that if the nominal exchange rate of each ASEAN6 country increases by 1%, the export of each ASEAN6 country to China decreases by 0.29%. The next variable is openness which according to the estimation results has a negative effect on exports of each ASEAN6 country to China and but has no significant effect because the probability of this variable is greater than 0.05. (logx) with an alpha of 5%, namely loggdp, logdist and logexc which are indicated by probability values less than 0.05 alpha. The loggdp variable has a positive effect on imports of 1.663232 which means that when the GDP of China and ASEAN6 countries has increased by 1%, the exports of each ASEAN6 country to China have increased by 1.67%. The second variable that has an effect on logx is logdist, which in this study is the distance between two countries as a proxy for transportation costs. Logdist has a significant negative effect on exports of each ASEAN6 country to China. The effect of logdist is 0.327274, which means that if the cost of transportation from each ASEAN6 country to China increases by 1%, the export of each ASEAN6 country to China decreases by 0.33%. The next independent variable that affects logx is logexc. This variable has a significant negative effect on exports of The classical assumption test used in this study is the multicollinearity and heteroscedasticity test. From the multicollinearity test, it can be concluded that the data does not experience multicollinearity, which means that there is no attachment between the independent variables used in the study. This can be seen from the values listed in Table 4, where the table shows that there are no values between variables smaller than 0.8. Table 4. Test Results Multicollinearity LOGGDP LOGDIST LOGEXC openess LOGGDP 1 0.0500776636729 0.621123320778 0.0115028304456 LOGDIST 0.0500776636729 1 0.119401910807 0.0546518595241 LOGEXC 0.621123320778 0.119401910807 1 -0.599506469682 openess 0.0115028304456 0.0546518595241 -0.599506469682 1 has an effect on international trade, especially in ASEAN6 countries and China which have established trade cooperation in ACFTA. has an effect on international trade, especially in ASEAN6 countries and China which have established trade cooperation in ACFTA. Panel Data Regression Test with Random Effect After the researcher selects the best model, then estimation is done by panel data regression test using the Random Effect. The results of the panel data Table 3. Regression Panel Data with Random Effect Model Variable Coefficient Std. Error t-Statistic Prob. C -66.23974 4.982195 -13.29529 0.0000 LOGGDP 1.663232 0.092921 17.89936 0.0000 LOGDIST -0.327274 0.154613 -2.116729 0.0384 LOGEXC -0.288815 0.048801 -5.918280 0.0000 850 \| Bilateral Trade Analysis of ASEAN and China Countries in ACFTA Cooperation (Gravity Model Approach) 850 \| Bilateral Trade Analysis of ASEAN and China Countries in ACFTA Cooperation (Gravity Model Approach) 850 \| Bilateral Trade Analysis of ASEAN and China Countries in ACFTA Cooperation (Gravity Model Approach) CONCLUSIONS Overall it can be concluded that the direction of the results of this study is in line with the Gravity Model and analysis can be formulated in several conclusions as follows: b. The distance between countries that have trade cooperation, which in this study is proxied in the form of transportation costs between the two a. GDP of origin and destination countries a. GDP of origin and destination countries Devi Tri Wulandari, Lilis Yuliati, Siti Komariyah \| 851 c. countries, has an effect on determining whether the value of international trade is large or small. c. countries, has an effect on determining whether the value of international trade is large or small. Christian M., & Mena, Jeannette A. (2012). An assessment of the use of partial least squares structural equation modeling in marketing research. Journal of the Academy of Marketing Science, 4(3), 414–433. Christian M., & Mena, Jeannette A. (2012). An assessment of the use of partial least squares structural equation modeling in marketing research. Journal of the Academy of Marketing Science, 4(3), 414–433. d. The exchange rate of the domestic currency against the US$ is still one of the influential variables in determining international trade. d. The exchange rate of the domestic currency against the US$ is still one of the influential variables in determining international trade. 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https://openalex.org/W4226085344	http://old.scielo.br/pdf/anp/v79n12/1678-4227-anp-0004-282x-anp-2021-0172.pdf	English	null	Professor Ângelo Machado: career, scientific contributions, and the iconic neuroanatomy book	Arquivos de Neuro-Psiquiatria	2,021	cc-by	3,153	https://doi.org/10.1590/0004-282X-ANP-2021-0172 https://doi.org/10.1590/0004-282X-ANP-2021-0172 HISTORICAL NOTES RESUMO O professor Ângelo Barbosa Monteiro Machado (22/05/1934–06/04/2020) foi um dos mais destacados e respeitados professores da história do Brasil. Ele trabalhou amplamente como professor, neurocientista, escritor, dramaturgo e entomologista. A publicação do livro de neuroanatomia é pioneira, revolucionária e icônica na história da educação médica acadêmica no Brasil. No campo da literatura, também escreveu diversos livros nos quais adaptou o conhecimento científico para as crianças. Neste artigo, os autores abordam a vida acadêmica do professor Ângelo Machado e as etapas que culminaram no mais conceituado livro brasileiro de neuroanatomia: Neuroanatomia Funcional. Palavras-chave: Neuroanatomia; Neurobiologia; Livro-Texto; Docentes. 1Universidade Federal da Paraíba, Departamento de Medicina Interna, Serviço de Neurologia, João Pessoa PB, Brazil. 2Pontifícia Universidade Católica, Departamento de Medicina Interna, Curitiba PR, Brazil. 3Universidade Federal de Minas Gerais, Hospital das Clínicas, Departamento de Medicina Interna, Serviço de Neurologia, Belo Horizonte MG, Brazil. 4Universidade Federal do Paraná, Hospital de Clínicas, Departamento de Medicina Interna, Serviço de Neurologia, Curitiba PR, Brazil. 5Universidade Federal de São Paulo, Departamento de Neurologia, São Paulo SP, Brazil. ATM https://orcid.org/0000-0002-6685-7491; GLF https://orcid.org/0000-0002-0207-3671; FC https://orcid.org/0000-0003-0808-0116; HAGT https://orcid.org/0000-0003-2305-1073; OGPB https://orcid.org/0000-0002-0107-0831; JLP https://orcid.org/0000-0002-1672-8894 Correspondence: Alex Tiburtino Meira; Email: alex.m.meira@gmail.com. Conflict of interest: There is no conflict of interest to declare. Authors’ contributions: ATM, JLP: conceptualization, data curation, formal analysis, investigation, methodology, resources, supervision, validation, visualization, writing-original draft, writing review and editing; GLF, FC, HAGT, OGPB: formal analysis, methodology, project administration, validation, visualization, writing review and editing. Received on May 02, 2021; Received in its final form on July 06, 2021; Accepted on July 27, 2021. ABSTRACT Professor Ângelo Barbosa Monteiro Machado (May 22, 1934 to April 6, 2020) was one of the most outstanding and respected professors in the Brazilian history. He worked broadly as a professor, neuroscientist, writer, dramaturgist, neurobiologist, and entomologist. The publication of the neuroanatomy book is pioneer, revolutionary, and iconic in the history of academic medical education in Brazil. In the literature field, he also wrote many books in which he adapted scientific knowledge to children. In this article, the authors approach the academic life of Professor Ângelo Machado and the steps that culminated in the most renowned Brazilian textbook of neuroanatomy: Neuroanatomia Funcional. Keywords: Neuroanatomy; Neurobiology; Textbook; Faculty. Professor Ângelo Machado: career, scientific contributions, and the iconic neuroanatomy book Professor Ângelo Machado: carreira, contribuições científicas e o icônico livro de neuroanatomia Alex Tiburtino MEIRA1, Gustavo Leite FRANKLIN2, Francisco CARDOSO3, Hélio Afonso Ghizoni TEIVE4, Orlando Graziani Povoas BARSOTTINI5, José Luiz PEDROSO5 INTRODUCTION Several Brazilian associations, such as the Brazilian Academy of Neurology (ABN), the Brazilian Society for the Progress of the Science, the Federal Council of Biology, and the Brazilian Society of Pediatric Neurosurgery, among others, mourned the death of the polymath of the contemporary era. INTRODUCTION beloved work1. His academic life can be divided into two peri- ods: the first as professor and neuroscientist, which resulted in his masterpiece, the book Neuroanatomia Funcional2,3, and the second as the professor of entomology at the same institution. He was married to Conceição Ribeiro da Silva Machado (1936–2007) — his “major scientific discovery” as he stated, with whom he shared not only a life of love and partnership but also an academic life, which resulted in the creation of the Neurobiology Laboratory at the UFMG1,2,4. Professor Machado was one of the most respected profes- sors in the history of Brazil, and whose expertise spanned a substantial number of subject areas. He died on April 6, 2020. Ângelo Barbosa Monteiro Machado was born on May 22, 1934 in Belo Horizonte, Minas Gerais, Brazil1. Since child- hood, Ângelo Machado demonstrated all the characteris- tics of a born scientist. His curiosity and greed for knowledge drew the attention of his family, who set up a laboratory for him in the backyard of their house equipped with a micro- scope, aquarium, dissection material, and all the books he wanted to buy (personal communication). He graduated in Medicine at the Universidade Federal de Minas Gerais (UFMG) in 1958, for which he later dedicated 67 years of 1149 with great repercussion in its teaching (personal communi- cation). His main contribution to the neurobiological field had been the formation of norepinephrine containing synap- tic vesicles by this time5. In 1963, he defended his Ph.D. thesis in Anatomy at the UFMG, and, 4 years later, his postdoctor- ate at the Northwestern University, Chicago (where he lived for 2.5 years)6. There, he and his wife learned histochemical techniques for identifying catecholaminergic pathways and neurons and electron microscopy techniques, which later culminated in the creation of the UFMG electron microscopy laboratory and made them pioneers in the study of catechol- aminergic pathways with several contributions to the study of the autonomic nervous system. He had taught as a profes- sor of neuroanatomy for almost 30 years before he retired and went a public contest becoming professor of the zoologist department, and then becoming professor of entomology at the same institution, a position in which he worked for more than 20 years, including as an emeritus professor after 20051,6,7. CAREER During high school, Professor Machado had done an internship at the Osvaldo Cruz Foundation, between 1951 and 1956, with the pathologist Lobato Paraense, with whom he also worked later during the medical course. He con- siders that he started his career as a scientist at that insti- tute. He had also been working as a trainee in the Anatomy Department of School of Medicine at UFMG. Although he had been graduated in Medicine at UFMG in 1958, he had never practiced as a physician2. He immediately started his lectures at the Anatomy Department, exerting good influence over medical students and becoming renowned for his remark- able didactic skills and his iconoclastic and sarcastic sense of humor2,5. In some of his jokes, when he was asked why he was interested in the pineal gland, he used to answer “my profes- sor of Anatomy thought some weird field I should research for: the pineal gland of the armadillo. So, I did, then I discov- ered that the armadillo doesn’t have pineal gland.” He chose the neuroscientific field by noticing that it has been a poten- tial area for research (Figure 1A); he transformed a neglected subject into a new and independent discipline at UFMG, He worked broadly as a professor, neuroscientist, writer, dramaturgist, and entomologist but was never enchanted by bureaucratic positions3. In the neurobiology field, not only he was crucial for the knowledge regarding the involve- ment of the autonomous nervous system in animal mod- els of Chagas disease, but also his time working as a pro- fessor of neuroanatomy was pivotal in the development of the book Neuroanatomia Funcional, launched in 1974, now- adays in its 3rd edition (2014)2,6. During his academic career, he substantially dedicated himself to the taxonomy of drag- onflies7, the popularization of science, children’s education, and nature conservation8. Figure 1. Professor Ângelo Machado. (A) Researching in the Anatomy Department of the School of Medicine, Universidade Federal de Minas Gerais, Brazil, in 1957. (B) Receiving the prestigious “Jabuti Literature Award” in 1993. (C) Some of the books in Angelo Machado’s library, which were used to prepare his classes (from left to right: Ramon y Cajal S., Histologie du système nerveux de l’homme & des vertébrés; Testut L. and Latarjet A., Traité d’anatomie humaine; Handbuch der mikroskopischen Anatomie des Menschen. BOOKS In 2021, the 4th edi- tion of the book is expected, with improvements mainly in the anatomoclinical correlations (personal communication).i correlations. It was first launched in 1974 (Figure 2B) and is still considered the standard reference for neuroanatomy courses in several universities in Brazil9. With the advances in the neurological field, mainly in the neuroimaging field, the 2nd edition was launched in 2004 (Figure 2C)10. In 2014, an entirely updated edition was published (Figure 2D), coau- thored by his daughter Lúcia Machado Haertel, child neu- rologist, who was responsible for enriching the anatomical and clinical aspects of the book, making the study even more attractive, by correlating the morphofunctional aspects with the clinical practice11. Since the beginning, the designer Val Moro from the UFMG department was in charge of the illus- trations (Figure 2E — original drawings)9. In 2021, the 4th edi- tion of the book is expected, with improvements mainly in the anatomoclinical correlations (personal communication). In 1987, he started a new field of knowledge: writing books in which he adapted scientific knowledge to chil- dren12. He wrote 37 books for them and other 3 romances3. He was also a dramaturgist, with 6 plays for children and 2 comedies4. He received many National or State awards as follows: (1) the Jabuti award (Figure 1C), the most famous and important award of Brazilian literature, in 1993, for the book “O velho da montanha, uma aventura amazônica;” (2) the SESC-SATED award of the best children’s play in 1996, for the play “O casamento da ararinha azul,” which was also adapted as a short film; (3) and the highest gross- ing in 2001, for the comedy play “Como sobreviver em festas com buffet escasso”4. In 2017, he was awarded with the title of Emeritus researcher of the National Council of Scientific and Technological Development (CNPq), which is among 150 others awards and tributes, including the commenda- tion of scientific merit and the Gram Cross of scientific merit delivered by the President of the Republic4. The last book written by Prof. Machado was “Cristóvão e os grandes descobrimentos” in 201913. Before his death, Prof. Ângelo Machado has been writing “Tratado de Guerra,” a comic book for adults, concluded but unpublished, which will be done by his offsprings7. BOOKS Neuroanatomia Funcional, the pioneer and the standard reference book in National Medical Education for Neuroanatomy courses in several universities in Brazil: in its 1st edition, published in 1974 (B); 2nd edition, 2004 (C); and 3rd edition, 2015 (D); and some original drawings for the book, made by Val Moro, a designer from the Universidade Federal de Minas Gerais department (E). correlations. It was first launched in 1974 (Figure 2B) and is still considered the standard reference for neuroanatomy courses in several universities in Brazil9. With the advances in the neurological field, mainly in the neuroimaging field, the 2nd edition was launched in 2004 (Figure 2C)10. In 2014, an entirely updated edition was published (Figure 2D), coau- thored by his daughter Lúcia Machado Haertel, child neu- rologist, who was responsible for enriching the anatomical and clinical aspects of the book, making the study even more attractive, by correlating the morphofunctional aspects with the clinical practice11. Since the beginning, the designer Val Moro from the UFMG department was in charge of the illus- trations (Figure 2E — original drawings)9. In 2021, the 4th edi- tion of the book is expected, with improvements mainly in the anatomoclinical correlations (personal communication). In 1987, he started a new field of knowledge: writing books in which he adapted scientific knowledge to chil- dren12. He wrote 37 books for them and other 3 romances3. He was also a dramaturgist, with 6 plays for children and 2 comedies4. He received many National or State awards as follows: (1) the Jabuti award (Figure 1C), the most famous and important award of Brazilian literature, in 1993, for the b k “O lh d t h t ô i ” correlations. It was first launched in 1974 (Figure 2B) and is still considered the standard reference for neuroanatomy courses in several universities in Brazil9. With the advances in the neurological field, mainly in the neuroimaging field, the 2nd edition was launched in 2004 (Figure 2C)10. In 2014, an entirely updated edition was published (Figure 2D), coau- thored by his daughter Lúcia Machado Haertel, child neu- rologist, who was responsible for enriching the anatomical and clinical aspects of the book, making the study even more attractive, by correlating the morphofunctional aspects with the clinical practice11. Since the beginning, the designer Val Moro from the UFMG department was in charge of the illus- trations (Figure 2E — original drawings)9. BOOKS Despite being less prominent than his wife in terms of neuroscientific curriculum, Professor Ângelo Machado was more embracing. His work as a professor of neuroanatomy associated with his great didactic and language skills was a landmark in the history of this field in Brazil. His students started collecting the knowledge that has been transmitted in his classes2. Soon, this material was gathered by the professor (Figure 2A), and he was strongly encouraged by his wife and friends to publish the first nationally published book about neuroanatomy2. Once the national book about the field was lacking, before starting as a professor, he moved away to the family farm to study English, French, and German books, pre- paring the entire content of his classes (personal communi- cation — Figure 1B). He found slides too monotonous, and that is why he preferred to create his drawings on large pan- els to illustrate his lessons. His masterpiece is in charge of many medical students having chosen neurology or neuro- surgery as a specialty to follow after graduating. The book is a practical guide for the anatomy course, with in-depth knowl- edge regarding structure, function, and anatomoclinical Figure 1. Professor Ângelo Machado. (A) Researching in the Anatomy Department of the School of Medicine, Universidade Federal de Minas Gerais, Brazil, in 1957. (B) Receiving the prestigious “Jabuti Literature Award” in 1993. (C) Some of the books in Angelo Machado’s library, which were used to prepare his classes (from left to right: Ramon y Cajal S., Histologie du système nerveux de l’homme & des vertébrés; Testut L. and Latarjet A., Traité d’anatomie humaine; Handbuch der mikroskopischen Anatomie des Menschen. 1150 Arq Neuropsiquiatr 2021;79(12):1149-1152 Figure 2. The handouts for the neuroanatomy course, which were later transformed into the book (A). Neuroanatomia Funcional, the pioneer and the standard reference book in National Medical Education for Neuroanatomy courses in several universities in Brazil: in its 1st edition, published in 1974 (B); 2nd edition, 2004 (C); and 3rd edition, 2015 (D); and some original drawings for the book, made by Val Moro, a designer from the Universidade Federal de Minas Gerais department (E). Figure 2. The handouts for the neuroanatomy course, which were later transformed into the book (A). BOOKS Two things are certain as follows: his conser- vationism, environmentalism, and education will remain in our memories, and his book, Neuroanatomia Funcional, will con- tinue encouraging and inspiring many medical students. In 1987, he started a new field of knowledge: writing books in which he adapted scientific knowledge to chil- dren12. He wrote 37 books for them and other 3 romances3. He was also a dramaturgist, with 6 plays for children and 2 comedies4. He received many National or State awards as follows: (1) the Jabuti award (Figure 1C), the most famous and important award of Brazilian literature, in 1993, for the book “O velho da montanha, uma aventura amazônica;” (2) the SESC-SATED award of the best children’s play in ACKNOWLEDGMENT The authors thank the family Machado, in the names of Flávia Machado and Lúcia Machado Haertel, who cooper- ated incredibly with this article. 1151 Meira AT, et al. Professor Ângelo Machado. References 1. Universidade Federal de Minas Gerais. 2021 [accessed on 2021 May 29]. Morre o professor, entomologista e escritor Ângelo Machado. Available from: https://ufmg.br/comunicacao/noticias/morre-o- professor-entomologista-e-escritor-angelo-machado 2. Cardoso F. In memoriam Dr. Ângelo Machado. Arq Neuro-Psiquiatr. 2020 May;78(5):316-7. https://doi.org/10.1590/0004-282X20200049 3. Mitre M. Professor Angelo Machado: The remarkable deeds of a polyvalent mind. Lundiana. 2005;6(Supplement):5-10. 4. Marcolin N. Angelo Machado: Entre livros e libélulas. Pesq FAPESP. 2007;132:10-15. [accessed on 2021 Apr 7th, 2021]. Available from: https://revistapesquisa.fapesp.br/entre-livros-e-libelulas/ 5. Machado AB, Machado CR, Wragg LE. Catecholamines and granular vesicles in adrenergic axons of the developing pineal body of the rat. Experientia. 1968 May;24(5):464-5. https://doi.org/10.1007/ bf02144394 6. Currículo do sistema Lattes. Angelo Barbosa Monteiro Machado. 17 may 2017 [accessed on 2021 May 29]. Available from: http:// lattes.cnpq.br/5343850000941639 7. Pinto AP. The dragonfly’s face of the multidimensional Dr. Angelo Barbosa Monteiro Machado: a short bio-bibliography. Zootaxa. 2016 Feb;4078(1):8. https://doi.org/10.11646/zootaxa.4078.1.4 1. Universidade Federal de Minas Gerais. 2021 [accessed on 2021 May 29]. Morre o professor, entomologista e escritor Ângelo Machado. Available from: https://ufmg.br/comunicacao/noticias/morre-o- professor-entomologista-e-escritor-angelo-machado 2. Cardoso F. In memoriam Dr. Ângelo Machado. Arq Neuro-Psiquiatr. 2020 May;78(5):316-7. https://doi.org/10.1590/0004-282X20200049 3. Mitre M. Professor Angelo Machado: The remarkable deeds of a polyvalent mind. Lundiana. 2005;6(Supplement):5-10. 4. Marcolin N. Angelo Machado: Entre livros e libélulas. Pesq FAPESP. 2007;132:10-15. [accessed on 2021 Apr 7th, 2021]. Available from: https://revistapesquisa.fapesp.br/entre-livros-e-libelulas/ 5. Machado AB, Machado CR, Wragg LE. Catecholamines and granular vesicles in adrenergic axons of the developing pineal body of the rat. Experientia. 1968 May;24(5):464-5. https://doi.org/10.1007/ bf02144394 6. Currículo do sistema Lattes. Angelo Barbosa Monteiro Machado. 17 may 2017 [accessed on 2021 May 29]. Available from: http:// lattes.cnpq.br/5343850000941639 7. Pinto AP. The dragonfly’s face of the multidimensional Dr. Angelo Barbosa Monteiro Machado: a short bio-bibliography. Zootaxa. 2016 Feb;4078(1):8. https://doi.org/10.11646/zootaxa.4078.1.4 8. Nunes M. Ângelo Machado, cientista, escritor e “um grande líder da conservação da biodiversidade”, morre aos 85 anos. [accessed on 2021 Apr 7th, 2021]. Available from: https://conexaoplaneta.com.br/ blog/angelo-machado-cientista-escritor-e-um-grande-lider-da- conservacao-da-biodiversidade-morre-aos-85-anos/ 9. Machado ABM. Neuroanatomia funcional. 1st ed. São Paulo: Atheneu; 1974. 10. Machado ABM. Neuroanatomia funcional. 2st ed. São Paulo: Atheneu; 2004. 11. Machado ABM, Haertel LM. Neuroanatomia funcional. 3st ed. São Paulo: Atheneu; 2014. 12. Higashi AMF. Ciência e literatura em textos infantis de Angelo Machado [dissertation]. ACKNOWLEDGMENT São Paulo: Universidade de São Paulo, Faculdade de Filosofia, Letras e Ciências Humanas; 2010 [accessed on 2021 May 29]. https://doi.org/10.11606/D.8.2010. tde-31012011-104437 13. Peixoto M. Ciência, arte e bom humor: conheça o imenso legado de Ângelo Machado [accessed on 2021 May 29]. Available from: https://www.em.com.br/app/noticia/cultura/2020/04/07/interna_ cultura,1136284/ciencia-arte-e-bom-humor-conheca-o-imenso- legado-de-angelo-machado.shtml 1. Universidade Federal de Minas Gerais. 2021 [accessed on 2021 May 29]. Morre o professor, entomologista e escritor Ângelo Machado. Available from: https://ufmg.br/comunicacao/noticias/morre-o- professor-entomologista-e-escritor-angelo-machado 8. Nunes M. Ângelo Machado, cientista, escritor e “um grande líder da conservação da biodiversidade”, morre aos 85 anos. [accessed on 2021 Apr 7th, 2021]. Available from: https://conexaoplaneta.com.br/ blog/angelo-machado-cientista-escritor-e-um-grande-lider-da- conservacao-da-biodiversidade-morre-aos-85-anos/ 2. Cardoso F. In memoriam Dr. Ângelo Machado. Arq Neuro-Psiquiatr. 2020 May;78(5):316-7. https://doi.org/10.1590/0004-282X20200049 9. Machado ABM. Neuroanatomia funcional. 1st ed. São Paulo: Atheneu; 1974. 3. Mitre M. Professor Angelo Machado: The remarkable deeds of a polyvalent mind. Lundiana. 2005;6(Supplement):5-10. 10. Machado ABM. Neuroanatomia funcional. 2st ed. São Paulo: Atheneu; 2004. 4. Marcolin N. Angelo Machado: Entre livros e libélulas. Pesq FAPESP. 2007;132:10-15. [accessed on 2021 Apr 7th, 2021]. Available from: https://revistapesquisa.fapesp.br/entre-livros-e-libelulas/ 11. Machado ABM, Haertel LM. Neuroanatomia funcional. 3st ed. São Paulo: Atheneu; 2014. 5. Machado AB, Machado CR, Wragg LE. Catecholamines and granular vesicles in adrenergic axons of the developing pineal body of the rat. Experientia. 1968 May;24(5):464-5. https://doi.org/10.1007/ bf02144394 12. Higashi AMF. Ciência e literatura em textos infantis de Angelo Machado [dissertation]. São Paulo: Universidade de São Paulo, Faculdade de Filosofia, Letras e Ciências Humanas; 2010 [accessed on 2021 May 29]. https://doi.org/10.11606/D.8.2010. tde-31012011-104437 13. Peixoto M. Ciência, arte e bom humor: conheça o imenso legado de Ângelo Machado [accessed on 2021 May 29]. Available from: https://www.em.com.br/app/noticia/cultura/2020/04/07/interna_ cultura,1136284/ciencia-arte-e-bom-humor-conheca-o-imenso- legado-de-angelo-machado.shtml 1152 Arq Neuropsiquiatr 2021;79(12):1149-1152 1152 1152 Arq Neuropsiquiatr 2021;79(12):1149-1152
https://openalex.org/W2136509799	https://dash.harvard.edu/bitstream/1/11878852/1/3683849.pdf	English	null	Using population admixture to help complete maps of the human genome	Nature genetics	2,013	cc-by	10,502	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Published Version doi:10.1038/ng.2565 http://nrs.harvard.edu/urn-3:HUL.InstRepos:11878852 Citation Citation Genovese, G., R. E. Handsaker, H. Li, N. Altemose, A. M. Lindgren, K. Chambert, B. Pasaniuc, et al. 2013. “Using population admixture to help complete maps of the human genome.” Nature genetics 45 (4): 406-414e2. doi:10.1038/ng.2565. http://dx.doi.org/10.1038/ng.2565. Genovese, G., R. E. Handsaker, H. Li, N. Altemose, A. M. Lindgren, K. Chambert, B. Pasaniuc, et al. 2013. “Using population admixture to help complete maps of the human genome.” Nature genetics 45 (4): 406-414e2. doi:10.1038/ng.2565. http://dx.doi.org/10.1038/ng.2565. Published Version doi:10.1038/ng.2565 Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility NIH-PA Author Manuscript NIH Public Access Author Manuscript NIH-PA Author Manuscript Published in final edited form as: Nat Genet. 2013 April ; 45(4): 406–414e2. doi:10.1038/ng.2565. Using population admixture to help complete maps of the human genome Giulio Genovese1,2,3,4, Robert E. Handsaker1,2,4, Heng Li1,2, Nicolas Altemose2, Amelia M. Lindgren5, Kimberly Chambert1,4, Bogdan Pasaniuc6, Alkes L. Price1,6, David Reich2, Cynthia C. Morton1,5,7, Martin R. Pollak1,3, James G. Wilson8, and Steven A. McCarroll1,2,4 1Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA Giulio Genovese1,2,3,4, Robert E. Handsaker1,2,4, Heng Li1,2, Nicolas Altemose2, Amelia M. Lindgren5, Kimberly Chambert1,4, Bogdan Pasaniuc6, Alkes L. Price1,6, David Reich2, Cynthia C. Morton1,5,7, Martin R. Pollak1,3, James G. Wilson8, and Steven A. McCarroll1,2,4 1Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA ript NIH-PA Author Manuscript 2Department of Genetics, Harvard Medical School, Boston, MA 3Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA NIH-PA Author Manuscript 4Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 5Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital, Boston, MA 6Department of Biostatistics, Harvard School of Public Health, Boston, MA 7Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 7Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 8Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS 8Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS Correspondence should be addressed to G.G. (giulio.genovese@gmail.com) and S.A.M. (mccarroll@genetics.med.harvard.edu). AUTHOR CONTRIBUTIONS G.G. and S.A.M. conceived the project, designed the analyses and wrote the manuscript. G.G. performed the analysis of the CARe, ICDB, JHS, and BodyMap 2.0 datasets. R.E.H. performed the sequence read depth analysis of selected regions. H.L. performed the alignments of HuRef scaffolds and GenBank clones. N.A. contributed the analysis of the HuRef unplaced scaffolds. A.M.L. performed the FISH experiments. K.C. organized and contributed to the design of the Sequenom experiment. B.P., A.P., and D.R. provided advice for the local ancestry inference. C.C.M. participated in interpretation of the FISH experiments. M.R.P. participated in planning discussions for the linkage analysis. J.G.W. participated in planning discussions, coordinated interactions with the Jackson Heart Study, and edited the manuscript. Correspondence should be addressed to G.G. (giulio.genovese@gmail.com) and S.A.M. (mccarroll@genetics.med.harvard.edu). AUTHOR CONTRIBUTIONS Abstract Tens of millions of base pairs of euchromatic human genome sequence, including many protein- coding genes, have no known location in the human genome. We describe an approach for localizing the human genome's missing pieces by utilizing the patterns of genome sequence variation created by population admixture. We mapped the locations of 70 scaffolds spanning four million base pairs of the human genome's unplaced euchromatic sequence, including more than a dozen protein-coding genes, and identified eight large novel inter-chromosomal segmental duplications. We find that most of these sequences are hidden in the genome's heterochromatin, particularly its pericentromeric regions. Many cryptic, pericentromeric genes are expressed in RNA and have been maintained intact for millions of years while their expression patterns diverged from those of paralogous genes elsewhere in the genome. We describe how knowledge of the locations of these sequences can inform disease association and genome biology studies. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Genovese et al. Page 2 Physical maps of the human genome, including the sequence of most of its euchromatic portions1,2, are basic resources in human genetics and genomics research: they provide the framework for analysis of sequence data; and they enable genome-scale analysis of single nucleotide polymorphisms (SNPs), copy number variants (CNVs), epigenetic phenomena, and gene expression. NIH-PA Author Manuscript NIH-PA A Yet physical maps of the human genome remain incomplete. Almost 30 million base pairs (Mbp) of euchromatic genome sequence that are apparently human – observed in human whole-genome sequence data3,4, containing human expressed sequence tags5,6 (ESTs), and homologous to other mammalian genome sequences – are either absent from, or have no assigned locations in, current assemblies of the human genome7,8. These “missing pieces” of the reference human genome are a likely source of mistaken inference in today's analyses of genome sequence data9. Sequence reads arising from the missing pieces may be discarded as non-alignable, or incorrectly assumed to arise from paralogous sequences in the known, assembled part of the human genome. Sequences missing from the reference human genome might also help answer questions in human genetics research, such as the source of the genetic signals that have been ascertained (but not yet fine-mapped to causal variation or causal genes) by linkage, association, and CNVs. Nat Genet. Author manuscript; available in PMC 2013 October 01. Abstract NIH-PA Author Manuscript Here we describe an approach for “admixture mapping” the human genome's missing pieces at megabase pair scales, by utilizing the patterns of sequence variation that have been created by isolation and subsequent re-mixture of human populations. We report the successful mapping of ~5Mbp of unplaced human euchromatic sequences, including many protein-coding genes. We find that most of these sequences are euchromatic islands within the genome's heterochromatic oceans, including centromeres and the short arms of the acrocentric chromosomes, and that they almost always consist of segmental duplications (sometimes recent, sometimes millions of years old) of sequence present elsewhere in the reference genome. An approach for admixture mapping unplaced sequence The construction of large-scale genome models (“assemblies”) utilizes physical sequence overlaps between genomic clones10. Clones are assembled, into larger scaffolds based on overlapping sequences at their ends. NIH-PA Author Manuscript By contrast, mapping based on statistical relationships among variants can provide information that is complementary to physical mapping, as it does not require a continuous path of sequences to be cloned and uniquely assembled. Before physical mapping was feasible, linkage among alleles was used to construct the first genetic maps of the human genome based on restriction fragment length polymorphisms11,12, and later to build and improve genetic maps based on microsatellite markers13,14. NIH-PA Author Manuscript A unique kind of long-range information – finer in resolution than linkage in families, yet longer in reach than linkage disequilibrium (LD) in populations – is present in many of the world's admixed populations. Whenever human populations have been reproductively isolated for long periods of time (such as Africans and Europeans) and then re-mixed (such as among African Americans), the genomes of the descendants are mosaics of segments that derive from ancestors from the two ancestral populations (Fig. 1a). The divergence of the sequences in the ancestral populations gives rise to sequence variation that is informative about the ancestry of each segment. Long-range “admixture LD” has been used to map genetic factors that segregate at different frequencies in different populations15,16 and to identify genomic sites of recombination in African Americans17,18. NIH-PA Author Manuscript Page 3 Genovese et al. We reasoned that population admixture could also be used to map the locations of unmapped human genome sequences. Provided that the sequence in a genomic missing piece is variable, that this variation was subject to genetic drift, and that the extent of this drift is known in the two ancestral populations, we could infer the ancestral origin of a missing piece – whether it has been inherited from each individual's European or African ancestors – with varying levels of statistical certainty, in a large panel of admixed individuals. By comparing such ancestry profiles for the genome's missing pieces to similar determinations across the known/mapped/assembled majority of these individuals’ genomes, each missing piece could in principle be connected to the genomic location at which it resides, even if we lack a continuous path of cloned, assembled sequence with which to make such a connection (Fig. 1b). Sources of the missing pieces We used three sources of unplaced genome sequence: (i) the current reference genome (hg19), which contains 59 unplaced contigs (~5Mbp of euchromatic sequence) for which the correct location is either only known at the chromosomal level or not known at all; (ii) the HuRef genome20, assembled by random shotgun sequencing of a single individual, containing an even larger number of unplaced scaffolds (~3.5Mbp of euchromatic sequence in 28 scaffolds >100kbp and ~7Mbp of euchromatic sequence in 698 scaffolds >10kbp); and (iii) sequence from bacterial artificial chromosome (BAC) and fosmid clones available from GenBank21 (Online Methods). NIH-PA Author Manuscript An approach for admixture mapping unplaced sequence NIH-PA Author Manuscript Manuscript NIH-PA Author Manuscript Specifically, we can test ancestry-informative SNPs for correlation between their genotypes and inferred local ancestry across the genome, estimated using available genome-wide genotypes19. This is different from, and potentially much more powerful than, detecting LD between genotypes at two SNPs, since the correlation between genotypes and local ancestry is expected to be much stronger (than that between SNPs) at genetic distances up to a few cM, and the distance between unmapped “missing pieces” and the nearest parts of the reference genome may be substantial. Furthermore, we estimated statistical mapping power from allele frequencies in the ancestral populations and found that it was substantial, even for admixed population samples of even a few hundred individuals (Supplementary Note and Supplementary Figs. 1–3). Thus, admixture mapping could in principle connect sequences that are physically farther apart than most genomic clones (20-180kbp) and LD blocks (15-50kbp). NIH-PA Author Manuscript Nat Genet. Author manuscript; available in PMC 2013 October 01. Identifying additional, cryptic missing pieces An additional set of cryptic missing pieces might be missing entirely from human genome reference sequences (i.e. might not even be described as unlocalized sequences, nor present in HuRef) but exist instead as cryptic segmental duplications (or paralogs) of known genomic sequences and have been incorrectly assumed to represent the same genomic sequence as their known paralogs. We reasoned that admixture mapping could also be used to identify cryptic segmental duplications. A SNP that is annotated in the assembled part of the human genome might in fact exist on a cryptic paralogous sequence elsewhere. Therefore, identification of SNPs that admixture-map to a different genomic location than their annotated location might indicate the presence of these SNPs at another genomic location on a cryptic segmental duplication. To identify mismapped SNPs we analyzed genome-wide SNP data from two large African American cohorts. Among the 906,703 SNPs from the Affymetrix 6.0 array genotyped in ~7,800 individuals from the Candidate gene Association Resource (CARe) cohort25 and the 566,714 SNPs from the Illumina HumanHap550 array genotyped in ~1,800 individuals from the ICDB cohort, we identified, respectively, 121 and 15 SNPs that admixture-mapped to genomic locations far from their HapMap26 annotations of physical location (Supplementary Note and Supplementary Table 3). Approximately half these mismapped SNPs belonged to a single region, a ~360kbp segmental duplication from 16q22.2 to 1q21.1 involving the HYDIN gene27–29, confirmed by fluorescence in situ hybridization (FISH), and previously found to give rise to false genome-wide association signals at 16q22.2, that in fact arise from true association at the Duffy locus at 1q23.230 (Supplementary Tables 4 and 5). Excluding the HYDIN paralog, incorrect mapping for ~30 SNPs can be explained by known segmental duplications31–37, while for the remaining ~40 mismapped SNPs, the most likely explanation is that they reside on sequence missing from the reference genome. (Among the ~30 SNPs that we simply re-mapped from one known segmental duplication copy to another, ten corresponded to sites previously used as single unique nucleotides38 (SUNs) to distinguish among known segmental duplications. By definition, none of the re-mapped SNPs with which we identified novel segmental duplications corresponded to SUNs.) NIH-PA Author Manuscript NIH-PA Author Manuscript To understand the relationships between these cryptic paralogs and unplaced scaffolds from large sequencing efforts, we cross-referenced the locations of these SNPs with alignments of unlocalized sequence from HuRef and GenBank. Mapping the human genome's missing pieces If an ancestry-informative SNP resides on an unmapped contig, we can map the location of the contig by admixture mapping the SNP. We (i) aligned all unmapped sequence reads from the 1000 Genomes Project22,23 to unplaced scaffolds from HuRef, (ii) identified polymorphic sites across these unplaced sequences, and (iii) computed genotypes at each locus in all European (CEU) and West African (YRI) samples (Online Methods). We selected 314 ancestry-informative SNPs whose genotypes had Pearson correlation r2>15% with local ancestry. We then genotyped these SNPs in a cohort of 380 African American participants from the Jackson Heart Study24 (JHS), selecting this sample size based on initial analyses of the predicted power to map each SNP as function of the number of available genotypes (Online Methods and Supplementary Fig. 3). We successfully admixture-mapped 139 SNPs (Supplementary Fig. 4 and Supplementary Table 1), assigning locations for 70 previously unlocalized scaffolds (Fig. 2 and Supplementary Table 2). We never observed SNPs from the same scaffold mapping to Nat Genet. Author manuscript; available in PMC 2013 October 01. Genovese et al. Page 4 NIH-PA Author Manuscript different locations, as could be the case if the scaffold were itself misassembled. Sequences mapped by this approach comprised a total of ~4Mbp of euchromatic sequence that had not been included or mapped in hg19. NIH-PA Author Manuscript We describe the properties of these mapped locations below. We describe the properties of these mapped locations below. Nat Genet. Author manuscript; available in PMC 2013 October 01. Identifying additional, cryptic missing pieces We identified 18 sequences >40kbp each containing one or more of the mismapped SNPs. Twelve of these 18 regions (spanning ~1.3Mbp of euchromatic sequence) could not be explained by segmental duplications already annotated in the reference genome; these indicate the presence of cryptic segmental duplications. To critically evaluate these findings by an independent method, we utilized the principle that cryptic segmental duplications should give rise, for SNPs called from sequencing data, to excess heterozygosity that does not follow simple models of Hardy Weinberg equilibrium between pairs of alleles. We searched for such a signal – annotated SNPs that behave more Genovese et al. Page 5 Page 5 like paralogous sequence variants (PSVs) – in data from the 1000 Genomes Project pilot, and were able to confirm all of these regions (Online Methods and Supplementary Table 6). For 8 of these 12 cryptic segmental duplications we could find no mention in the literature. We further confirmed six of them by inter-chromosomal LD analysis using HapMap genotypes (Table 1). NIH-PA Author Manuscript We determined for each region whether the alternate allele for any of the mismapped SNPs was present in any of the BAC clones aligning to that region, by aligning sequences from BAC clones retrieved from GenBank to the hg19 reference genome. For SNPs in six of these regions we could identify BAC clones carrying the alternate allele, suggesting that these clones harbor the sequence where these SNPs actually reside (Table 1). For one of these regions containing the gene PRIM2, further analysis indicated an intra-chromosomal duplication in the pericentromeric region of chromosome 6 and an additional inter- chromosomal duplication in the pericentromeric region of chromosome 3 (Supplementary Note). We confirmed the existence of this triplication by the existence of excess sequence read depth across this region in low-coverage data from the 1000 Genomes Project (Fig. 3a and Supplementary Fig. 5) and fluorescence in situ hybridization (FISH) analysis (Fig. 3b). We also observed that the copy in the reference genome is a hybrid of the two copies on chromosome 6 due to a misassembly (Supplementary Note and Supplementary Fig. 6). anuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Nat Genet. Author manuscript; available in PMC 2013 October 01. Pericentromeric locations of the missing pieces Despite the fact that most of the 300 or so gaps8 in the reference human genome exist in interstitial regions, most of the sequence we were able to localize mapped not to interstitial gaps but to cytogenetically-defined heterochromatic regions of the human genome. Among the mapped scaffolds, 57 of 70 mapped to pericentromeric regions (Fig. 2 and Supplementary Table 2). Among the re-mapped SNPs identifying cryptic segmental duplications, 40 of 70 mapped to pericentromeric regions. (In all these cases the resolution of the mapping was limited to the pericentromeric region identified.) We sought to confirm these pericentromeric mappings using both published and new cytogenetic data. Among the 70 scaffolds we mapped successfully, 17 were among 29 scaffolds that were previously analyzed by FISH (Supplementary Information of 39 and Supplementary Table S8 of 20). All 17 of these admixture-based mappings were consistent with one of the often multiple locations suggested by FISH (Fig. 2 and Supplementary Table 2). While confirmatory, this result also emphasizes the discerning power of admixture mapping over techniques based on hybridization, as the latter can yield ambiguous results when clones contain segmental duplications or other kinds of repeats. NIH-PA Author Manuscript NIH-PA Author Manuscript We also performed additional FISH experiments to critically evaluate the mappings of five novel cryptic paralogous sequences for which no previous FISH data existed. In all (5/5) cases, FISH confirmed the presence of the additional copy in the predicted pericentromeric region (Fig. 4 and Online Methods). A further prediction of these mappings to pericentromeric regions involves the sequence content of the respective scaffolds. If these genomic missing pieces are indeed euchromatic islands within heterochromatic oceans, then they might frequently contain heterochromatic beaches consisting of the satellite sequences associated with human centromeres. To evaluate this prediction, we measured the amount of sequence classified as heterochromatic satellite on each scaffold. The great majority (50/57) of the scaffolds that admixture-mapped to pericentromeric regions contained more than 5% satellite sequence (Online Methods, Supplementary Fig. 4, and Supplementary Table 2), compared with almost none (1/13) of the scaffolds that admixture-mapped to interstitial regions (p=0.003). NIH-PA Author Manuscript Genovese et al. Page 6 Another prediction of these pericentromeric mappings is that, given earlier data indicating that recombination within centromeres is likely to be heavily repressed40, scaffolds mapping to the same pericentromeric regions might show LD with one another. Are the missing pieces copy number variable? Although the cryptic, pericentromeric euchromatic regions described here have not been purposefully interrogated in earlier CNV studies, they may have been indirectly interrogated via assays that targeted paralogous sequences in the known, assembled parts of the human genome. This seems the likely scenario, as almost all of the mismapped SNPs we identified from genotyping arrays (63 of 70, not including the HYDIN locus) fall within CNVs reported in the Database of Genomic Variants (DGV)43 (Supplementary Table 3), despite the fact that DGV CNVs together cover less than a third of the human genome. Given the sequence divergence over the identified cryptic paralogs (often greater than 2%), these additional copies are likely to have fixed in the ancestors of all humans. Identifying CNVs over these sequences at a greater rate than for the rest of the genome may therefore indicate the instability of sequences in pericentromeric regions rather than a persistent state of polymorphism of these additional copies in the human population after the duplication event. To evaluate CNV of four selected paralogous region-pairs, we analyzed read depth of coverage and paralogous sequence variation using data from the 1000 Genomes Project (Online Methods). We identified common CNVs affecting the segmental duplications from the 2p22.2, 4q35.2, and DUSP22 loci (Supplementary Figs. 7–9), and we found evidence for CNVs affecting either of the PRIM2 cryptic paralogs (Fig. 3a and Supplementary Fig. 5). In each case we could confirm using PSVs that the cryptic paralogs, rather than the paralogs present in the reference genome, account for the observed copy number variation (Supplementary Note), consistent with CNVs having arisen in the pericentromeric paralogs. NIH-PA Author Manuscript Pericentromeric locations of the missing pieces We identified pairs of SNPs (from distinct scaffolds) with significant linkage disequilibrium not due to admixture, and were able to identify ~500 SNPs from distinct scaffolds mapping to same genomic regions (Supplementary Table 7). In no instance did these LD-based relationships among scaffolds disagree with our mappings from admixture. To understand how the pericentromeric missing pieces relate to the known human genome, we aligned their sequences to hg19; virtually all scaffolds mapping to pericentromeric regions were found to consist of one or more segmental duplications of mapped euchromatic sequence, with 2-5% sequence divergence (Supplementary Table 2). This suggests that a large fraction of these sequences arrived at their current locations by a process of segmental duplication in primate ancestors41. Our mapping of these cryptic segmental duplications to centromeric regions is consistent with an earlier finding that most chromosome arms (35 of 43) have an increasing number of known interchromosomal duplications in the proximity of the centromeres42; both results appear to reflect a tendency of interchromosomal duplications to deposit sequence at and around centromeres. NIH-PA Author Manuscript Nat Genet. Author manuscript; available in PMC 2013 October 01. Expression of protein-coding genes from pericentromeric regions Cryptic, pericentromeric paralogs of known protein-coding genes could in principle be either pseudogenes or expressed, intact genes. To test whether cryptic paralogs of coding genes are expressed at an RNA level, we analyzed RNA-seq data from the Human BodyMap 2.0 project. We focused on reads aligning to the genes DUSP22, PRIM2, HYDIN, MAP2K3, and KCNJ12, all of which appear to have cryptic paralogs in pericentromeric regions (Fig. 3 and Supplementary Figs. 5, 9, and 10). To distinguish RNA arising from Nat Genet. Author manuscript; available in PMC 2013 October 01. Genovese et al. Page 7 reference gene copies from RNA arising from the cryptic paralogs, we focused on reads covering PSVs identifiable from genomic DNA sequence (many of which were previously mis-annotated as SNPs); this makes it extremely likely that sequence differences observed in RNA have a genomic origin (Fig. 5 and Online Methods). We identified expressed RNA for all of the paralogs except MAP2K3 (Fig. 5). NIH-PA Author Manuscript The expression of cryptic, pericentromeric gene copies showed several kinds of relationship to expression of their paralogs. Both DUSP22 and its recently duplicated paralog were expressed and exhibited similar distribution across tissues. In contrast, the cryptic paralogs of PRIM2, which contain only exons 6-14 of the original transcript (as shown in Fig. 3a), give rise to shorter transcripts that are expressed exclusively in brain and testes (Fig. 5). For HYDIN, which is expressed in brain and several other tissues, this analysis indicated that the cryptic paralog at 1q21.1 is expressed in the brain, consistent with its earlier observation in a brain cDNA library28. For KCNJ12 we could detect expression of the paralog KCNJ18 in testis tissue (Supplementary Fig. 11), though we did not detect this KCNJ12 paralog in skeletal muscle where KCNJ18 is known to be expressed at a level sufficient to cause a phenotype44. The tissue specificity observed for paralogous copies is also evidence that these observations are not the result of sequencing errors at putative PSV sites. uscript NIH-PA Author Manuscript These results suggest that many of these cryptic, pericentromeric gene paralogs are expressed genes, and that their expression patterns can differ from those of their known paralogs. NIH-PA Author Manuscript DISCUSSION We have described a population-based approach for helping to assemble the rest of the euchromatic human genome, even when missing pieces are separated from known euchromatic sequence by extensive heterochromatic sequence. Because our approach uses data that are widely available or are quickly becoming so, its power will increase quickly in the coming years. We anticipate that this approach will help complete physical maps of the human genome. Analysis of ancestry-informative markers in unlocalized scaffolds can be used to map the genomic locations of these scaffolds with a physical resolution comparable to that of FISH but with unambiguous mapping to individual loci, and in a highly scalable way that will become inherently more powerful as sequence data sets grow. (Many aspects of the genome assembly will continue to require other methods – for example, our approach does not determine the physical orientation of novel sequence with respect to the chromosome.) Using this approach we mapped ~4Mbp of unplaced euchromatic sequence, most of which we found to be embedded in the heterochromatic regions of the genome. These regions are not included in the current human reference genome and, with two exceptions, they do not overlap with any of the current patches included in the latest revision (Supplementary Table 8). NIH-PA Author Manuscript NIH-PA Author Manuscript One limitation of our approach is that it relies on novel sequence having been correctly assembled and distinguished from paralogous sequence. Most sequences from HuRef unplaced scaffolds have a divergence greater than 2% with their closest paralogs; due to limitations of shotgun sequencing assembly, paralogous segments with <2% sequence divergence are likely to be under-represented in human genome assemblies45. Unfortunately, due to their short read lengths, current whole-genome next-generation sequencing approaches do not provide better assemblies for such regions than those obtained i h ill b d i h 46 N h l h d h d i One limitation of our approach is that it relies on novel sequence having been correctly assembled and distinguished from paralogous sequence. Most sequences from HuRef unplaced scaffolds have a divergence greater than 2% with their closest paralogs; due to limitations of shotgun sequencing assembly, paralogous segments with <2% sequence divergence are likely to be under-represented in human genome assemblies45. Unfortunately, due to their short read lengths, current whole-genome next-generation sequencing approaches do not provide better assemblies for such regions than those obtained with capillary-based sequencing approaches46. Nat Genet. Author manuscript; available in PMC 2013 October 01. DISCUSSION Nonetheless, we showed that admixture Genovese et al. Page 8 NIH-PA Author Manuscript mapping the SNPs ascertained in such regions can still allow the discovery and mapping of these cryptic paralogous sequences. Our results have several potential implications for disease gene mapping in humans, particularly wherever genetic signals map near pericentromeric regions, assembly gaps, and segmental duplications. • Copy number variations (CNVs) frequently straddle or are flanked by ambiguous regions of the genome assembly. For example, deletions and duplications at 1q21.1 reported to affect ~1.5Mbp of genomic sequence associate with cardiac developmental defects47, schizophrenia48,49, mental retardation, autism, congenital anomalies50, and abnormal head size51. Fully defining the gene content of these CNVs will require interrogating the missing sequence hidden in the assembly gaps at 1q21.1. nuscript NIH-PA Author Manuscript • Some regions implicated in genome-wide association studies may require re- analysis in light of the results here. For example, human height associates with rs17511102 and other markers in a lincRNA-containing segment of 2p22.252 for which we found a cryptic segmental duplication (and paralogous lincRNA) in the pericentromeric region of chromosome 22. Following up this association will require that markers throughout the region be re-assigned to the correct paralogous gene copies. NIH-PA Author Manuscript • Gene SERPINB6 was associated with a clinical phenotype through homozygosity mapping by the identification of an homozygous region terminated by the heterozygous genotype of SNP rs776281153, which our results suggest being incorrectly assigned to 6p25.3 while in fact residing at 16p11.2, leading to a slight underestimation of the correct homozygous region. • The genes affected by cryptic segmental duplications may be functionally important and critical to include and explicitly model in exome sequencing studies. For example mutations in KCNJ18, a gene missing from the reference genome, have been shown to cause thyrotoxic hypokalemic periodic paralysis44. • An admixture mapping study found that African Americans with multiple sclerosis have elevated European ancestry around the centromere of chromosome 115, a region to which our work has assigned more than a megabase of novel sequence. We showed that CNVs are more common over cryptic paralogs missing from the reference genome, most likely due to the physical instability of pericentromeric regions. We also showed that paralogous genes in these cryptic pericentromeric duplications are transcribed, sometimes with patterns of expression that diverge from those of their paralogs, and therefore potentially serving unique biological functions. Nat Genet. Author manuscript; available in PMC 2013 October 01. Alignment of HuRef genome and GenBank BAC and fosmid clones To align the HuRef genome and sequenced BAC and fosmid clones to the hg19 reference genome we first downloaded all available sequence from The Institute for Genomic Research and GenBank websites (by downloading the scaffold-not-in-chromosome.fasta file from ftp://ftp.tigr.org/pub/data/huref/ and all gbpri* files from ftp://ftp.ncbi.nih.gov/ genbank/), and then used BWA57 (using bwa bwasw) for the alignments against hg19. We identified repeats classified as satellite sequences over HuRef unplaced scaffolds using RepeatMasker58. Satellite sequence consists of large arrays of tandemly repeated units of non-coding DNA. The amount of satellite and missing sequence is reported for each unplaced scaffold (Supplementary Fig. 4 and Supplementary Table 2). To identify within these resources the presence of cryptic segmental duplications, that is, sequence missing from the current reference genome but present in a diverged duplicated form, we aligned all available contigs from HuRef and GenBank clones against hg19 (Supplementary Table 2). NIH-PA Author Manuscript DISCUSSION NIH-PA Author Manuscript The presence of duplicated regions complicates genome assemblies, SNP and CNV discovery (Supplementary Figs. 12–24). Notably, HYDIN and PRIM2 are among the most difficult genes to reconstruct using de novo assembly from short sequence reads54. PRIM2 and KCNJ12 are among the genes with the largest number of mis-identified non- synonymous SNPs55, most likely due to identification of PSVs as SNPs. Approximately 6% of the human genome reference is currently considered unreliable for variant discovery by the 1000 Genomes Project23, due to dearth or excess read coverage or poor alignment of sequence reads. Most of the regions we identified as harboring a cryptic segmental duplication (Table 1 and Supplementary Table 6) fall in this inaccessible part of the human genome. While waiting for a more complete version of the human genome reference, the 1000 Genomes Project now aligns sequence data to an expanded genome Genovese et al. Genovese et al. Page 9 Page 9 reference that includes additional unlocalized sequences (termed “decoy sequences”), to reduce false alignments in regions with cryptic segmental duplications. These additional sequences consist mainly of sequenced clones discarded by the Human Genome Project and sequence from the HuRef assembly (~30% of decoy sequences consist of HuRef unlocalized scaffolds). Of course, the eventual goal of such projects will be the alignment of all human sequence reads to their actual physical locations. NIH-PA Author Manuscript In completing maps of the human genome, the important remaining challenges include mapping the human genome's structure at all scales, fully cataloging the genome's sequence content, and appreciating how sequences are ordered and arranged along chromosomes. As the scientific community works toward a complete reference assembly of the human genome56, analysis of genome-wide data from admixed populations will add unique value and help complete our understanding of the human genome's structure and evolution. anuscript NIH-PA Author Manuscript URLs. HuRef unplaced scaffolds, ftp://ftp.tigr.org/pub/data/huref/ GenBank database: ftp://ftp.ncbi.nih.gov/genbank/ Database of Genotypes and Phenotypes (dbGaP): http://www.ncbi.nlm.nih.gov/gap Database of Genotypes and Phenotypes (dbGaP): http://www.ncbi.nlm.nih.gov/gap NIH-PA Author Manuscript Illumina iControlDB: http://www.illumina.com/science/icontroldb.ilmn HapMap inter-chromosomal LD: ftp://ftp.ncbi.nlm.nih.gov/hapmap/inter_chr_ld/ HapMap inter-chromosomal LD: ftp://ftp.ncbi.nlm.nih.gov/hapmap/inter_chr_ld/ Illumina Human BodyMap 2.0 data: http://www.ncbi.nlm.nih.gov/projects/geo/query/ acc.cgi?acc=GSE30611 Illumina Human BodyMap 2.0 data: http://www.ncbi.nlm.nih.gov/projects/geo/query/ acc.cgi?acc=GSE30611 Decoy sequences: ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/technical/reference/ phase2_reference_assembly_sequence/ Nat Genet. Author manuscript; available in PMC 2013 October 01. Alignment and variant calls for 1000 Genomes Project data For genotyping from sequence reads, we selected all the CEU and YRI samples available in the 1000 Genomes Project22,23. Unmapped reads were aligned against the HuRef unplaced scaffolds using BWA59 (using bwa aln/sampe). Genotype calling in the unplaced contigs was performed using the Genome Analysis Toolkit60 (GATK), using default settings for the UnifiedGenotyper walker. Nat Genet. Author manuscript; available in PMC 2013 October 01. Page 10 Page 10 Genovese et al. Strategy for admixture mapping NIH-PA Author Manuscript To map the location of a SNP, the genotypes were first adjusted by regressing for amount of global West African ancestry for each sample. The adjusted genotypes were then tested for correlation with local ancestry across the genome using a one-tailed Pearson correlation test. If the correlation of the genotypes with global West African ancestry is positive, a right- tailed test is chosen, otherwise a left-tailed test is chosen. The location corresponding to the smallest p-value is then recorded for each SNP, together with the location corresponding to the smallest p-value in a different chromosome. All these steps were performed using custom MATLAB (2011b, The MathWorks, Natick, MA) scripts. It is intuitive to expect that genotyping SNPs over paralogous sequences, only one of which will be expected to be polymorphic, will be often incorrect as it won't be possible to correctly infer the homozygous state for the alternate allele, leading to failure of Hardy- Weinberg equilibrium among other things. This is not always so for genotyping arrays however, as genotyping of SNPs is often based on a two-dimensional Gaussian mixture model over summarized probe intensities for each of the two alleles61, enabling the correct distinction of the three possible genotypes even without modeling the presence of a cryptic paralog. SNP selection, sample selection, and Sequenom genotyping From all detected SNPs in hg19 unplaced contigs and HuRef unplaced scaffolds we filtered out SNPs at loci for which the number of reads with mapping quality 0 was at least 4 and at least 10%. We also filtered out clusters of four SNPs within a window size of 10bp. The rationale is that in loci with ambiguous alignment, it is possible to call SNPs which actually belong to a paralogous region of the genome. Variants called in loci where many SNPs cluster together have a higher chance to be an artifact of misaligned reads originating from paralogous regions that are not present in the reference genome used for the alignment. This methodology maximizes the chances that a SNP belongs to the unplaced scaffold where it is called. Among the filtered list, up to 7 ancestry-informative SNPs were chosen for each contig for which genotype was estimated to have Pearson correlation coefficient with amount of local European ancestry satisfying r2>15%. SNPs were further filtered to fit within 10 Sequenom plexes, prioritizing degree of correlation with ancestry. We selected 380 samples from the Jackson and Heart Study (JHS)24, which had been genotyped with the Affymetrix 6.0 array and analyzed with HAPMIX62. To achieve the maximum possible mapping resolution, we exclusively selected samples with at least 62 detected crossovers between ancestries (maximum was 115). NIH-PA Author Manuscript Most likely due to the repetitiveness of the flanking sequences for which primers were designed, 86 assays failed completely; of the remainder, 53 failed the Hardy-Weinberg equilibrium test (p<10-6), and 175 passed. Nevertheless, we could still reliably identify the location of 139 SNPs (Pearson correlation test p<10-6), 106 of which had passed and 33 of which had failed Hardy-Weinberg equilibrium test, showing that SNPs with unreliable genotypes can still be informative for mapping purposes (Supplementary Fig. 4 and Supplementary Table 1). By analyzing for each successfully mapped SNP the best correlation between adjusted genotype and local ancestry on chromosomes other than the one where the SNP mapped, we estimated that the selected conservative threshold of 10-6 for the p-value gives a false discovery rate lower than 1%. Nat Genet. Author manuscript; available in PMC 2013 October 01. Fluorescence in situ hybridization Peripheral blood mononuclear cells were stimulated with phytohemagglutinin and harvested. Metaphase spreads were prepared by standard protocols. Fosmid clones spanning the regions of interest were selected for FISH mapping using the University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu/). Fosmids were labeled with either SpectrumOrange or SpectrumGreen conjugated dUTP using a nick translation kit (Abbott Molecular, Des Plaines, IL). Labeled pairs were hybridized overnight to metaphase chromosome preparations. Following 4x SSC/0.1% Tween, 2xSSC/0.3% Tween, and phosphate-buffered detergent washes, chromosomes were counterstained with DAPI and analyzed by epi-fluorescence with a Zeiss Axioplan2 microscope (Thornwood, NY) and Applied Imaging CytoVision software (Santa Clara, CA). Analysis of cryptic paralogs from 1000 Genomes Project pilot data To identify regions with an excess of PSVs suggesting the presence of large cryptic segmental duplications, we searched for SNPs across the reference genome whose probabilistic genotype from 1000 Genomes Project pilot low-pass sequencing data failed the Genovese et al. Page 11 Hardy-Weinberg equilibrium test63 (using bcftools view -c). We identified variants that failed the equilibrium test (p<10-6) in CEU and YRI samples, grouped them together if they were <5kbp apart (using custom MATLAB scripts), and listed all resulting regions >40kbp (Supplementary Table 6). NIH-PA Author Manuscript Analysis of sequence read depth from 1000 Genomes Project data To assess the copy number variability of the missing reference segments, we used an updated version of Genome STRiP64 to analyze read depth. Normalized read depth was measured by comparing the number of DNA fragments with sequencing reads aligned to the reference genome in a given region to the expected read depth per haploid copy based on (i) the total sequencing depth for each sample, (ii) the alignability of each position, based on whether it would be uniquely mapped by a perfect 36bp read, and (iii) sequencing bias due to GC content. We performed normalization for GC-bias empirically, similar to 38. We first identified a 588Mbp subset of the autosomal reference sequence with no known evidence of copy number variation to use as a baseline. We removed all positions within 200bp of annotated CNV regions listed in DGV, segmental duplications listed in the UCSC browser, repeats annotated by RepeatMasker58 and assembly gaps, yielding a subset that is highly likely to be copy number invariant in the majority of people. This reference subset is divided into 400bp windows, stratified by GC fraction within each window, and the observed read depth at each GC fraction is compared to the total read depth across all windows to yield a GC- normalization curve for each sequencing library. NIH-PA Author Manuscript Given a genomic locus, estimation of diploid copy number for each sample was done by fitting a Gaussian mixture model with sample-specific variance to the observed and expected read depth for each sample64, allowing the model to fit as many copy number classes as needed at each locus. To analyze genome regions with known paralogs in sequences not in the hg19 reference (notably 2p22.2), we used BWA59 (using bwa aln/sampe) to realign the 1000 Genomes reads from the genomic region to a synthetic reference containing the original reference sequence plus the sequence for the extra paralog. Estimation of copy number was then carried out as described above. Nat Genet. Author manuscript; available in PMC 2013 October 01. Analysis of RNA sequence expression data To compare expression of different paralogs of genes DUSP22, PRIM2, HYDIN, MAP2K3, and KCNJ12, we first identified PSVs over the predicted mRNA for these genes looking at all heterozygous loci called for 1000 Genomes Project pilot high coverage samples NA12878/CEU, NA12891/CEU, NA12892/CEU, NA19238/YRI, NA19239/YRI, and Nat Genet. Author manuscript; available in PMC 2013 October 01. Genovese et al. Page 12 Page 12 NIH-PA Author Manuscript NA19240/YRI and then determined, when possible, which allele belongs to each paralog (Supplementary Tables 9–13). Once we obtained a list of all PSVs, we counted reads from the Illumina Human BodyMap 2.0 project for each of the alleles observed at the locus using the GATK60 (using default settings for the UnifiedGenotyper walker and custom scripts). To validate the findings and filter out possible artifacts, sequence reads were further manually analyzed using the Integrative genomics viewer65 (IGV). Supplementary Material Refer to Web version on PubMed Central for supplementary material. Nat Genet. Author manuscript; available in PMC 2013 October 01. Acknowledgments script NIH-PA Author Manuscript This study was supported by grants RC1 GM091332-01 (S.A.M. and J.G.W.) and R01DK54931 (G.G. and M.R.P.) from the National Institutes of Health, and by a Smith Family Foundation Award for Excellence in Biomedical Research (S.A.M.). The Jackson Heart Study is supported and conducted in collaboration with Jackson State University (N01- HC-95170), University of Mississippi Medical Center (N01-HC-95171), and Touglaoo College (N01-HC-95172) contracts from the National Heart, Lung, and Blood Institute (NHLBI) and the National Institute for Minority Health and Health Disparities (NIMHD), with additional support from the National Institute on Biomedical Imaging and Bioengineering (NIBIB). NIH-PA Author Manuscript The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts (HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, and HHSN268201100012C). The Coronary Artery Risk Development in Young Adults Study (CARDIA) is conducted and supported by the National Heart, Lung, and Blood Institute (NHLBI) in collaboration with the University of Alabama at Birmingham (N01-HC95095 & N01-HC48047), University of Minnesota (N01-HC48048), Northwestern University (N01- HC48049), and Kaiser Foundation Research Institute (N01-HC48050). MESA, MESA Family, and the MESA SHARe project are conducted and supported by the National Heart, Lung, and Blood Institute (NHLBI) in collaboration with MESA investigators. Support for MESA is provided by contracts N01-HC-95159 through N01-HC-95169 and RR-024156. Funding for MESA Family is provided by grants R01-HL-071051, R01-HL-071205, R01-HL-071250, R01-HL-071251, R01-HL-071252, R01-HL-071258, R01-HL-071259. MESA Air is funded by the US EPA - Science to Achieve Results (STAR) Program Grant # RD831697. Funding for genotyping was provided by NHLBI Contract N02-HL-6-4278 and N01-HC-65226. 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Admixture mapping the human genome's missing pieces. Nat Genet. Author manuscript; available in PMC 2013 October 01. References (a) Chromosomes of West Afric descent (red) have recombined with chromosomes of European descent (blue) through admixture to form mosaic genomes in African Americans. (b) Localization of genomic “missing pieces”, including unlocalized scaffolds and cryptic segmental duplications, by admixture mapping. Wherever allele frequencies have been influenced by genetic drift in ancestral populations, significant correlation between genotype and local ancestry allows unplaced genomic sequence to be mapped to its correct location. Pag Pag Page 16 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Autho NIH-PA Author Manuscript Figure 1. Nat Genet. Author manuscript; available in PMC 2013 October 01. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Au Figure 1. gu e . Admixture mapping the human genome's missing pieces. (a) Chromosomes of West African descent (red) have recombined with chromosomes of European descent (blue) through admixture to form mosaic genomes in African Americans. (b) Localization of genomic “missing pieces”, including unlocalized scaffolds and cryptic segmental duplications, by admixture mapping. Wherever allele frequencies have been influenced by genetic drift in the ancestral populations, significant correlation between genotype and local ancestry allows the unplaced genomic sequence to be mapped to its correct location. g Admixture mapping the human genome's missing pieces. (a) Chromosomes of West African descent (red) have recombined with chromosomes of European descent (blue) through admixture to form mosaic genomes in African Americans. (b) Localization of genomic “missing pieces”, including unlocalized scaffolds and cryptic segmental duplications, by admixture mapping. Wherever allele frequencies have been influenced by genetic drift in the ancestral populations, significant correlation between genotype and local ancestry allows the unplaced genomic sequence to be mapped to its correct location. Nat Genet. Author manuscript; available in PMC 2013 October 01. Genovese et al. Nat Genet. Author manuscript; available in PMC 2013 October 01. Figure 2. Figure 2. Approximate locations of previously unplaced genome-sequence scaffolds that were mapped by our approach. Contigs from hg19 are labeled with three digits and stand for GL000###, while scaffolds from HuRef are labeled with five digits and stand for SCAF_11032791#####. Scaffolds with available chromosomal assignment or FISH data are denoted in blue; other scaffolds are denoted in red. Green indicates BAC clones that we mapped through SNPs from the CARe, ICDB, or HapMap datasets. No scaffold was mapped to a location incompatible with FISH data. Mappings in the pericentromeric regions of acrocentric chromosomes indicate any location either in the pericentromeric regions or the short arms. Figure 2. Approximate locations of previously unplaced genome-sequence scaffolds that were mapped by our approach. Contigs from hg19 are labeled with three digits and stand for GL000###, while scaffolds from HuRef are labeled with five digits and stand for SCAF_11032791#####. Scaffolds with available chromosomal assignment or FISH data are denoted in blue; other scaffolds are denoted in red. Green indicates BAC clones that we mapped through SNPs from the CARe, ICDB, or HapMap datasets. No scaffold was mapped to a location incompatible with FISH data. Mappings in the pericentromeric regions of acrocentric chromosomes indicate any location either in the pericentromeric regions or the short arms. NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 3. Cryptic paralogs of the PRIM2 gene. (a) Analysis of sequencing coverage depth in data from the 1000 Genomes Project suggests the presence of three segments (blue arrows) with elevated copy number. While the copy number of each segment appears to be fixed in most genomes, at least two genomes show extra copy number gain at two of the three segments (HG00155/GBR at region1 + region2, blue, and NA18541/CHB at region2 + region3, red), suggesting a model in which there are two additional copies of this locus in most human genomes, one copy containing region1 + region2 and another copy containing region2 + region3. (b) FISH analysis of PRIM2 and its cryptic paralogs. Fosmid clone WI2-0569M11 overlapping PRIM2 (G248P8956G6 aligned to chr6:57,417,677-57,467,167) hybridized to two distinct locations in the pericentromeric region of chromosome 6, 6p11.2 and 6q11.1, and to a third location in the pericentromeric region of chromosome 3, confirming the two additional partial copies of the PRIM2 gene missing from the reference genome. Genovese et al. Page 18 Page 18 Genovese et al. NIH-PA Author Manuscript Nat Genet. Author manuscript; available in PMC 2013 October 01. Figure 3. Figure 3. Cryptic paralogs of the PRIM2 gene. (a) Analysis of sequencing coverage depth in data from the 1000 Genomes Project suggests the presence of three segments (blue arrows) with elevated copy number. While the copy number of each segment appears to be fixed in most genomes, at least two genomes show extra copy number gain at two of the three segments (HG00155/GBR at region1 + region2, blue, and NA18541/CHB at region2 + region3, red), suggesting a model in which there are two additional copies of this locus in most human genomes, one copy containing region1 + region2 and another copy containing region2 + region3. (b) FISH analysis of PRIM2 and its cryptic paralogs. Fosmid clone WI2-0569M11 overlapping PRIM2 (G248P8956G6 aligned to chr6:57,417,677-57,467,167) hybridized to two distinct locations in the pericentromeric region of chromosome 6, 6p11.2 and 6q11.1, and to a third location in the pericentromeric region of chromosome 3, confirming the two additional partial copies of the PRIM2 gene missing from the reference genome. NIH-PA Author Manuscript Page 19 Genovese et al. Figure 4. FISH analysis confirmed the presence of cryptic segmental duplications. (a) Fosmid clone WI2-1750D05 (G248P87673B3 aligned to chr2:133,062,362-133,104,847); (b) WI2-1656E10 (G248P83226C5 aligned to chr3:613,680-650,737) hybridized to the centromeric/acrocentric region of chromosome 14, as predicted by admixture mapping; (c) WI2-0903H06 (G248P8635D3 aligned to chr4:3,573,606-3,614,890) hybridized to the centromere of chromosome 9, as predicted by admixture mapping; (d) WI2-1022I06 (G248P82546E3 aligned to chr5:21,531,026-21,568,722) hybridized to 6p11.2. NIH-PA Author Manuscript Figure 4. FISH analysis confirmed the presence of cryptic segmental duplications. (a) Fosmid clone WI2-1750D05 (G248P87673B3 aligned to chr2:133,062,362-133,104,847); (b) WI2-1656E10 (G248P83226C5 aligned to chr3:613,680-650,737) hybridized to the centromeric/acrocentric region of chromosome 14, as predicted by admixture mapping; (c) WI2-0903H06 (G248P8635D3 aligned to chr4:3,573,606-3,614,890) hybridized to the centromere of chromosome 9, as predicted by admixture mapping; (d) WI2-1022I06 (G248P82546E3 aligned to chr5:21,531,026-21,568,722) hybridized to 6p11.2. NIH-PA Author Manuscript Figure 4. Figure 4. FISH analysis confirmed the presence of cryptic segmental duplications. (a) Fosmid clone WI2-1750D05 (G248P87673B3 aligned to chr2:133,062,362-133,104,847); (b) WI2-1656E10 (G248P83226C5 aligned to chr3:613,680-650,737) hybridized to the centromeric/acrocentric region of chromosome 14, as predicted by admixture mapping; (c) WI2-0903H06 (G248P8635D3 aligned to chr4:3,573,606-3,614,890) hybridized to the centromere of chromosome 9, as predicted by admixture mapping; (d) WI2-1022I06 (G248P82546E3 aligned to chr5:21,531,026-21,568,722) hybridized to 6p11.2. Figure 5. Expression of cryptic gene paralogs from pericentromeric regions of the human genome. Nat Genet. Author manuscript; available in PMC 2013 October 01. Figure 3. Paralogous sequence variants were used to distinguish expression of the DUSP22, PRIM2, HYDIN, and MAP2K3 genes and from expression of thier cryptic paralogs, in RNA-seq data from diverse human tissues. Variants in the UTR regions are represented by blue text; variants predicted to change the protein product of the paralog are shown in red; synonymous variants are shown in green. Page 20 Genovese et al. Page 20 Page 20 Pa NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Autho Figure 5. Expression of cryptic gene paralogs from pericentromeric regions of the human genome. Paralogous sequence variants were used to distinguish expression of the DUSP22, PRIM2, HYDIN, and MAP2K3 genes and from expression of thier cryptic paralogs, in RNA-seq data from diverse human tissues. Variants in the UTR regions are represented by blue text; variants predicted to change the protein product of the paralog are shown in red; synonymous variants are shown in green. NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 5. g Expression of cryptic gene paralogs from pericentromeric regions of the human genome. Paralogous sequence variants were used to distinguish expression of the DUSP22, PRIM2, HYDIN, and MAP2K3 genes and from expression of thier cryptic paralogs, in RNA-seq data from diverse human tissues. Variants in the UTR regions are represented by blue text; variants predicted to change the protein product of the paralog are shown in red; synonymous variants are shown in green. g Expression of cryptic gene paralogs from pericentromeric regions of the human genome. Paralogous sequence variants were used to distinguish expression of the DUSP22, PRIM2, HYDIN, and MAP2K3 genes and from expression of thier cryptic paralogs, in RNA-seq data from diverse human tissues. Variants in the UTR regions are represented by blue text; variants predicted to change the protein product of the paralog are shown in red; synonymous variants are shown in green. NIH-PA Author Manuscript Genovese et al. Page 21 Page 21 NIH-PA Author Manuscript NIH-PA Au Segmental duplications localized by admixture mapping. Figure 5. CHR FROM TO BAND GENE SIZE CHR’ FROM’ TO’ BAND’ SCAFFOLD DIV CARE ICDB HAPMAP FISH chr1 83,598,160 83,955,427 1p31.1 POMZP3 ~400kbp chr7 76,182,346 76,575,579 7q11.23 NA ~1.4% 6 1 yes no chr1 206,072,708 206,558,788 1q32.1 FAM72/SRGAP2 ~240kbp chr1 143,880,004 144,095,783 1q21.1 NA ~0.6% 3 0 no no chr2 37,958,019 38,003,219 2p22.2 NA ~45kbp chr22 NA NA 22q11.1 SCAF_1103279187616 ~4.0% 3 0 yes no chr2 91,737,476 91,880,745 2p11.1 OTOP1 ~140kbp chr1 NA NA 1q21.1 RP11-247L13 ~1.2% 2 0 yes no chr2 133,005,020 133,120,083 2q21.2 NA ~115kbp chr20 NA NA 20q11.21 RP11-462H3 >2.0% 1 1 yes yes chr3 612,223 663,367 3p26.3 NA ~50kbp chr22 NA NA 22q11.1 GL000217 ~2.0% 1 0 no yes chr3 75,761,051 75,871,577 3p12.3 ZNF717 >110kbp chr21 NA NA 21q11.2 RP4-813B7 >5.0% 1 0 no no chr4 25,709 68,702 4p16.3 ZNF595 ~40kbp chr22 NA NA 22q11.1 RP11-85C8 ~0.5% 1 0 no no chr4 3,536,207 3,636,136 4p16.3 FLJ35424 ~100kbp chr9 NA NA 9p11.2 SCAF_1103279188214 ~3.0% 1 0 yes yes chr4 190,470,115 190,684,480 4q35.2 NA ~215kbp chr21 NA NA 21q11.2 GL000193 >2.0% 2 0 no no chr5 21,506,326 21,573,437 5p14.3 NA ~65kbp chr6 58,137,660 58,139,549 6p11.2 CH17-92N24 ~1.5% 0 0 yes yes chr6 256,518 382,461 6p25.3 DUSP22 ~125kbp chr16 NA NA 16p11.2 NA ~0.1% 0 1 no no chr6 57,204,729 57,435,462 6p11.2 PRIM2 ~230kbp chr6 NA NA 6p11.2 SCAF_1103279188350 ~2.0% 0 0 no yes chr6 57,204,729 57,608,453 6p11.2 PRIM2 ~400kbp chr6 NA NA 6q11.1 SCAF_1103279188263 ~2.0% 0 0 no yes chr6 57,369,236 57,608,453 6p11.2 PRIM2 ~240kbp chr3 NA NA 3p11.1 SCAF_1103279180085 ~2.0% 3 0 yes yes chr6 57,401,565 57,570,618 6p11.2 PRIM2 >170kbp chr3 NA NA 3p11.1 RP1-216J23 ~2.0% 3 0 yes yes chr6 57,447,574 57,575,919 6p11.2 PRIM2 ~130kbp chr6 NA NA 6p11.2 SCAF_1103279188406 ~2.0% 0 0 no yes chr12 147,380 188,194 12p13.33 FAM138 >40kbp chr20 62,947,067 62,965,512 20q13.33 SCAF_1103279187960 ~1.2% 1 0 no no chr13 19,020,001 19,167,977 13q11 ANKRD30BP2 ~200kbp chr21 14,447,204 14,594,419 21q11.2 NA ~0.8% 3 0 yes no chr14 19,817,857 20,194,548 14q11.2 POTEH/POTEM ~400kbp chr22 16,085,071 16,459,525 22q11.1 NA ~0.6% 8 0 yes no chr16 70,845,287 71,202,573 16q22.2 HYDIN ~360kbp chr1 146,341,167 146,400,000 1q21.1 GL000192 ~0.6% 58 8 yes no chr21 10,971,951 11,032,242 21p11.1 TPTE >60kbp chr13 NA NA 13q11 RP5-1039L24 ~0.2% 1 1 no no chr21 11,083,847 11,156,072 21p11.1 BAGE >80kbp chr13 NA NA 13q11 NA NA 2 0 no no CHR, FROM, TO, BAND: chromosome, hg19 coordinates, and localization of the ancestral copy of the duplication; GENE: protein coding gene(s) overlapping the duplication; SIZE: estimated size of the duplication; CHR’, FRO coordinates, and localization of the derived copy of the duplication; SCAFFOLD: genomic scaffold containing the sequence in the derived copy of the duplication; DIV: estimated sequence divergence between the ancestral and th number of Affymetrix 6.0 SNPs re-mapped in the CARe dataset; ICDB: number of Illumina SNPs re-mapped in the ICDB dataset; HAPMAP: whether independent evidence of the cryptic duplication was confirmed by inter-chrom whether a FISH experiment was performed to validate the duplication. Nat Genet. Author manuscript; available in PMC 2013 October 01. Figure 5. Nat Genet. Author manuscript; available in PMC 2013 October 01. Nat Genet. Author manuscript; available in PMC 2013 October 01.
https://openalex.org/W4313407379	https://link.springer.com/content/pdf/10.1007/s10163-022-01570-y.pdf	English	null	Managing post-conflict demolition wastes in Gaza Strip: a case study on May 2021 conflict	Journal of material cycles and waste management	2,022	cc-by	6,147	Abstract Gaza Strip is considered as one of the armed conflicts prone areas in Middle East. Several intensive conflicts occurred in Gaza Strip in 2008, 2012, 2014 and 2021. These conflicts caused massive destroying the infrastructures, facilities, and buildings, which affected all services and activities in Gaza Strip. One of the major post-conflict issues in Gaza strip is the management of resulted demolition waste including its removal, sorting, recycling, and material recovery. In May 2021, over than 370,000 tons of demolition waste composed of rubbles and debris was generated during 11 days of armed conflict. The accumulated previous experience of rubbles and debris removal and recycling in Gaza Strip supported to perform a quick management approach for safe removal of the post-conflict demolition waste and reuse/recycle the resulted waste materials in various applications. The sorting and transporting process of concreate and non-concreate rubble elements of the waste were carried out in cooperation between local and international agencies as emergency recovery-funded projects. The most proportion of rubbles are concrete aggregates, thus, the material recovery was conducted through crushing process for con- crete rubbles and then reusing it for road rehabilitation or producing concrete building blocks. The large concrete blocks reused to be placed for shoreline protection for specific area along Gaza beach. The recycling of post-conflict demolition waste management projects in Gaza Strip brought economic and social benefits through the reuse and recycle of resources and creation of job opportunities. In conclusion, although the post-conflict demolition waste management is quite different from municipal/industrial waste management in Gaza Strip, it is conducted through applying similar techniques of disaster waste management in waste removal, and those of construction and demolition (C&D) waste management in sorting, crush- ing, and sieving for recycling. Keywords Post-conflict demolition waste management · Rubbles removal & recycling · Material recovery · Construction & demolition waste * Hatem AbuHamed hatem.abuhamed1@gmail.com Managing post‑conflict demolition wastes in Gaza Strip: a case study on May 2021 conflict Hatem AbuHamed1 · Waheed Al Bursh2 · Suleiman Abu Mfarreh3 · Mitsuo Yoshida4,5 Received: 13 August 2022 / Accepted: 27 November 2022 / Published online: 19 December 2022 © The Author(s) 2022 Journal of Material Cycles and Waste Management (2023) 25:684–693 https://doi.org/10.1007/s10163-022-01570-y Journal of Material Cycles and Waste Management (2023) 25:684–693 https://doi.org/10.1007/s10163-022-01570-y Introduction Defining “war” and “conflict” is vital, and in this paper we use the term conflict based on the diction of UNDP (2000) [25]. The Gaza Strip is located in the Middle East, along the east- ern coast of the Mediterranean Sea, bordering Egypt on the southwest and Israel on east and north and, together with the West Bank is part of the Palestinian Territories. The Strip is 40 km long and 6–12 km wide and covers an area of 365 km2. With a population of more than 2.16 million inhab- itants, it is one of the highest densely populated areas in the word [1]. Gaza Strip is divided into 5 governorates: North- ern Gaza, Gaza, Deir Al Balah, Khan Yunis, and Rafah.i * Hatem AbuHamed hatem.abuhamed1@gmail.com 1 MoLG-JICA Project for Capacity Development in Solid Waste Management in Palestine, Ramallah, Palestine 2 Joint Service Council for Solid Waste Management in Gaza and North Gaza Governorates, Gaza, Palestine 3 General Directorate of Joint Service Councils, Ministry of Local Government, Ramallah, Palestine 4 International Network for Environmental and Humanitarian Cooperation Nonprofit Inc., Tokyo, Japan 5 Global Environment Department, Japan International Cooperation Agency (JICA), Tokyo, Japan 1 MoLG-JICA Project for Capacity Development in Solid Waste Management in Palestine, Ramallah, Palestine The Palestinian Territories in general, specifically Gaza Strip is considered as one of armed conflicts prone areas in Middle East. Concerning the sensitive political situations in this hot spot area, several military attacks conducted by Vol:.1 :.(123456789 3 685 Journal of Material Cycles and Waste Management (2023) 25:684–693 Israeli authorities during the last 15 years over Gaza strip, intensive conflicts occurred in 2008, 2012, 2014 and 2021. The Israeli force attacked on Gaza caused massive destroy- ing the infrastructures, facilities and buildings, which affected all services and activities in Gaza. Furthermore, as blockade and isolations have been applied on Gaza Strip since 2007 that lead deteriorated economic conditions of Gaza Strip as most of infrastructure facilities became inad- equate to the function. The blockade and the armed conflicts, together, prevented or delayed the implementation of many development projects, and that Gaza Strip currently is still heavily dependent on the international humanitarian sup- port [2].l basis and is collected, transported, and disposed in prede- termined place. Introduction However post-conflict waste is suddenly generated debris and rubbles at an unpredictable time and place, in which a large amount of waste is generated all at once and blocks/damages roads that are used for waste collection and transportation activities. Therefore, in the operation of post-conflict demolition waste management, at first, it is necessary to secure the access to piles of the post-conflict debris/rubbles for collection and transporta- tion and to establish soon an emergency treatment and disposal system. On the other hand, since post-conflict demolition waste is mostly composed of debris and rub- bles, there are aspects similar to construction and demo- lition (C&D) waste treatment methods that target these types of materials [26]. Moreover, at the generation stage, it has characteristics similar to those of disaster waste. One of the major post-conflict issues in Gaza Strip is the appropriate management of massive amounts of demoli- tions wastes composed of debris and rubbles, which were resulted from damaged infrastructure, housing, commercial buildings, governmental buildings, and others. Report- edly, an amount of 600,000 tons of rubbles generated from 2008/2009 conflict in Gaza [3], while it is estimated that 2 million tons of debris resulted in Gaza as a consequences of 2014 conflict, as this conflict lasted for 51-day [4]. The most recent conflict record, which occurred in May 2021 revealed that approximately over than 370,000 tons of rub- bles, resulted from 11 days of military operation [5]. In addition to that, unexploded ordinances (UXOs) have been reported at the main post-conflict demolition waste disposal sites and in the rubble of destroyed buildings, which pose potential risks [6].l In the light of above, this article aims to present the overview of post-conflict demolition waste management in Gaza Strip with surveying the data on destination of rubble recycling caused by most recent conflict occurred in May 2021, including describing the on-ground actions. It is also included summary of collaborative efforts imple- mented by different agencies involved in rubble removal and recycling activities in the Gaza Strip. Management process and rubbles destinations Regarding solid waste management operations during con- flict emergencies, the waste management service provid- ers such as municipalities and JSCs defined special action measures concerning the accessibility to waste disposal sites and waste accumulation, re-organization of waste collection fleet management and collection operations on-ground and use emergency sites to take the immediate response actions. In regards to post-conflict debris/rubbles management issue during the conflicts time in Gaza strip, the immediate actions taken for the priority of saving people life's once buildings targeted by attacks, through securing heavy machines for specific response actions towards recuse people. Further- more, some fleets assigned to remove partial of debris/ rubbles scattered amounts, which locked the streets for the purpose of roads movements' facilitation and/or transferring them to specific emergency sites. However, generally the solid waste management service providers possess insuffi- cient number of heavy machineries for the removal of post- conflict debris/rubbles, and the need to coordinate with the MoPWH for deploying emergency its heavy machineries. Many of waste management fleets also engage the opera- tion of heavy machineries such as wheel loader and trucks during the emergencies for the post-conflict debris/rubbles removal.Posteriorly, the process of the recovery of debris/ rubbles resulting from conflict in Gaza strip follow common procedures starting from damage assessments, debris/rub- bles removal and material recovery. Generally, the process of debris removal and recovery carried out through private sector contractors who have a certain capacity for machines and transporters used for such process. The final disposal sites for debris/rubbles were selected in coordination with At the same context of assessing other damaged infra- structure, 77 of public buildings were partially and totally damaged, in which 58% of the totally damaged buildings were used for public services. Also, it has been recorded that around 273 educational buildings, 35 health facilities, 239 energy locations, 240 roads, 76 WASH (Water, Sanitation, and Hygiene) facilities and total 116.6 km length of water and wastewater networks, 107 workshops, 38 municipal machinery and 2528 private sector facilities were affected at varying degrees ranging from severe to moderate. The cost estimated for reconstruction efforts to cover entire affected infrastructure were over than 108 million USD [9]. Infrastructure damage assessment in Gaza 2021 Mentioning the infrastructure damage resulting from the attack on Gaza on May 2021, the on-ground survey revealed that the housing sector has experienced the largest propor- tion of damages. The assessment recorded 11,894 housing units as partially damaged distributed among 8,241 build- ings in which 88% of them are concreate buildings [9]. While 664 housing units were totally damaged included within 164 buildings, where 78.2% were concrete buildings and thus resulting of more than 80,000 square meters totally damaged. Gaza and North Gaza governorates have the high- est numbers of totally damaged buildings, with 56% in Gaza and 25% in the North of Gaza. As of October 2021, and according to the latest United Nations Office for the Coor- dination of Humanitarian Affairs (OCHA) Situation Report, about 8,250 internally displaced people (IDPs) across the Gaza Strip [9]. Figures 1 and Fig. 2 show location maps of housing sectors damages in Gaza Strip during May 2021 conflict distributed geographically in terms of totally and partially damage level respectfully, which were produced by UNDP-PAPP. In addition, United Nations Satellite Centre (UNOSAT) provides satellite images for damaged infrastruc- tures in Gaza Strip up to 28th May 2021 [10]. l United Nations Mine Action Service (UNMAS) is involved for related risks regarding the clearance and safe destruction of UXOs for remove explosive items and under- take destruction, assist stakeholders in the rubble removal operations and providing site-specific risk assessments [4] [8]. Materials and methods General solid waste is generated in a certain amount on a daily or weekly 1 3 3 686 Journal of Material Cycles and Waste Management (2023) 25:684–693 As aforementioned, the rubbles removal and recycling in Gaza depends on international agencies’ interventions, as UNDP is the primary agency that considered the rub- ble management during emergencies in Gaza Strip. At the same context, other international agencies were involved for rubbles removal in Gaza after May 2021 attack, such as Egyptian Committee for Gaza Reconstruction and others [5]. In same context, a coordination channels established between Environmental Quality Authority (EQA), Ministry of Local Government (MoLG), Joint Service Councils for Solid Waste Management (JSCs) and municipalities for set- ting up the required planning response interventions towards all aspects of post-conflict solid waste issues. Materials and methods Interviews and records are applied for the purpose of data collection from the concerned authorities and agencies in charge of conflict debris removal and recycling involved in rubble and debris management projects in Gaza strip, the data for 11-day, May 2021 conflict attack on Gaza Strip. Primary data collection has been performed through inter- views with focal persons in charge with such projects in the Ministry of Public Works and Housing (MoPWH) and UNDP-PAPP during the period January to May 2022. Fur- thermore, collective information has been considered from attending stakeholder meetings and workshops concern- ing the rubbles removal projects progress, achievements, and challenges organized by UNDP-PAPP in Gaza Strip in June 2022 [17]. The gathered data included overall of rub- bles management approaches, quantities of debris removed and recycled, materials recovery, compositions of demoli- tions wastes and destination of treated demotions wastes.f The main concern following each conflict is how and where to dispose this massive volume of concrete rubble taking into consideration the fact of oversaturation of exist- ing landfills. Accordingly, several post emergencies and recovery projects in Gaza Strip undertook the immediate removal of the rubble and debris that were accumulated as result of the military operation and transfer it to a safe place for further processing. The major of these projects were implemented by the United Nations Development Pro- gramme—Programme of Assistance to the Palestinian Peo- ple (UNDP-PAPP) [7] to manage of resulted post-conflict demolition wastes in Gaza Strip including rubble removal, sorting, recycling and material recovery though divert funds from several donors, notably Government of Japan, under the overall supervision of local government ministries. The main activity components of these project are to (i) Unex- ploded Ordnance (UXOs) clearance, safe disposal and (ii) Removal of post-conflict demolition waste from destroyed sites, and (iii) Crushing, sorting and recycling of collected rubble and debris [8].l It is worth to mention that part of efforts of rubbles removal and recycling under different projects are still ongoing, up to this reporting period. However, many records are obtained from the damage assessments reports and the announced bidding documents of relevant projects, which implemented/or still implementing by UNDP-PAPP and relevant ministries. Management of post-conflict demolition waste presents a significantly different aspect from general (municipal and industrial) solid waste management. Institutional arrangements for conducting post‑conflict demolition waste management in Gaza Based on experience and lessons learned from previ- ous confliction event occurred in Gaza Strip, a common protocol has been established joining all involved actors for better to coordinate and supervise the operations of managing the post-conflict demolition waste, debris and rubbles, in Gaza Strip. The Ministry of Public Works and Housing (MoPWH) is the leading government agency for such sector starting from the damage assessment to the final recycling of the debris. 1 3 687 Journal of Material Cycles and Waste Management (2023) 25:684–693 Totally Damaged Housing Units, May 2021 Escalation in Gaza Strip (Source: UNDP/PAPP [9]) Fig. 1 Totally Damaged Housing Units, May 2021 Escalation in Gaza Strip (Source: UNDP/PAPP [9]) 1 3 Journal of Material Cycles and Waste Management (2023) 25:684–693 688 Partially Damaged Housing Units, May 2021 Escalation in Gaza Strip (Source: UNDP/PAPP [9]) Fig. 2 Partially Damaged Housing Units, May 2021 Escalation in Gaza Strip (Source: UNDP/PAPP [9]) 1 3 Journal of Material Cycles and Waste Management (2023) 25:684–693 689 relevant authorities, and usually located in landfills sites. The Juhr AlDeek landfill have been selected for the process of placing the transferred post-conflict debris/rubbles and for the process of crushing and recovery rubbles in May, 2021 conflict. resulting from the conflictions on the Gaza Strip from 11 to 21 May 2021 with full coordination with relevant min- istries/entities [11]. In particular, UNDP coordinated with MOPWH to receive a list of buildings that proposed to be removed through the recovery interventions. In the same line, a quality assessments were carried out which include identification of asbestos and other hazard- ous components, and identification of UXOs possible loca- tions [12]. After the 2009 conflict, the quality assessment for rubbles’ hazardous components was carried out by UNEP [13]. Risk Assessment reports prepared by UNMAS for each building before commencement of the demolition activity of damaged buildings. In May 2021 conflict, 30 risk assessments were identified for urgent removal of Explo- sive Remnants of War (ERW) during the period of conflict by UNMAS [6] during the period of conflict. Afterwards in post-conflict period, UNMAS, an integral component of the UNDP-PAPP Rubble Removal project, conducted 291 risk assessments as requested during damage assessment stage. Based on the assessment results, UNMAS in coordi- nation with local partners carried out the operation of direct explosive ordnance disposal (EOD) at designated sites. On the other hand, EQA assessment report reveals that enor- mous amount of agricultural hazardous waste, in particular chemical wastes of pesticides and fertilizers are resulted from direct targeting a fertilizer factory in North Gaza [14]. Urgent removal actions were considered for an amount of 886 tons of hazardous wastes though UNDP intervention in l The recovery of debris resulting from May 2021 conflict in Gaza strip follows specific procedures as per following subsections. Figure 3 shows different field pictures for man- aging post-conflict demolitions wastes in Gaza Strip starting from removal process to the final reuse of debris, which adopted in May, 2021 conflict. Table 1 The amount of rubbles linked with number of damaged buildings, which have been generated in Gaza Strip during the May 2021 conflict segregated by different agencies that handled the rubbles removal [5] Collecting damage information and its assessments The process starts with collecting damage information and conducting its assessment to identify on-ground damages resulting related to infrastructure and classifying such dam- age to levels of severity, which allow rehabilitation, recon- struction, and recovery interventions. As the process of the damage assessment, it is not only targeted the fully damaged building but also some of par- tially damaged building. Evaluating such issue covered detailed engineered and economic assessment regarding the recovery to the state it was before the damages, and recovery of damaged buildings towards a better physical construction [9]. The UNDP granted a fund from the Government of Japan to conduct detailed assessment of infrastructure damages Fig. 3 Field pictures for the major process for post-conflict demo- litions wastes in Gaza Strip for May 2022 conflict. a Demolition process of attacked building in Gaza [17], b Crushing equipment used for concrete rubbles materials at Juhr Al Deek Landfill, 2021. c Resulted crushed materials to be reused for roads rehabilitation [17]. d Removed reinforced concreate foundations and large concre- ate blocks from damaged buildings. e Reusing the rubble foundation blocks for shoreline protection at Gaza beach [17] c Resulted crushed materials to be reused for roads rehabilitation [17]. d Removed reinforced concreate foundations and large concre- ate blocks from damaged buildings. e Reusing the rubble foundation blocks for shoreline protection at Gaza beach [17] Fig. 3 Field pictures for the major process for post-conflict demo- litions wastes in Gaza Strip for May 2022 conflict. a Demolition process of attacked building in Gaza [17], b Crushing equipment used for concrete rubbles materials at Juhr Al Deek Landfill, 2021. c Resulted crushed materials to be reused for roads rehabilitation [17]. d Removed reinforced concreate foundations and large concre- ate blocks from damaged buildings. e Reusing the rubble foundation blocks for shoreline protection at Gaza beach [17] Fig. 3 Field pictures for the major process for post-conflict demo- litions wastes in Gaza Strip for May 2022 conflict. a Demolition process of attacked building in Gaza [17], b Crushing equipment used for concrete rubbles materials at Juhr Al Deek Landfill, 2021. c Resulted crushed materials to be reused for roads rehabilitation [17]. d Removed reinforced concreate foundations and large concre- ate blocks from damaged buildings. Removal of concrete rubble and non‑concrete debris The major quantities of rubbles removed either by the citi- zens themselves through contracting with a private sector, or by UNDP and other agencies. Considering a lack of fleet capacity and equipment for implementing such activities through Local Government Units (LGU's) or ministries, the local private sector (con- tactors) carried out demolition of destroyed buildings, the transportation of the debris to the crushing site and crushing the material though contracting with the UNDP or MoPWH. Table 1 describe the amount of rubbles, which have been generated in Gaza Strip during the May 2021 conflict and that have been assigned for removal and processing of recov- ery with relation to its number of damaged buildings. In addition, Table 1 linked the amounts of removed rubbles to the different responsible agencies involved with rubbles removal projects in Gaza strip. Therefore, the citizens and UNDP handled 75% of rubbles removal. The amount of rubbles have been reported according to MoPWH internal reports of removal rubbles progressing up to beginning of January 2022. Exact amounts records have been reported for UNDP-PAPP rubbles removal project in Gaza Strip. Material recovery for concrete debris/rubble aggregates The process of recycling the concrete debris/rubbles as per following steps; (1) Preparation of crushing site including leveling works and laying of polythene sheets, (2) Crushing transported concrete materials, (3) Stockpiling of crushed concrete materials at the prepared area at the crushing site, and (4) Lab tests for the crushed concrete materials [17]. Regarding the rubble removal and recycling project conducted by UNDP, the demolition process were defined through classify the type of rubbles to concrete and non-con- crete elements in regards to the level of damaged buildings heightens as per followings; (i) Demolishing of buildings with height less than 12 m and (ii) Demolishing of build- ings with height more than 12 m. A set of specific instruc- tions regarding the safety measurements were defined for the contractors carried out the demolition and rubbles removals for buildings with high height as towers[16] [17]. Figure 3a shows an example demolition activity for a damage building from May, 2021 conflict [17]. The UNDP-PAPP project provided a crushing equipment, through contractor, at Juhr Alddek landfill (Fig. 3b). The total rubble transported to crushing site were 122,525 tons of rubble resulted from May 2021 conflict and assigned for UNDP intervention, in which around 111,621 tons were crushed by this equipment (Fig. 3c) [17]. Similarly, the citizens and other organization were involved in rubbles removal, and contacted other private sector conducted crush- ing at the sites in Gaza for further recycling the removed rubbles. International Labour Organization (ILO) in 2012, estimated that 30–50 small rubble crusher were available in Gaza, while about 200 to 300 people were involved in the collection of rubbles and blocks from destroyed buildings [18]. l The demolishing, sorting and transporting process of con- create materials for rubbles have specific producers prior the recycling process; according to instructions reported, the major components instructions for this activity is to; excava- tion for rubbles, cutting concreate elements to small parts (maximum 50 cm × 50 cm × 50 cm), extracting and cutting reinforced concreate bars, demolishing damage building The test for the crushed materials revealed some size differences in some types of granules after conducting Sieve Analysis test. Collecting damage information and its assessments e Reusing the rubble foundation blocks for shoreline protection at Gaza beach [17] 1 3 3 Journal of Material Cycles and Waste Management (2023) 25:684–693 690 full coordination with relevant ministries, in particular EQA and Joint Service Council in Gaza and North Governorates (JSC-GNG) [15]. Safety measures were taken amid imple- menting the removal, transport and stockpiling the collected hazard waste to the special cell at the Juhr Aldeek landfill. and walls, loading, transporting and unloading of concrete rubble to final location for further recycling process. While the large reinforced concrete foundations are only extracted from damage buildings to be reused after that without cut- ting into small parts. On the other hand, the non-concrete materials were demolished and sorted such as wood, alu- minum, steel, and any other items from existing damaged structures and rubble stockpiles [16] [17].l Material recovery for concrete debris/rubble aggregates In same context, the California Bearing Ratio of Soil (CBR) also applied for testing the strength of Agency handle the rubbles removal Rubbles amount (tons) # of buildings removed % of removal involvement Citizens themselves 155,415 311 42% UNDP (Fund from Japanese Government) 122,525 151 33% Egyptians Support 63,504 13 17% Joint coordination between agencies 18,521 16 5% Arabian Committee for Reconstruction 13,120 16 4% Total 373,085 507 100% 1 3 Journal of Material Cycles and Waste Management (2023) 25:684–693 691 In addition, one of the very important useful applica- tions for reusing the demolitions wastes in Gaza Strip is utilizing the reinforced foundation blocks resulting from rubbles (Fig. 3d) for shoreline protection. The current situ- ation indicated that the coast of Gaza is suffering from sand erosion problems and sedimentation pattern and building roads adjacent to the shoreline raising a serious stability problem in some locations on Gaza Beach area [19] Accordingly, a proximately of 4000 tons were placed to protect the shoreline in Rafah, Al-Zahra, and Gaza (Fig. 3e). The process of this reusing technique is to lay the big concrete elements horizontally and/or in inclined way according to site investigation [17]. aggregated resulting from the crushing, a range from 80 to 120% were reported. The labs results varied due to many factors; as way of mixing crushed materials, differences in type of crushed materials, and variation in soil surrounded the demolition buildings [17]. These results were performed for assessing the applicability of using crushing debris for paving roads. Based on the projects data for rubbles removals, it has been noticed that concrete elements represent around 88% of the total rubbles removed, while 8.6% are non-concrete elements. Reinforced concrete foundations blocks represent 3.4%. Overall post‑conflict demolition waste management observed in Gaza Strip The post-conflict demolition waste management proce- dures is well established in Gaza Strip through the different national and international agencies, where rubbles can be managed applying similar techniques of disaster waste man- agement [23] in contingency planning, identifying harm- ful materials, and emergency waste removal, and those of construction and demolition (C&D) waste management [24], such as sorting, crushing, and sieving for recycling. The accumulated experience of armed conflicts in Gaza Strip revealed good dealing with rubble and debris removal including the unexploded ordnance clearance, safe disposal, and crushing and reuse of collected rubble for various appli- cations under close coordination with relevant local and international agencies. Concrete debris were removed and recycled in Gaza mostly by buildings owners and recovery by international projects but also other supports from local and international organizations. For the technical side, some on-ground field challenges were observed during the removal and recycling process of rub- bles from the damages buildings; such as (but not limited): (i) uncertainty of the damages levelness from the physical investigation, thus, further engineered exploration were needed, in which lead to delay the rubbles removal process. For example; damage can be observed at the structure of partial damaged buildings, but more observations needed for the its infrastructure (ii) limitations of reusing the recy- cled items from rubbles concreate materials in which that its physical parameters is changed or modified; e.g. reused steel reinforcements shall be for secondary concrete elements that should not be addressed to stresses. Furthermore, and the reuse of crushed concreate material as base course is not recommended in roads with high slope that need specific levelness of tensile strength which cannot be achieved by the properties of reused crushed materials [17]. The rubble concrete materials represent the most propor- tion of debris, which removed according to specific engi- neering instructions, and then it crushed to produce recy- cled aggregates used for paving specific types of roads, or it transported to private crushing sites for produce buildings blocks. Reusing recovered concreate demolition wastes Regarding to steel bars extracted from rubble reinforced concrete items, as columns, slabs. etc., it is important to mention that such steel bars modified and/or adjusted to be reused for only non-major constructions elements and secondary concrete elements because it lose a significant percentage of its tensile strength. For example, the reused bars can be utilized in door shoulders, infills and lintels. In either case, engineering tests shall be considered to assess the applicability of reusing any extracted items from rubbles. A part of crushed materials was used to paving agricul- tural roads though laying of crushed materials as subgrade replacement. Total of 72,400 tons of crushed materials were used for paving 50 road in Gaza Strip, for an over than 26,000 m length though the funds of Swedish International Development Cooperation Agency (Sida) and Government of Japan [17]. It is worth to mention that several local uni- versities researchers and international agencies verified the opportunities for using crushed rubbles in the road reha- bilitation and construction industry in general [19]. For Gaza Strip context, several tests were performed to ensure possibility and potential of producing recycled aggregates from the construction and demolition wastes and concrete rubbles through laboratory tests and the results revealed the applicability of reusing the crushed materials in construction industry and for road subbases [12] [20] [21]. Furthermore, when citizens (house owners) managed to remove the rubbles, they sell the concreate materials to pri- vate sector crushing sites to produce small aggregate for building blocks industry. The sands materials utilized for leveling matters. Figure 4 shows a schematic overview diagram for the entire management process for post-conflict rubbles and debris in Gaza war during May 2021. Fig. 4 Schematic diagram for management process for post war rubbles and debris after the Gaza, May 2021 conflict Fig. 4 Schematic diagram for management process for post war rubbles and debris after the Gaza, May 2021 conflict 1 3 692 Journal of Material Cycles and Waste Management (2023) 25:684–693 Cost description for rubbles removal and recycling support (though international funds) in Gaza Strip for war recovery programs.l The current fleet and equipment capacity of concerned ministries, LGUs and JSCs for handling the removal and recycling process is very limited, even not exists, thus, managing the post demolition wastes implemented through international support under the supervision and coordination with local authorities. The average cost was determined based on the previous experience of the entire rubbles management processes. The cost might have small variations among the conflicts occur- rence years. However, according to latest bids of rubbles removal and recycling in Gaza Strip during 2021 conflict, the operation cost of demolishing, sorting and transporting activities is approx. 12 USD/ton, while the crushing process costs were about 7 USD/ton. Overall post‑conflict demolition waste management observed in Gaza Strip The rubbles foundations blocks were reused to be placed for shoreline protections in specific areas in Gaza Strip.l On the other side, the consequences experience of the demolition wastes projects in Gaza Strip confirms its socio- economic feasibility, thus, it opens more widen eyes towards the demolition wastes value through compensating the short- age of construction materials that are not available on the local market, providing new job opportunities and encourag- ing the public private partnership companies. Although the post-conflict demolition waste management is different from the management of municipal and industrial wastes, both share common points of view: urgent collec- tion and transport, appropriate treatment, proper disposal, and recovery and recycling of as many resource materials as possible in the process. From the institutional setting side, it is obvious that most of protocols, measures, contingency plans, operational pro- ducers for demolitions wastes removal and recycling have been set as a result of accumulated experience of govern- mental and international institutions for these types of pro- jects since 2005 in Gaza strip. The activities of removal and recycling of debris were optimized according to Gaza context. The relevant ministries, international agencies and other local partners experienced formulating technical and management committees for dealing the sector of disaster waste at the time of crisis and recovery period [22]. While there is a gap of formulating such kind of national regula- tions for rubbles management and recycling resulting from conflicts/wars. However, it worth to mention about the availability of experience, technical capability and financial Acknowledgements The authors thank Mr. Mohammed Abu Mezyed from UNDP's Programme of Assistance to the Palestinian People (UNDP/PAPP) and Mr. Mohammed Alaskary from MoPWH-Gaza for their great support in providing the data regarding the post-conflict debris/rubbles removal and recycling in Gaza strip, which was the basis of this overview research. The authors also thank the International Net- work for Environmental and Humanitarian Cooperation, Nonprofit Inc., Tokyo and MoLG-JICA Project for Capacity Development in Solid Waste Management in Palestine Phase-III, for their encouragement and supports in various ways. The authors are grateful to anonymous reviewers for their valuable comments that have helped us to revise 1 3 3 693 Journal of Material Cycles and Waste Management (2023) 25:684–693 the manuscript. The views expressed in this article are given under the responsibility of authors and do not necessarily represent the official positions of institutions to which they belong. 10. Declarations 14. EQA-Gaza survey for damage assessment on public health and environment during Gaza aggression, May 2021. Conflict of interest The authors declare no potential conflicts of in- terest with respect to the research, authorship, and/or publication of this article. The Publication cost (APC) for this article was financially supported by Japan International Cooperation Agency, Tokyo, Japan. 15. Joint Service Council for Solid Waste Management in Gaza and North Gaza Governorates, JSC GNG internal report 'removal of hazardous wastes resulted from Gaza May 2021 attack'. Unpublished. 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Workshop Presentation, 'Rubbles Removal Project Achievements and Challenges', UNDP/PAPP. Sunday, 26/06/2022, Gaza city. 18. A. Muhaisen and J. Ahlback, “Towards sustainable construction and green jobs in the Gaza Strip,” 2012. 19. Al-Mabhouh MA, Musalam AMA, Shukri A, Al-Najjar H, Abu- namous RES Requirements for Managing the Recycling of War Debris in the Gaza Strip. Israa University Journal of Applied Sci- ence Volume 5: Issue1, October 2021 ISSN: 2523–0522. 20. United Nation Industrial Development Programme (UNIDO): Testing Program to Investigate the Application of Construction and Demolition Wastes in Construction Industry in Gaza Strip: Analysis Report, Gaza 2005. Overall post‑conflict demolition waste management observed in Gaza Strip UNOSAT, “Damage assessment in the Gaza strip—UNOSTAT satellite derived geospatial analysis as 28 May 2021” https://www. unitar.org/maps/map/3301. Accessed 12 January 2022. 11. 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https://openalex.org/W4386060703	https://www.epj-conferences.org/articles/epjconf/pdf/2023/13/epjconf_eosam2023_13001.pdf	English	null	Analysing interaction and localization dynamics in modulation instability via data-driven dominant balance	EPJ web of conferences	2,023	cc-by	1,695	Analysing interaction and localization dynamics in modulation instability via data-driven dominant balance Andrei V. Ermolaev1,, Mehdi Mabed1, Christophe Finot2, Goëry Genty3, and John M. Dudley1 1Université de Franche-Comté, Institut FEMTO-ST, CNRS UMR 6174, Besançon, France 2Université de Bourgogne, Laboratoire Interdisciplinaire Carnot de Bourgogne, CNRS UMR 6303, 21078 Dijon, France 3Photonics Laboratory, Tampere University, Tampere, FI-33104, Finland 1Université de Franche-Comté, Institut FEMTO-ST, CNRS UMR 6174, Besançon, France 2Université de Bourgogne, Laboratoire Interdisciplinaire Carnot de Bourgogne, CNRS UMR 6303, 21078 Dijon, France 3Photonics Laboratory, Tampere University, Tampere, FI-33104, Finland Abstract. We report the first application of the Machine Learning technique of data-driven dominant balance to optical fiber noise-driven Modulation Instability, with the aim to automatically identify local regions of dispersive and nonlinear interactions governing the dynamics. We first consider the analytical solutions of Nonlinear Schrödinger Equation – solitons on finite background – where it is shown that dominant balance distinguishes two particularly different dynamical regimes: one where the nonlinear process is dominating the dispersive propagation, and one where nonlinearity and second order dispersion act together driving the localization of breathers. By means of numerical simulations, we then analyse the spatio-temporal dynamics of noise-driven Modulation Instability and demonstrate that data-driven dominant balance can successfully identify the associated dominating physical regimes even within the turbulent dynamics. 1 Introduction where ξ is the distance, τ is comoving time and u is the field envelope. It has been recently demonstrated that the data-driven dominant balance technique based on unsupervised learning algorithms can be used in the identification of dominating physical regimes. Generally, this involves the following steps: (i) determining the evolution map of u(ξ,τ) by means of numerical simulations of Eq. 1 or using the analytical solutions; (ii) analysing the obtained map in the associated “equation space,” applying the cluster detection algorithm to identify regions where combinations of the NLSE terms (iuξ, uττ, \|u\|2u) dominate the interaction, i.e. selecting ensembles of points in the three-dimensional equation space that have a significantly reduced variance with respect to some of its directions; (iii) finally, re-mapping these identified clusters back onto the (ξ,τ) domain allows us to directly compare the selected subregions associated with the dominant physical processes with the initial spatio-temporal field distribution. We present an application of a fundamentally new approach for interpreting noise-driven Modulation Instability (MI) dynamics in optical fiber propagation via the Machine Learning (ML) technique of data-driven dominant balance [1]. We aim to automate the problem of identifying which particular physical processes drive propagation in different regimes by means of unsupervised learning algorithms, a task usually performed using intuition and/or asymptotic limits [2]. Here we apply the first use of dominant balance to interpret continuous wave MI in the Nonlinear Schrödinger Equation (NLSE), allowing us to readily associate different interactions with various structures seen during the evolution. By using analytic results for MI-related breather solutions, we automatically distinguish purely nonlinear propagation from regions where nonlinearity and dispersion combine to drive localization. We then use numerical simulations to apply the technique to the more complex case of noise-driven chaotic MI evolution. 2 Methods Figures 1(a-c) illustrate the application of the technique to the analytic solutions for the Peregrine soliton and the Akhmediev and Kuznetsov-Ma breathers - strongly localized structures known to emerge from noise-driven MI [3]. For each case, subfigure (i) shows the corresponding spatio-temporal distribution of the \|u(ξ,τ)\|2, subfigure (ii) illustrates the equation space cluster MI describes the breakup of a continuous wave injected into optical fiber in the anomalous dispersion regime. The governing NLSE is written in normalized form as: iuξ + uττ + \|u\|2u = 0, (1) iuξ + uττ + \|u\|2u = 0, (1) , 13001 (2023) EPJ Web of Conferences EOSAM 2023 287 , 13001 (2023) EPJ Web of Conferences EOSAM 2023 287 https://doi.org/10.1051/epjconf/202328713001 © 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/). s DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 nses/by/4.0/). iuξ + uττ + \|u\|2u = 0, (1) Corresponding author: andrei.ermolaev@femto-st.fr © 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/). s , 13001 (2023) EPJ Web of Conferences EOSAM 2023 287 https://doi.org/10.1051/epjconf/202328713001 Fig. 1 Application of dominant balance analysis for the (a) Peregrine soliton, (b) Akhmediev breather and (c) Kuznetsov- Ma breather: (i) represents the spatio-temporal map of \|u(ξ,τ)\|2, (ii) shows an example of the cluster assignment algorithm performed in the equation space, and (iii) depicts the clusters mapped back to the (ξ,τ) coordinates. The colormaps on the bottom apply to all figures. Blue subregions represent the d i f li it hil h b i Fig. 2 (i) the evolution map and (ii) the remapped clustering results for noise-driven MI. Features PS and AB in plot (ii) show signatures of the Akhmediev breather and Peregrine soliton. and dispersive (orange) effects are playing the dominant role. Moreover, we readily see how dominant balance identifies the characteristic interaction signatures of Peregrine solitons and Akhmediev breathers that we saw earlier from the analytic results in Fig. 1(a) and (b). There are many such signatures visible in the figure, and two are shown as examples as marked features PS and AB i l i Fi 2(ii) Fig. 1 Application of dominant balance analysis for the (a) Peregrine soliton, (b) Akhmediev breather and (c) Kuznetsov- Ma breather: (i) represents the spatio-temporal map of \|u(ξ,τ)\|2, (ii) shows an example of the cluster assignment algorithm performed in the equation space, and (iii) depicts the clusters mapped back to the (ξ,τ) coordinates. The colormaps on the bottom apply to all figures. Blue subregions represent the dominancy of nonlinearity, while orange shows subregions where dispersion and nonlinearity contribute equally. Fig. 2 (i) the evolution map and (ii) the remapped clustering results for noise-driven MI. Features PS and AB in plot (ii) show signatures of the Akhmediev breather and Peregrine soliton. Fig. 2 (i) the evolution map and (ii) the remapped clustering results for noise-driven MI. Features PS and AB in plot (ii) show signatures of the Akhmediev breather and Peregrine soliton. and dispersive (orange) effects are playing the dominant role. Moreover, we readily see how dominant balance identifies the characteristic interaction signatures of Peregrine solitons and Akhmediev breathers that we saw earlier from the analytic results in Fig. 1(a) and (b). References 1. J. L. Callaham, J. V. Koch, B. W. Brunton, J. N. Kutz, and S. L. Brunton, Nat. Commun. 12, 1016 (2021) * Corresponding author: andrei.ermolaev@femto-st.fr There are many such signatures visible in the figure, and two are shown as examples as marked features PS and AB respectively in Fig. 2(ii). Fig. 1 Application of dominant balance analysis for the (a) Peregrine soliton, (b) Akhmediev breather and (c) Kuznetsov- Ma breather: (i) represents the spatio-temporal map of \|u(ξ,τ)\|2, (ii) shows an example of the cluster assignment algorithm performed in the equation space, and (iii) depicts the clusters mapped back to the (ξ,τ) coordinates. The colormaps on the bottom apply to all figures. Blue subregions represent the dominancy of nonlinearity, while orange shows subregions where dispersion and nonlinearity contribute equally. 3 Discussion & Conclusion detection by showing the identified cluster in (iuξ, uττ)- plane – one of the planes of three-dimensional equation space, – and subfigure (iii) shows these clusters re- mapped back to (ξ,τ) enabling us to directly compare it with the initial evolution map. The color scales are on the bottom, and are identical for all figures. The key physical insight that appears from the subfigures (iii) is that the blue regions are associated only with dominant (iuξ, \|u\|2u) nonlinear interactions, whilst the orange region are prescribed to the interplay of all three NLSE terms (iuξ, uττ, \|u\|2u). These results clearly indicate that in the blue "continuous wave" region, where temporal localization is absent, dispersive effects are not playing the dominant role. Conversely, in the orange region where there is significant temporal localization, it is shown that both dispersion and nonlinearity contribute equally. detection by showing the identified cluster in (iuξ, uττ)- plane – one of the planes of three-dimensional equation space, – and subfigure (iii) shows these clusters re- mapped back to (ξ,τ) enabling us to directly compare it with the initial evolution map. The color scales are on the bottom, and are identical for all figures. The key physical insight that appears from the subfigures (iii) is that the blue regions are associated only with dominant (iuξ, \|u\|2u) nonlinear interactions, whilst the orange region are prescribed to the interplay of all three NLSE terms (iuξ, uττ, \|u\|2u). These results clearly indicate that in the blue "continuous wave" region, where temporal localization is absent, dispersive effects are not playing the dominant role. Conversely, in the orange region where there is significant temporal localization, it is shown that both dispersion and nonlinearity contribute equally. Taken together, these results show the ability of data- driven dominant balance techniques to automatically attribute contributing physical processes to specific stages of MI evolution and are another example of how machine learning can complement traditional approaches to analysis of nonlinear dynamics. While the current results were obtained using analytic results and simulations, advancements in experimental full-field characterization techniques suggest that future research may explore the application of dominant balance to laboratory data in order to discover underlying physical principles. [4]. 4. A. V. Ermolaev, A. Sheveleva, G. Genty, C. Finot, and J. M. Dudley, Sci. Rep. 12, 12711 (2022). 3.2. Noise-driven MI 2. G. P. Agrawal, Nonlinear Fiber Optics, 4th. Ed., (Academic Press, Boston, 2007) 3. J. M. Dudley, F. Dias, M. Erkintalo, and G. Genty, Nat. Photon. 8, 755-764 (2014) Figure 2 shows more complex evolution for chaotic MI driven by an input continuous wave with low amplitude noise (corresponding to a one photon per mode noise background in dimensional terms). Yet even for this highly random dynamics, data-driven dominant balance can successfully distinguish the spatio-temporal regions where only the nonlinear (blue) and combined nonlinear 4. A. V. Ermolaev, A. Sheveleva, G. Genty, C. Finot, and J. M. Dudley, Sci. Rep. 12, 12711 (2022). 2
https://openalex.org/W4288871302	https://bmccancer.biomedcentral.com/counter/pdf/10.1186/s12885-022-09940-3	English	null	Changing trends in the disease burden of non-melanoma skin cancer globally from 1990 to 2019 and its predicted level in 25 years	BMC cancer	2,022	cc-by	8,395	Hu et al. BMC Cancer (2022) 22:836 https://doi.org/10.1186/s12885-022-09940-3 © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Changing trends in the disease burden of non‑melanoma skin cancer globally from 1990 to 2019 and its predicted level in 25 years Wan Hu, Lanlan Fang, Ruyu Ni, Hengchuan Zhang and Guixia Pan* Wan Hu, Lanlan Fang, Ruyu Ni, Hengchuan Zhang and Guixia Pan* Abstract Background: The disease burden of non-melanoma skin cancer (NMSC) has become a significant public health threat. We aimed to conduct a comprehensive analysis to mitigate the health hazards of NMSC. Methods: This study had three objectives. First, we reported the NMSC-related disease burden globally and for dif- ferent subgroups (sex, socio-demographic index (SDI), etiology, and countries) in 2019. Second, we examined the temporal trend of the disease burden from 1990 to 2019. Finally, we used the Bayesian age-period-cohort (BAPC) model integrated nested Laplacian approximation to predict the disease burden in the coming 25 years. The Norpred age-period-cohort (APC) model and the Autoregressive Integrated Moving Average (ARIMA) model were used for sensitivity analysis. Results: The disease burden was significantly higher in males than in females in 2019. The results showed significant differences in disease burden in different SDI regions. The better the socio-economic development, the heavier the disease burden of NMSC. The number of new cases and the ASIR of basal cell carcinoma (BCC) were higher than that of squamous cell carcinoma (SCC) in 2019 globally. However, the number of DALYs and the age-standardized DALYs rate were the opposite. There were statistically significant differences among different countries. The age-standardized incidence rate (ASIR) of NMSC increased from 54.08/100,000 (95% uncertainty interval (UI): 46.97, 62.08) in 1990 to 79.10/100,000 (95% UI: 72.29, 86.63) in 2019, with an estimated annual percentage change (EAPC) of 1.78. Other indicators (the number of new cases, the number of deaths, the number of disability-adjusted life years (DALYs), the age-standardized mortality rate (ASMR), and the age-standardized DALYs rate) showed the same trend. Our predic- tions suggested that the number of new cases, deaths, and DALYs attributable to NMSC would increase by at least 1.5 times from 2020 to 2044. Conclusions: The disease burden attributable to NMSC will continue to increase or remain stable at high levels. Therefore, relevant policies should be developed to manage NMSC, and measures should be taken to target risk fac- tors and high-risk groups. Keywords: Non-melanoma skin cancer, Global, Burden, Predict, Trend Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, 81 Meishan Road, Hefei 230032, Anhui, China Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, 81 Meishan Road, Hefei 230032, Anhui, China Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, 81 Meishan Road, Hefei 230032, Anhui, China Data sourcesh Skin cancer is a malignant tumor of the skin, and it has become a prominent public health threat [1]. It could be divided into fatal malignant melanoma and less deadly non-melanoma [2]. Non-melanoma skin cancer (NMSC) is the most common type, representing about 1/3 of all malignancies diagnosed worldwide yearly [3]. NMSC is the most common malignancy in people with fair skin, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) [4]. It is not suitable for surgical treat- ment, adjunctive care, or palliative care. It is usually treated with radiotherapy [5, 6]. With an aging popula- tion, the incidence attributable to NMSC is increasing [7, 8]. It was estimated that the national health service spent 180 million pounds in 2020 [3]. The average annual cost of treating melanoma in the United States was estimated at $3.3 billion from 2007 to 2011, and the average annual cost of treating NMSC was estimated at $4.8 billion, for a total of $8.1 billion [9]. Current research shows that pre- vention helps reduce the disease burden, so prevention efforts are positive from every perspective [10]. There- fore, it is urgent to understand the global trend of NMSC. The purpose is to help formulate relevant health policies and guide the practice, prevention, and management of NMSC. The data on deaths, disability-adjusted life years (DALYs), and incidence of NMSC from 1990 to 2019 were extracted from the GBD Study 2019 website (http://www. globalburden.org/). This was free of charge provided by the Institute for Health Metrics and Evaluation (IHME) [15–17]. The GBD study 2019 was a systematic survey that assesses the health effects of diseases, injuries, and risk factors based on age, sex, and GBD region [18]. The details of the methodology had been described in pre- vious publications [19–21]. The following was a brief introduction to the GBD study 2019. First, the period of this study was from 1990 to 2019. Second, the scope of the study was global. All countries and territories were divided into seven super-regions and 21 regions based on geographic contiguity and epidemiological homogeneity. At the same time, all countries and terri- tories were divided into five areas according to the socio- demographic index (SDI) indicator. They were high SDI, high-middle SDI, middle SDI, low-middle SDI, and low SDI. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 11 Hu et al. BMC Cancer (2022) 22:836 Capsule summary assessment of health losses caused by NMSC, with detailed analysis by sex, Global Burden of Disease (GBD) region, economic level, and types.h • This is the first systematic assessment and prediction of the disease burden of non-melanoma skin cancer worldwide. The Bayesian age-period-cohort model integrated nested Laplacian approximation is used to predict the disease burden. Therefore, this study aimed to assess the global disease burden of NMSC and predict the global disease burden in the future. The objective was to provide an evidence- based assessment of the effectiveness of current preven- tion and treatment strategies, make recommendations for future prevention and control policies, and reduce the disease burden of NMSC. • The disease burden will continue to increase or remain relatively stable at high levels in the future. Data sourcesh The SDI indicator was a comprehensive measure of developmental level based on average education level, total fertility, and per capita income, ranging from worst zero to best one hundred.h The data of the age-period-cohort (APC) model were as follows. The population forecast data came from the 2019 revised edition of the population of the world out- look (https://population.un.org/wpp/Download/Stand ard/CSV/). The standardization of the World Health Organization (WHO) in 2000–2025 demographic data came from a public website (https://seer.cancer.gov/ stdpopulations/world.who.html/). Currently, studies on NMSC mainly focus on clinical treatment [5, 11, 12], and few studies measures the dis- ease burden of NMSC. Aggarwal assessed the burden of skin cancer in the United States from 1990 to 2019. The results showed an increase in the incidence and preva- lence of melanoma, BCC, and SCC [9]. Pondicherry reported the incidence of NMSC in the Auckland region of New Zealand [13]. Cakir presented incidence and health care costs for NMSC from Australia, the United States, and Europe. Additionally, he noted that NMSC ranked fifth in cost of care after prostate, lung, colon, and breast cancers [14]. However, these studies were only limited to some countries, and no studies had com- prehensively assessed the global disease burden caused by NMSC. When population health measurements are more complex, it is essential to provide a comprehensive Global disease burden assessment for NMSC in 2019h The number of new cases attributable to NMSC was 6,353,687 (95% uncertainty interval (UI): 5,805,441, 6,952,145) in 2019, the number of deaths was 56,054 (95% UI: 50,415, 59,792), and the number of DALYs was 1,183,233 (95% UI: 1,085,365, 1,264,545). The ASIR was 79.10/100,000 (95% UI: 72.29, 86.63), the ASMR was 0.73/100,000 (95% UI: 0.65, 0.78), and the age-standard- ized DALYs rate was 14.67/100,000 (95% UI: 13.45, 15.67) (Supplementary Table 1–3). In subgroup analyses, the EAPC values of the age- standardized rates had significant differences between the genders from 1990 to 2019. The increase in disease burden was more significant for males than females (Supplementary Table 1–3). From 1990 to 2019, the number of new cases, deaths, and DALYs attributable to NMSC increased in all regions regardless of the level of SDI. The ASIR of NMSC also increased in the five SDI regions (high SDI, high-middle SDI, middle SDI, low- middle SDI, and low SDI). However, the ASMR decreased in the high-middle SDI region and remained stable in the high SDI region from 1990 to 2019. The age-standardized DALYs rate decreased in the high-middle SDI region. There were significant differences in NMSC-related dis- ease burden between the etiologies. Over time, all disease burden indicators (the number of new cases, the ASIR, the number of deaths, the ASMR, the number of DALYs, and the age-standardized DALYs rate) of SCC showed an upward trend from 1990 to 2019. Furthermore, some of the disease burden indicators (the number of new cases, the ASIR, the number of DALYs, and the age-standard- ized DALYs rate) of BCC showed an upward trend (Sup- plementary Table 1–3). Regarding geographical GBD ( pp y ) The disease burden was significantly higher in males than in females in 2019. The results of the Kruskal Wal- lis test showed significant differences in disease burden among different SDI regions (Supplementary Table 1– 3). We found that regions with better socioeconomic development had a greater disease burden from NMSC in 2019. As for the subgroup analysis of etiology, the number of new cases and the ASIR of BCC were higher than that of SCC in 2019 globally. The number of DALYs and the age-standardized DALYs rate of BCC were lower than SCC in 2019. We could not compare the number of deaths and the ASMR between the two etiologies due to the lack of data for BCC in the database. Global disease burden assessment for NMSC from 1990 to 2019h to 2019 The number of new cases attributable to NMSC increased from 1,951,299 (95% UI: 1,692,794, 2,237,075) in 1990 to 6,353,687 (95% UI: 5,805,441, 6,952,145) in 2019, and the number of deaths of NMSC increased from 23,222 (95% UI: 21,441, 24,436) to 56,054 (95% UI: 50,415, 59,792) between 1990 and 2019. The number of DALYs also exhibited a upward trend, which increase significantly from 561,854 (95% UI: 518,874, 599,141) in 1990 to 1,183,233 (95% UI: 1,085,365, 1,264,545) in 2019. The age-standardized incidence rate (ASIR) of NMSC increased from 54.08/100,000 (95% UI: 46.97, 62.08) in 1990 to 79.10/100,000 (95% UI: 72.29, 86.63) in 2019, with an EAPC of 1.78 (95% confidence interval (CI): 1.35, 2.21). The age-standardized mortality rate (ASMR) of NMSC increased significantly from 0.69/100,000 (95% UI: 0.63, 0.73) to 0.73/100,000 (95% UI: 0.65, 0.78) between 1990 and 2019, with an EAPC of 0.41 (95% CI: 0.34 to 0.49), and the age-standardized DALYs rate sig- nificantly increased from 14.44/100,000 (95% UI: 13.31, 15.42) to 14.67/100,000 (95% UI: 13.45, 15.67) during the same period, with an EAPC of 0.20 (95% CI: 0.10 to 0.30) (Supplementary Table 1–3). All data collation and analysis were performed by R (version 4.0.2) software. Statistical analysis Thi d Finally, we used the Bayesian age-period-cohort (BAPC) model integrated nested Laplace approximations [24] to predict the disease burden from 2019 to 2044. We applied the Norpred APC model [25] and the Autore- gressive Integrated Moving Average (ARIMA) model [26] for sensitivity analysis to verify the stability of the prediction. the highest number of deaths and DALYs of all countries and territories. The ASMR was severe in Australia and a few countries in South America. Furthermore, the age- standardized DALYs rate was highest in Canadian, the Arctic, and Australia. Statistical analysis Thi d This study was a secondary analysis of GBD research results and had three objectives. First, we assessed the NMSC-related disease burden in 2019 and analyzed it by subgroups, including sex, SDI, etiology, and coun- tries. We described the NMSC-related disease burden by using the number and the age-standardized rates of inci- dence, death, and DALYs. Nonparametric rank-sum tests, Hu et al. BMC Cancer (2022) 22:836 Page 3 of 11 including the Mann-Whitney U test and the Kruskal Wallis test, were used to analyze differences in disease burden among subgroups [22]. The significance level was set at 0.05 [22]. Second, we evaluated the trend for dis- ease burden from 1990 to 2019. To reflect the trend of NMSC burden, we used linear regression analysis to cal- culate the estimated annual percentage change (EAPC) of the age-standardized rates globally and in all sub- groups, including sex, SDI, etiology, and GBD regions. The age-standardized rates were based on the GBD ref- erence population. In addition, 21 GBD regions were divided into four categories (a: significant growth; b: a slight increase; c: basically stable or decrease slightly; d: significantly decreased) through cluster analysis [23] to compare the disease burden of NMSC in different GBD regions. Finally, we used the Bayesian age-period-cohort (BAPC) model integrated nested Laplace approximations [24] to predict the disease burden from 2019 to 2044. We applied the Norpred APC model [25] and the Autore- gressive Integrated Moving Average (ARIMA) model [26] for sensitivity analysis to verify the stability of the prediction. including the Mann-Whitney U test and the Kruskal Wallis test, were used to analyze differences in disease burden among subgroups [22]. The significance level was set at 0.05 [22]. Second, we evaluated the trend for dis- ease burden from 1990 to 2019. To reflect the trend of NMSC burden, we used linear regression analysis to cal- culate the estimated annual percentage change (EAPC) of the age-standardized rates globally and in all sub- groups, including sex, SDI, etiology, and GBD regions. The age-standardized rates were based on the GBD ref- erence population. In addition, 21 GBD regions were divided into four categories (a: significant growth; b: a slight increase; c: basically stable or decrease slightly; d: significantly decreased) through cluster analysis [23] to compare the disease burden of NMSC in different GBD regions. Global disease burden assessment for NMSC in 2019h The number of deaths will increase from 33,244 to 60,575 between 2019 and 2044, an increase of about 1.82 times. The number of DALYs will increase from 742,528 to 1,688,448 during the same period, an increase of 2.27 times. The number of new cases among females will increase approximately 15.57-fold, from 2,670,753 in regions, the disease burden of NMSC varied significantly between GBD regions (Supplementary Table 1–3). The results of cluster analysis are shown in Supplementary Fig. 1. The regions which had significant growth in EAPC of AMIR from 1990 to 2019 were East Asia and High- income North America. The Caribbean and Southeast Asia were in the group of significantly decreased EAPC values. The EAPC of AMDR increased most in Central Asia and decreased most in Central Europe. As for the age-standardized DALYs rate, the region which had sig- nificant growth in EAPC of AMIR from 1990 to 2019 was Central Asia. Central Europe and High-income Asia Pacific were in the group of significantly decreased EAPC values. Global disease burden assessment for NMSC in 2019h The global dis- ease burden for different countries in 2019 is shown in Fig. 1. We found that the United States had the highest number of new cases attributable to NMSC. The ASIR was high in North America except in Mexico. China had Hu et al. BMC Cancer (2022) 22:836 Page 4 of 11 Fig. 1 Numbers and age-standardized rates of NMSC-related incidence (a and b), deaths (c and d), and DALYs (e and f) across countries. Abbreviations: DALYs: disability-adjusted life years; NMSC: non-melanoma skin cancer Fig. 1 Numbers and age-standardized rates of NMSC-related incidence (a and b), deaths (c and d), and DALYs (e and f) across countries. Abbreviations: DALYs: disability-adjusted life years; NMSC: non-melanoma skin cancer next 25 years, but the ASMR and the age-standardized DALYs rate will decrease (Fig. 2 and Supplementary Table 4). The number of new cases, deaths, and DALYs will increase over the next 25 years due to population growth and aging. The shadows in the figures show that if the corresponding rates increase or decrease by 1% per year, the number of new cases and deaths could change dramatically. This further highlights the impor- tance of NMSC prevention and treatment. The results show that the number of new cases of NMSC for males will increase from 3,682,933 in 2019 to 73,642,458 in 2044, an approximately 20-fold increase. The number of deaths will increase from 33,244 to 60,575 between 2019 and 2044, an increase of about 1.82 times. The number of DALYs will increase from 742,528 to 1,688,448 during the same period, an increase of 2.27 times. The number of new cases among females will increase approximately 15.57-fold, from 2,670,753 in next 25 years, but the ASMR and the age-standardized DALYs rate will decrease (Fig. 2 and Supplementary Table 4). The number of new cases, deaths, and DALYs will increase over the next 25 years due to population growth and aging. The shadows in the figures show that if the corresponding rates increase or decrease by 1% per year, the number of new cases and deaths could change dramatically. This further highlights the impor- tance of NMSC prevention and treatment. The results show that the number of new cases of NMSC for males will increase from 3,682,933 in 2019 to 73,642,458 in 2044, an approximately 20-fold increase. Global disease burden prediction for NMSCh The BAPC model predicts that the ASIR attributable to NMSC will increase slightly for both sexes over the Hu et al. BMC Cancer (2022) 22:836 Page 5 of 11 Fig. 2 Trends in the NMSC-related ASIR (a and b), ASMR (c and d), and the age-standardized DALYs (e and f) by sex globally: observed (dashed lines) and predicted rates of the BAPC model (solid lines). The blue region shows the upper and lower limits of the 95% UIs. Abbreviations: DALYs: disability-adjusted life years; NMSC: non-melanoma skin cancer; BAPC: Bayesian age-period-cohort; UIs: uncertainty intervals Fig. 2 Trends in the NMSC-related ASIR (a and b), ASMR (c and d), and the age-standardized DALYs (e and f) by sex globally: observed (dashed lines) and predicted rates of the BAPC model (solid lines). The blue region shows the upper and lower limits of the 95% UIs. Abbreviations: DALYs: disability-adjusted life years; NMSC: non-melanoma skin cancer; BAPC: Bayesian age-period-cohort; UIs: uncertainty intervals 2019 to 41,587,505 in 2044. The number of deaths will increase from 22,809 to 40,550, an increase of about 1.78 times. The number of DALYs will increase from 440,704 to 680,152, approximately 1.54 times (Fig. 3 and Supplementary Table 4). The results of the Nor- pred APC model are consistent with the above results. The results show that the ASIR of both genders shows an uptrend and the age-standardized DALYs rate shows a downtrend in the next 25 years. The ASMR of males shows a decreasing trend, while the ASMR of females remains unchanged (Fig. 4-5). As shown in the figure, the predicted results of the ARIMA model show that the age-standardized rate remains relatively stable. This is different from the prediction of the BAPC model. However, in terms of the quantitative burden of dis- ease indicators, the number of new cases and deaths for both males and females will increase over the next 25 years. This is consistent with the prediction of the APC model. The inconsistent results may be because the data is summarized yearly and is somewhat crude Hu et al. BMC Cancer (2022) 22:836 Page 6 of 11 Fig. 3 Trends in NMSC-related numbers of incidence cases (a and b), deaths cases (c and d), and DALYs cases (e and f) by sex globally: observed (before 2019) and predicted (after 2019) numbers. and sparse (Supplementary Fig. 2 and Supplemen- tary Fig. 3). global human health. Our results are consistent with some other studies [27, 28]. Ferlay shows that worldwide estimated mortality rates for all types of NMSC were higher than the corresponding mortality rates for mela- noma, mesothelioma, oropharyngeal, and thyroid can- cers [29]. NMSC causes a substantial economic burden worldwide [30, 31]. It was the most expensive cancer in Australia, with an expenditure of $511 million in 2010 Global disease burden prediction for NMSCh Shading indicates if the rate remained stable (baseline reference), decreased by 1% per year (optimistic reference, lower limit), and increased by 1% per year (pessimistic reference, upper limit) based on the observed rate in 2019. The curve formed by the triangle is the prediction result of the BAPC model. Abbreviations: DALYs: disability-adjusted life years; NMSC: non-melanoma skin cancer; BAPC: Bayesian age-period-cohort Fig. 3 Trends in NMSC-related numbers of incidence cases (a and b), deaths cases (c and d), and DALYs cases (e and f) by sex globally: observed (before 2019) and predicted (after 2019) numbers. Shading indicates if the rate remained stable (baseline reference), decreased by 1% per year (optimistic reference, lower limit), and increased by 1% per year (pessimistic reference, upper limit) based on the observed rate in 2019. The curve formed by the triangle is the prediction result of the BAPC model. Abbreviations: DALYs: disability-adjusted life years; NMSC: non-melanoma skin cancer; BAPC: Bayesian age-period-cohort Discussionh This study comprehensively assesses and predicts the global NMSC-related disease burden and makes nota- ble discoveries. Globally, the disease burden of NMSC is severe, underscoring that NMSC is a severe threat to Hu et al. BMC Cancer (2022) 22:836 Page 7 of 11 Fig. 4 Trends in NMSC-related number of incidence cases (a and b), deaths cases (c and d), and DALYs cases (e and f) by sex globally: observed (before 2019) and predicted (after 2019) numbers. Shading indicates if the rate remained stable (baseline reference), decreased by 1% per year (optimistic reference, lower limit), and increased by 1% per year (pessimistic reference, upper limit) based on the observed rate in 2019. Three methods are used in the prediction. The red line is calculated by the predicted rate of each 5 years group and the average population size of the 5 year group. The blue line method is to calculate the rate of each group in terms of the predicted rate of each 5 years group and the average population situation of the 5 year group. The yellow line is calculated by the predicted rate of each 5 years group and the annual population situation. Abbreviations: DALYs: disability-adjusted life years; NMSC: non-melanoma skin cancer eaths cases (c and d), and DALYs cases (e and f) by sex globally: observed the rate remained stable (baseline reference), decreased by 1% per year mistic reference, upper limit) based on the observed rate in 2019. Three edicted rate of each 5 years group and the average population size of the p in terms of the predicted rate of each 5 years group and the average y the predicted rate of each 5 years group and the annual population on-melanoma skin cancer l d b f d d b d Fig. 4 Trends in NMSC-related number of incidence cases (a and b), deaths cases (c and d), and DALYs cases (e and f) by sex globally: observed (before 2019) and predicted (after 2019) numbers. Shading indicates if the rate remained stable (baseline reference), decreased by 1% per year (optimistic reference, lower limit), and increased by 1% per year (pessimistic reference, upper limit) based on the observed rate in 2019. Three methods are used in the prediction. The red line is calculated by the predicted rate of each 5 years group and the average population size of the 5 year group. Discussionh The blue line method is to calculate the rate of each group in terms of the predicted rate of each 5 years group and the average population situation of the 5 year group. The yellow line is calculated by the predicted rate of each 5 years group and the annual population situation. Abbreviations: DALYs: disability-adjusted life years; NMSC: non-melanoma skin cancer [32]. In the United States, it is estimated that the total annual spending related to NMSC was 650 million US dollars, and the cost of health insurance is 6–7 times that of treating melanoma [9]. Furthermore, the disease bur- den has increased in recent years [32, 33]. Between 1997 and 2010, the treatment of NMSC increased by 86% [34]. Madan reports that NMSC incidence and death rates are rising [35]. The incidence of BCC shows a continuous lin- ear increase [36]. This phenomenon might be related to several factors. First, our society presents a trend of an aging population, and the elderly are a high-risk group for NMSC [37]. Additionally, exposure to UV radiation at work and play has a significant impact [38–40]. In females younger than 25 years, activity-induced tanning Hu et al. BMC Cancer (2022) 22:836 Page 8 of 11 Fig. 5 Trends in NMSC-related incidence (a and b), deaths (c and d), and DALYs rates (e and f) by sex globally: observed (solid lines) and predicted rates of the Norpred APC model (dashed lines). Abbreviations: DALYs: disability-adjusted life years; NMSC: non-melanoma skin cancer; APC: age-period-cohort Fig. 5 Trends in NMSC-related incidence (a and b), deaths (c and d), and DALYs rates (e and f) by sex globally: observed (solid lines) and predicted rates of the Norpred APC model (dashed lines). Abbreviations: DALYs: disability-adjusted life years; NMSC: non-melanoma skin cancer; APC: age-period-cohort is associated with a significantly increased risk of BCC and SCC in this population [41]. However, the current research focuses on some traditional cancers, so a full assessment of the global burden of NMSC is necessary. were twice as likely to die as females in the United States [9]. This might be because males and females have differ- ent ways of working and living. Males tend to work out- doors, exposing them to more ultraviolet rays. Males are also less likely than females to use sunscreen, hats, and other protective gear. Occupational ultraviolet exposure is strongly associated with the disease burden of NMSC [51]. Discussionh Sex differences in the onset of many diseases have been documented, such as diabetic cardiomyopathy [42], mul- tiple scleroses [43], and myeloid leukemia [44]. There also appears to be gender differences in disease burden attrib- utable to NMSC. Previous studies and the latest results from the National Cancer Institute show that cancer inci- dence and death rates are higher in males than females in all GBD regions [45]. Epidemiological studies have reported that the disease burden of NMSC is significantly higher in males than in females [46–49]. According to the American Cancer Society, NMSC is twice as common in males as in females, with SCC three times more common in males than females [50]. A population-based study assessing trends in NMSC mortality shows that males The risk of NMSC varies with social indicators, work environment, occupational class, and education level [52–54]. In a national study of NMSC incidence and sur- vival, results show that higher socioeconomic status is strongly associated with a higher risk of BCC in the pop- ulation. The higher the socioeconomic status, the higher the risk of BCC [55]. This is consistent with the results of this study. This might be because tanning is more and more favored by society and regarded as a symbol of hap- piness and success with the development of community Hu et al. BMC Cancer (2022) 22:836 Hu et al. BMC Cancer (2022) 22:836 Page 9 of 11 the ASMR between the two etiologies. Therefore, we will further assess the trend of the disease burden in differ- ent GBD regions of countries. Beyond that, it is neces- sary for us to better translate our research into action and develop public policies. and economy. In addition, people have more leisure time to do outdoor activities and even go to the seaside for holidays. Another possible explanation is that in areas with a higher SDI, people know more about NMSC and are more likely to be checked out. This leads to more detection and reporting. Since NMSC is only categorized as two types of BCC and SCC in the GBD study 2019, we assess the disease burden caused by these two types. Other studies show that BCC has the highest incidence but is rarely fatal [29, 56], which is consistent with our research. This might be because that SCC is primarily associated with total and occupational sun exposure. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12885-022-09940-3. The online version contains supplementary material available at https://doi. org/10.1186/s12885-022-09940-3. The online version contains supplementary material available at https://doi. org/10.1186/s12885-022-09940-3. Additional file 1 Supplementary Table 1. The number of new cases and the ASIR of NMSC in 1990 and 2019, and its temporal trends from 1990 to 2019. Abbreviations: NMSC, non-melanoma skin cancer; ASIR, age-standardized incidence rate. Supplementary Table 2. The number of deaths and the ASMR of NMSC in 1990 and 2019, and its temporal trends from 1990 to 2019. Abbreviations: NMSC, non-melanoma skin cancer; ASMR, age-standardized mortality rate. Supplementary Table 3. The number of DALYs rate and the age-standardized DALYs rate of NMSC in 1990 and 2019, and its temporal trends from 1990 to 2019. Abbreviations: NMSC, non-melanoma skin cancer; DALYs, disability-adjusted life years. Supplementary Table 4. Global trends in the number of new cases, the number of deaths, the number of DALYs, the ASIR, the ASMR, and the age- standardized DALYs rate by sex from 2019 to 2044 predicted by the BAPC model. Abbreviations: ASIR, age-standardized incidence rate; ASMR, age-standardized mortality rate; DALYs, disability-adjusted life years; BAPC, Bayesian age-period-cohort. Additional file 2 Supplementary Fig. 1. Results of cluster analysis (a: significant growth; b: a slight increase; c: basically stable or decrease slightly; d: significantly decreased) based on the EAPC values of the ASIR (A), the ASMR (B), and the age-standard DALYs rate (C) from 1990 to 2019. Abbreviations: EAPC, estimated annual percentage change; ASIR, age- standardized incidence rate; ASMR, age-standardized mortality rate; DALY, disability-adjusted-life-year. Additional file 2 Supplementary Fig. 1. Results of cluster analysis (a: significant growth; b: a slight increase; c: basically stable or decrease slightly; d: significantly decreased) based on the EAPC values of the ASIR (A), the ASMR (B), and the age-standard DALYs rate (C) from 1990 to 2019. Abbreviations: EAPC, estimated annual percentage change; ASIR, age- standardized incidence rate; ASMR, age-standardized mortality rate; DALY, disability-adjusted-life-year. However, this study has some limitations. First, the assessment of the disease burden is carried out at the country and GBD region levels. However, some countries are vast, and the burden of disease could vary signifi- cantly between different provinces in a country. Second, the GBD database has defects such as data quality assur- ance. Abbreviations NMSC: Non-melanoma skin cancer; BCC: Basal cell carcinoma; SCC: Squa- mous cell carcinoma; DALYs: Disability-adjusted life years; WHO: World Health Organization; BAPC: Bayesian age-period-cohort; APC: Age-period-cohort; GBD: Global Burden of Disease; EAPC: Estimated annual percentage change; ASIR: Age-standardized incidence rate; ASMR: Age-standardized mortality rate; IHME: The Institute for Health Metrics and Evaluation; ARIMA: Autoregressive Integrated Moving Average; UI: Uncertainty interval; CI: Confidence interval; SDI: Socio-demographic index. In this study, we present that the number of new cases, prevalence, deaths, and DALYs will continue to increase. This is likely the combined result of increasing high-risk behaviors (including outdoor recreation) and changing demographics over the following years. Studies show that NMSC increases by 2–3% annually in the United States [59]. Globally, the incidence rate of BCC has been grow- ing and is predicted to continue to grow until at least 2040 [16]. It is speculated that this might be due to more exposure to the outdoors for recreational and social rea- sons. In addition, Mushtaq points out that the incidence of NMSC is increasing, which is consistent with our study [60]. He also points out that this is attributed to the increased use of sunbeds, recreational sun exposure, and the aging population. Although the current measures and strategies of new medical management have achieved specific results. But overall, the disease burden remains severe. Furthermore, due to the aging trend of the popu- lation, high priority should be given to NMSC. Conclusion Th d h This study shows that NMSC poses a substantial global disease burden and predicts that the future disease bur- den of NMSC will remain severe. We call on health poli- cymakers to act and intervene. They also could develop more targeted and effective policies and measures to reduce adverse health effects associated with NMSC. These policies include enhancing the management and prevention of NMSC-related risk factors and focusing on high-risk groups. We find that the NMSC-related disease burden varies in different GBD regions. NMSC appears to be directly related to skin types in Caucasians [57]. NMSC is the most common malignancy in people with fair skin [58]. Residents are at significantly higher risk of skin cancer in some areas with high solar exposure [19]. Studies have shown a higher disease burden in European descent, such as in Australia, New Zealand, North America, and Northern Europe [29]. 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https://openalex.org/W4385303243	https://bmcgastroenterol.biomedcentral.com/counter/pdf/10.1186/s12876-023-02905-1	English	null	Correction: Limited wedge resection for T1 colon cancer (LIMERIC-II trial) – rationale and study protocol of a prospective multicenter clinical trial	BMC gastroenterology	2,023	cc-by	998	Correction: Limited wedge resection for T1 colon cancer (LIMERIC-II trial) – rationale and study protocol of a prospective multicenter clinical trial Julia Hanevelt1, Jelle F. Huisman1, Laura W. Leicher1, Miangela M. Lacle2, Milan C. Richir3, Paul Didden4, Joost M. J. Geesing5, Niels Smakman6, Jochim S. Terhaar Sive Droste7, Frank ter Borg8, A. Koen Talsma9, Ruud W. M. Schrauwen10, Bob J. van Wely11, Ingrid Schot12, Maarten Vermaas13, Philip Bos14, Colin Sietses15, Wouter L. Hazen16, Dareczka K. Wasowicz17, David E. Ploeg18, Dewkoemar Ramsoekh19, Jurriaan B. Tuynman20, Yasser A. Alderlieste21, Rutger-Jan Renger22, Ramon-Michel Schreuder23, Johanne G. Bloemen24, Ineke van Lijnschoten25, Esther C. J. Consten26, Daan J. Sikkenk26, Matthijs P. Schwartz27, Annelotte Vos28, Jordy P. W. Burger29, Bernhard W. M. Spanier30, Nikki Knijn31, Wouter H. de Vos Tot Nederveen Cappel11, Leon M. G. Moons4 and Henderik L. van Westreenen32 4Department of Gastroenterology and Hepatology, University Medical Center Utrecht, Utrecht, The Netherlands 5Department of Gastroenterology & Hepatology, Diakonessenhuis Hospital, Utrecht, The Netherlands 6Department of Surgery, Diakonessenhuis Hospital, Utrecht, The Netherlands 7Department of Gastro- enterology & Hepatology, Jeroen Bosch Ziekenhuis, Den Bosch, The Netherlands 8Department of Gastroenterology & Hepatology, Deventer Ziekenhuis, Deventer, The Netherlands 9Department of Surgery, Deventer Ziekenhuis, Deventer, The Netherlands 10Department of Gastroenterology & Hepatology, Ziekenhuis Bernhoven, Uden, The Netherlands 11Department of Surgery, Ziekenhuis Bernhoven, Uden, The Netherlands 12Department of Gastroenter ology & Hepatology, IJsselland Ziekenhuis, Capelle a/d Ijssel, The Netherlands 13Department of Surgery, IJsselland Ziekenhuis, Capellle a/d Ijssel, The Netherlands 14Department of Gastroenterology & Hepatology, Ziekenhuis Gelderse Vallei, Ede, The Netherlands 15Department of Surgery, Ziekenhuis Gelderse Vallei, Ede, The Netherlands 16Department of Gastroenterology & Hepatology, Elisabeth-Tweesteden Ziekenhuis, Tilburg, The Netherlands 17Department of Surgery, Elisabeth-Tweesteden Ziekenhuis, Tilburg, The Netherlands 18Department of Pathology, Elisabeth-Tweesteden Ziekenhuis, Tilburg, The Netherlands 19Department of Gastroenterology & Hepatology, Amsterdam UMC Location VUmc, Amsterdam, The Netherlands CORRECTION Open Access Correction: BMC Gastroenterol 23, 214 (2023). https://doi.org/10.1186/s12876-023-02854-9. Following publication of the original article [1], it was reported that David E. Ploeg’s name was incorrectly pub lished as David E. van der Ploeg. The correct authorship list is given in this Correction article and the original article has been updated. BMC Gastroenterology BMC Gastroenterology Hanevelt et al. BMC Gastroenterology (2023) 23:256 https://doi.org/10.1186/s12876-023-02905-1 Author details 1 1Department of Gastroenterology and Hepatology, Dokter Van Heesweg 2, Isala, Zwolle 28025 AB, The Netherlands 1Department of Gastroenterology and Hepatology, Dokter Van Heesweg 2, Isala, Zwolle 28025 AB, The Netherlands 2Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands 3 3Department of Surgery, University Medical Center Utrecht, Utrecht, The Netherlands 3Department of Surgery, University Medical Center Utrecht, Utrecht, The Netherlands 15Department of Surgery, Ziekenhuis Gelderse Vallei, Ede, The Netherlands 16Department of Gastroenterology & Hepatology, Elisabeth-Tweesteden Ziekenhuis, Tilburg, The Netherlands Ziekenhuis, Tilburg, The Netherlands 17 The online version of the original article can be found at https://doi. org/10.1186/s12876-023-02854-9. 17Department of Surgery, Elisabeth-Tweesteden Ziekenhuis, Tilburg, The Netherlands 18Department of Pathology, Elisabeth-Tweesteden Ziekenhuis, Tilburg, The Netherlands Correspondence: Julia Hanevelt j.hanevelt@isala.nl 19Department of Gastroenterology & Hepatology, Amsterdam UM Department of Gastroenterology & Hepatolo Location VUmc, Amsterdam, The Netherlands Location VUmc, Amsterdam, The Netherlands Full list of author information is available at the end of the article References References 1. Hanevelt J, Huisman JF, Leicher LW, et al. Limited wedge resection for T1 colon cancer (LIMERIC-II trial) – rationale and study protocol of a prospec tive multicenter clinical trial. BMC Gastroenterol. 2023;23:214. https://doi. org/10.1186/s12876-023-02854-9. References 1. Hanevelt J, Huisman JF, Leicher LW, et al. Limited wedge resection for T1 colon cancer (LIMERIC-II trial) – rationale and study protocol of a prospec tive multicenter clinical trial. BMC Gastroenterol. 2023;23:214. https://doi. org/10.1186/s12876-023-02854-9. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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 2 Hanevelt et al. BMC Gastroenterology (2023) 23:256 (2023) 23:256 Hanevelt et al. BMC Gastroenterology (2023) 23:256 31Pathology DNA, Location Arnhem, The Netherlands 32Department of Surgery, Isala, Zwolle, The Netherlands 20Department of Surgery, Amsterdam UMC Location VUmc, Amsterdam, The Netherlands 21Department of Gastroenterology & Hepatology, Beatrixziekenhuis - Rivas, Gorinchem, The Netherlands 22Department of Surgery, Beatrixziekenhuis - Rivas, Gorinchem, The Netherlands 23Department of Gastroenterology & Hepatology, Catharina Ziekenhuis, Eindhoven, The Netherlands 24Department of Surgery, Catharina Ziekenhuis, Eindhoven, The Netherlands 25Eurofns/PAMM NL, Veldhoven, The Netherlands 26Department of Surgery, Meander Medisch Centrum, Amersfoort, The Netherlands 27Department of Gastroenterology & Hepatology, Meander Medisch Centrum, Amersfoort, The Netherlands 28Department of Pathology, Meander Medisch Centrum, Amersfoort, The Netherlands 29Department of Surgery, Rijnstate Hospital, Arnhem, The Netherlands 30Department of Gastroenterology & Hepatology, Rijnstate Hospital, Arnhem, The Netherlands 20Department of Surgery, Amsterdam UMC Location VUmc, Amsterdam, The Netherlands 21Department of Gastroenterology & Hepatology, Beatrixziekenhuis - Rivas, Gorinchem, The Netherlands 22Department of Surgery, Beatrixziekenhuis - Rivas, Gorinchem, The Netherlands 23Department of Gastroenterology & Hepatology, Catharina Ziekenhuis, Eindhoven, The Netherlands 24Department of Surgery, Catharina Ziekenhuis, Eindhoven, The Netherlands 25Eurofns/PAMM NL, Veldhoven, The Netherlands 26Department of Surgery, Meander Medisch Centrum, Amersfoort, The Netherlands 27Department of Gastroenterology & Hepatology, Meander Medisch Centrum, Amersfoort, The Netherlands 28Department of Pathology, Meander Medisch Centrum, Amersfoort, The Netherlands 29Department of Surgery, Rijnstate Hospital, Arnhem, The Netherlands 30Department of Gastroenterology & Hepatology, Rijnstate Hospital, Arnhem, The Netherlands Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Citation for published version (Harvard): LHCb Collaboration 2018, 'Erratum to: Measurement of the CKM angle γ using B± → DK± with D → KS0π+π−, KS0K+K− decays', Journal of High Energy Physics, vol. 2018, no. 10, 107. https://doi.org/10.1007/JHEP10(2018)107 Erratum to LHCb Collaboration DOI: 10.1007/JHEP10(2018)107 License: Creative Commons: Attribution (CC BY) DOI: 10.1007/JHEP10(2018)107 License: Creative Commons: Attribution (CC BY) Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): LHCb Collaboration 2018, 'Erratum to: Measurement of the CKM angle γ using B± → DK± with D → KS0π+π−, KS0K+K− decays', Journal of High Energy Physics, vol. 2018, no. 10, 107. https://doi.org/10.1007/JHEP10(2018)107 Link to publication on Research at Birmingham portal Publisher Rights Statement: Published in Journal of High Energy Physics on 16/10/2018 ArXiv ePrint: 1806.01202 The B+ and B−labels of the conﬁdence regions in ﬁgure 10 of the original paper [1] were erroneously swapped. 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Oct. 2024 Published for SISSA by Springer Received: September 21, 2018 Accepted: October 8, 2018 Published: October 16, 2018 Erratum: Measurement of the CKM angle γ using B± →DK± with D →K0 Sπ+π−, K0 SK+K−decays JHEP10(2018)107 The LHCb collaboration E-mail: mikkel.bjoern@physics.ox.ac.uk E-mail: mikkel.bjoern@physics.ox.ac.uk Erratum to: JHEP08(2018)176 References [1] LHCb collaboration, Measurement of the CKM angle γ using B± →DK± with D →K0 Sπ+π−, K0 SK+K−decays, JHEP 08 (2018) 176 [arXiv:1806.01202] [INSPIRE]. [2] LHCb collaboration, Update of the LHCb combination of the CKM angle γ using B →DK decays, LHCb-CONF-2017-004 (2017) [INSPIRE]. [2] LHCb collaboration, Update of the LHCb combination of the CKM angle γ using B →DK decays, LHCb-CONF-2017-004 (2017) [INSPIRE]. [2] LHCb collaboration, Update of the LHCb combination of the CKM angle γ using B →DK decays, LHCb-CONF-2017-004 (2017) [INSPIRE]. Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP10(2018)107 https://doi.org/10.1007/JHEP10(2018)107 JHEP10(2018)107 ± x ± y 0.2 − 0.1 − 0 0.1 0.2 0.2 − 0.1 − 0 0.1 0.2 2015 & 2016 data LHCb Combination LHCb − B + B Figure 10. Two-dimensional 68.3 %, 95.5 % and 99.7 % conﬁdence regions for (x±, y±) obtained in this measurement, as well as for the LHCb combination in ref. [2], taking statistical and systematic uncertainties, as well as their correlations, into account. ± x ± y 0.2 − 0.1 − 0 0.1 0.2 0.2 − 0.1 − 0 0.1 0.2 2015 & 2016 data LHCb Combination LHCb − B + B Figure 10. Two-dimensional 68.3 %, 95.5 % and 99.7 % conﬁdence regions for (x±, y±) obtained in this measurement, as well as for the LHCb combination in ref. [2], taking statistical and systematic uncertainties, as well as their correlations, into account. – 2 – The LHCb collaboration R. Aaij27, B. Adeva41, M. Adinolﬁ48, C.A. Aidala73, Z. Ajaltouni5, S. Akar59, P. Albicocco18, j , , , , j , , J. Albrecht10, F. Alessio42, M. Alexander53, A. Alfonso Albero40, S. Ali27, G. Alkhazov33, P. Alvarez Cartelle55, A.A. Alves Jr59, S. Amato2, S. Amerio23, Y. Amhis7, L. An3, L. Anderlini17, G. Andreassi43, M. Andreotti16,g, J.E. Andrews60, R.B. Appleby56, F. Archilli2 P. d’Argent12, J. Arnau Romeu6, A. Artamonov39, M. Artuso61, K. Arzymatov37, E. Aslanides6, P. d’Argent12, J. Arnau Romeu6, A. Artamonov39, M. Artuso61, K. Arzymatov37, E. Aslanides6 M. Atzeni44, S. Bachmann12, J.J. Back50, S. Baker55, V. Balagura7,b, W. Baldini16, A. Baranov Atzeni44, S. Bachmann12, J.J. Back50, S. Baker55, V. Balagura7,b, W. Baldini16, A. Baranov37, J. Barlow56, S. Barsuk7, W. Barter56, F. Baryshnikov70, V. Batozskaya31, B. Batsukh61, R.J. Barlow56, S. Barsuk7, W. Barter56, F. Baryshnikov70, V. Batozskaya31, B. Batsukh6 V. Battista43, A. Bay43, J. Beddow53, F. Bedeschi24, I. Bediaga1, A. Beiter61, L.J. Bel27, Battista43, A. Bay43, J. Beddow53, F. Bedeschi24, I. Bediaga1, A. Beiter61, L.J. Bel27, JHEP10(2018)107 N. Beliy63, V. Bellee43, N. Belloli20,i, K. Belous39, I. Belyaev34,42, E. Ben-Haim8, G. Benc Beliy63, V. Bellee43, N. Belloli20,i, K. Belous39, I. Belyaev34,42, E. Ben-Haim8, G. Bencivenni18, S. Benson27, S. Beranek9, A. Berezhnoy35, R. Bernet44, D. Berninghoﬀ12, E. Bertholet8, Benson27, S. Beranek9, A. Berezhnoy35, R. Bernet44, D. Berninghoﬀ12, E. Bertholet8, A. Bertolin23, C. Betancourt44, F. Betti15,42, M.O. Bettler49, M. van Beuzekom27, A. Bertolin23, C. Betancourt44, F. Betti15,42, M.O. Bettler49, M. van Beuzekom27, Ia. Bezshyiko44, S. Bhasin48, J. Bhom29, S. Bifani47, P. Billoir8, A. Birnkraut10, A. Bizzeti17 Bezshyiko44, S. Bhasin48, J. Bhom29, S. Bifani47, P. Billoir8, A. Birnkraut10, A. Bizzeti17,u, M. Bjørn57, M.P. Blago42, T. Blake50, F. Blanc43, S. Blusk61, D. Bobulska53, V. Bocci26, Bjørn57, M.P. Blago42, T. Blake50, F. Blanc43, S. Blusk61, D. Bobulska53, V. Bocci26, O. Boente Garcia41, T. Boettcher58, A. Bondar38,w, N. Bondar33, S. Borghi56,42, M. Borisya O. Boente Garcia41, T. Boettcher58, A. Bondar38,w, N. Bondar33, S. Borghi56,42, M. Borisya M. Borsato41,42, F. Bossu7, M. Boubdir9, T.J.V. Bowcock54, C. Bozzi16,42, S. Braun12, M. Brodski42, J. Brodzicka29, D. Brundu22, E. Buchanan48, A. Buonaura44, C. Burr56, M. Brodski42, J. Brodzicka29, D. Brundu22, E. Buchanan48, A. Buonaura44, C. Burr56, R. Calladine47, M. Calvi20,i, M. Calvo Gomez40,m, A. Camboni40,m, P. Campana18, R. Calladine47, M. Calvi20,i, M. Calvo Gomez40,m, A. Camboni40,m, P. Campana18, D.H. Campora Perez42, L. Capriotti56, A. Carbone15,e, G. Carboni25, R. Cardinale19,h Campora Perez42, L. Capriotti56, A. Carbone15,e, G. Carboni25, R. Cardinale19,h, A. Cardini22, P. The LHCb collaboration Carniti20,i, L. Carson52, K. Carvalho Akiba2, G. Casse54, L. Cassina20, rdini22, P. Carniti20,i, L. Carson52, K. Carvalho Akiba2, G. Casse54, L. Cassina20, Cattaneo42, G. Cavallero19,h, R. Cenci24,p, D. Chamont7, M.G. Chapman48, M. Charles8, M. Cattaneo42, G. Cavallero19,h, R. Cenci24,p, D. Chamont7, M.G. Chapman48, M. Charles8 Ph. Charpentier42, G. Chatzikonstantinidis47, M. Chefdeville4, V. Chekalina37, C. Chen3, Ph. Charpentier42, G. Chatzikonstantinidis47, M. Chefdeville4, V. Chekalina37, C. Chen3, Chen22, S.-G. Chitic42, V. Chobanova41, M. Chrzaszcz42, A. Chubykin33, P. Ciambrone18, S. Chen22, S.-G. Chitic42, V. Chobanova41, M. Chrzaszcz42, A. Chubykin33, P. Ciambrone18 Cid Vidal41, G. Ciezarek42, P.E.L. Clarke52, M. Clemencic42, H.V. Cliﬀ49, J. Closier42, X. Cid Vidal41, G. Ciezarek42, P.E.L. Clarke52, M. Clemencic42, H.V. Cliﬀ49, J. Closier42 V. Coco42, J. Cogan6, E. Cogneras5, L. Cojocariu32, P. Collins42, T. Colombo42, Coco42, J. Cogan6, E. Cogneras5, L. Cojocariu32, P. Collins42, T. Colombo42, Comerma-Montells12, A. Contu22, G. Coombs42, S. Coquereau40, G. Corti42, M. Corvo16,g, A. Comerma-Montells12, A. Contu22, G. Coombs42, S. Coquereau40, G. Corti42, M. Cor C.M. Costa Sobral50, B. Couturier42, G.A. Cowan52, D.C. Craik58, A. Crocombe50, M. Costa Sobral50, B. Couturier42, G.A. Cowan52, D.C. Craik58, A. Crocombe50, M. Cruz Torres1, R. Currie52, C. D’Ambrosio42, F. Da Cunha Marinho2, C.L. Da Silva74, Cruz Torres1, R. Currie52, C. D’Ambrosio42, F. Da Cunha Marinho2, C.L. Da Silva74, E. Dall’Occo27, J. Dalseno48, A. Danilina34, A. Davis3, O. De Aguiar Francisco42, K. De Bruyn42 Dall’Occo27, J. Dalseno48, A. Danilina34, A. Davis3, O. De Aguiar Francisco42, K. De Bruyn42, S. De Capua56, M. De Cian43, J.M. De Miranda1, L. De Paula2, M. De Serio14,d, P. De Simone De Capua56, M. De Cian43, J.M. De Miranda1, L. De Paula2, M. De Serio14,d, P. De Simone18, C.T. Dean53, D. Decamp4, L. Del Buono8, B. Delaney49, H.-P. Dembinski11, M. Demmer10, C.T. Dean53, D. Decamp4, L. Del Buono8, B. Delaney49, H.-P. Dembinski11, M. Demmer10, P. Di Nezza18, S. Didenko70, H. Dijkstra42, F. Dordei42, M. Dorigo42,y, A. Dosil Su´arez41, P. Di Nezza18, S. Didenko70, H. Dijkstra42, F. Dordei42, M. Dorigo42,y, A. Dosil Su´arez41, L. Douglas53, A. Dovbnya45, K. Dreimanis54, L. Dufour27, G. Dujany8, P. Durante42, J.M. Durham74, D. Dutta56, R. Dzhelyadin39, M. Dziewiecki12, A. Dziurda29, A. Dzyuba33 S. Easo51, U. Egede55, V. Egorychev34, S. Eidelman38,w, S. Eisenhardt52, U. Eitschberger10, R. Ekelhof10, L. Eklund53, S. Ely61, A. Ene32, S. Escher9, S. Esen27, T. Evans59, A. Falabella15, N. Farley47, S. Farry54, D. Fazzini20,42,i, L. Federici25, G. Fernandez40, P. Fernandez Declara A. Fernandez Prieto41, F. Ferrari15, L. Ferreira Lopes43, F. The LHCb collaboration Ferreira Rodrigues2, M. Ferr S. Filippov36, R.A. Fini14, M. Fiorini16,g, M. Firlej30, C. Fitzpatrick43, T. Fiutowski30, F. Fleuret7,b, M. Fontana22,42, F. Fontanelli19,h, R. Forty42, V. Franco Lima54, M. Frank42 C. Frei42, J. Fu21,q, W. Funk42, C. F¨arber42, M. F´eo Pereira Rivello Carvalho27, E. Gabriel52, Y. Gao3, L.M. Garcia Martin72, B. Garcia Plana41, J. Garc´ıa Pardi˜nas44, J. Garra Tico49, , , , , , L. Garrido40, D. Gascon40, C. Gaspar42, L. Gavardi10, G. Gazzoni5, D. Gerick12, E. Gersabeck5 M. Gersabeck56, T. Gershon50, D. Gerstel6, Ph. Ghez4, S. Gian`ı43, V. Gibson49, O.G. Girard43, – 3 – Giubega32, K. Gizdov52, V.V. Gligorov8, D. Golubkov34, A. Golutvin55,70, A. Gomes1,a, L. Giubega32, K. Gizdov52, V.V. Gligorov8, D. Golubkov34, A. Golutvin55,70, A. Gomes1,a, I.V. Gorelov35, C. Gotti20,i, E. Govorkova27, J.P. Grabowski12, R. Graciani Diaz40, L.A. Granado Cardoso42, E. Graug´es40, E. Graverini44, G. Graziani17, A. Grecu32, R. Greim P. Griﬃth22, L. Grillo56, L. Gruber42, B.R. Gruberg Cazon57, O. Gr¨unberg67, C. Gu3, E. Gushchin36, Yu. Guz39,42, T. Gys42, C. G¨obel62, T. Hadavizadeh57, C. Hadjivasiliou5, G. Haefeli43, C. Haen42, S.C. Haines49, B. Hamilton60, X. Han12, T.H. Hancock57, S. Hansmann-Menzemer12, N. Harnew57, S.T. Harnew48, T. Harrison54, C. Hasse42, M. Hatc J. He63, M. Hecker55, K. Heinicke10, A. Heister9, K. Hennessy54, L. Henry72, E. van Herwijnen42, Z.C. Huard59, W. Hulsbergen27, T. Humair55, M. Hushchyn37, D. Hutchcroft54, D. Hynds27, , g , , y , , y P. Ibis10, M. Idzik30, P. Ilten47, K. Ivshin33, R. Jacobsson42, J. Jalocha57, E. Jans27, JHEP10(2018)107 JHEP10(2018)107 A. Jawahery60, F. Jiang3, M. John57, D. Johnson42, C.R. Jones49, C. Joram42, B. Jost42, N. Jurik57, S. Kandybei45, M. Karacson42, J.M. Kariuki48, S. Karodia53, N. Kazeev37, M. Kecke12, F. Keizer49, M. Kelsey61, M. Kenzie49, T. Ketel28, E. Khairullin37, B. Khanji12 e12, F. Keizer49, M. Kelsey61, M. Kenzie49, T. Ketel28, E. Khairullin37, B. Khanji12, C. Khurewathanakul43, K.E. Kim61, T. Kirn9, S. Klaver18, K. Klimaszewski31, T. Klimkovich11 ewathanakul43, K.E. Kim61, T. Kirn9, S. Klaver18, K. Klimaszewski31, T. Klimkovich11, S. Koliiev46, M. Kolpin12, R. Kopecna12, P. Koppenburg27, I. Kostiuk27, S. Kotriakhova33, v46, M. Kolpin12, R. Kopecna12, P. Koppenburg27, I. Kostiuk27, S. Kotriakhova33, ozeiha5, L. Kravchuk36, M. Kreps50, F. Kress55, P. Krokovny38,w, W. Krupa30, M. Kozeiha5, L. Kravchuk36, M. Kreps50, F. Kress55, P. Krokovny38,w, W. Krupa30, W. Krzemien31, W. Kucewicz29,l, M. Kucharczyk29, V. Kudryavtsev38,w, A.K. Kuonen43, rzemien31, W. Kucewicz29,l, M. Kucharczyk29, V. Kudryavtsev38,w, A.K. Kuonen43, T. Kvaratskheliya34,42, D. Lacarrere42, G. Laﬀerty56, A. Lai22, D. Lancierini44, G. Lanfran C. Langenbruch9, T. Latham50, C. M. Poli Lener18, A. Poluektov50, N. Polukhina70,c, I. Polyakov61, E. Polycarpo2, G.J. Pomery48 The LHCb collaboration Lazzeroni47, R. Le Gac6, A. Leﬂat35, J. Lefran¸cois7, R. Lef`evre5, F. Lemaitre42, O. Leroy6, T. Lesiak29, B. Leverington12, P.-R. Li63, T. Li3, Z. Li61, X. Liang61, T. Likhomanenko69, R. Lindner42, F. Lionetto44, V. Lisovskyi7, X. Liu3, D. Loh A. Loi22, I. Longstaﬀ53, J.H. Lopes2, G.H. Lovell49, D. Lucchesi23,o, M. Lucio Martinez41, A. Loi22, I. Longstaﬀ53, J.H. Lopes2, G.H. Lovell49, D. Lucchesi23,o, M. Lucio Martinez4 A. Lupato23, E. Luppi16,g, O. Lupton42, A. Lusiani24, X. Lyu63, F. Machefert7, F. Maciuc32 V. Macko43, P. Mackowiak10, S. Maddrell-Mander48, O. Maev33,42, K. Maguire56, V. Macko43, P. Mackowiak10, S. Maddrell-Mander48, O. Maev33,42, K. Maguire56, G. Manca22,f, G. Mancinelli6, D. Marangotto21,q, J. Maratas5,v, J.F. Marchand4, U. Marconi15 G. Manca22,f, G. Mancinelli6, D. Marangotto21,q, J. Maratas5,v, J.F. Marchand4, U. Marconi15, C. Marin Benito40, M. Marinangeli43, P. Marino43, J. Marks12, G. Martellotti26, M. Martin6, arin Benito40, M. Marinangeli43, P. Marino43, J. Marks12, G. Martellotti26, M. Martin6, M. Martinelli42, D. Martinez Santos41, F. Martinez Vidal72, A. Massaﬀerri1, R. Matev42, M. Martinelli42, D. Martinez Santos41, F. Martinez Vidal72, A. Massaﬀerri1, R. Matev42, A. Mathad50, Z. Mathe42, C. Matteuzzi20, A. Mauri44, E. Maurice7,b, B. Maurin43, A. Mazu athad50, Z. Mathe42, C. Matteuzzi20, A. Mauri44, E. Maurice7,b, B. Maurin43, A. Mazurov47, M. McCann55,42, A. McNab56, R. McNulty13, J.V. Mead54, B. Meadows59, C. Meaux6, ann55,42, A. McNab56, R. McNulty13, J.V. Mead54, B. Meadows59, C. Meaux6, F. Meier10, N. Meinert67, D. Melnychuk31, M. Merk27, A. Merli21,q, E. Michielin23, Meier10, N. Meinert67, D. Melnychuk31, M. Merk27, A. Merli21,q, E. Michielin23, D.A. Milanes66, E. Millard50, M.-N. Minard4, L. Minzoni16,g, D.S. Mitzel12, A. Mogini8, Milanes66, E. Millard50, M.-N. Minard4, L. Minzoni16,g, D.S. Mitzel12, A. Mogini8, J. Molina Rodriguez1,z, T. Momb¨acher10, I.A. Monroy66, S. Monteil5, M. Morandin23, Molina Rodriguez1,z, T. Momb¨acher10, I.A. Monroy66, S. Monteil5, M. Morandin23, Morello18, M.J. Morello24,t, O. Morgunova69, J. Moron30, A.B. Morris6, R. Mountain61, G. Morello18, M.J. Morello24,t, O. Morgunova69, J. Moron30, A.B. Morris6, R. Mountain61, F. Muheim52, M. Mulder27, C.H. Murphy57, D. Murray56, D. M¨uller42, J. M¨uller10, K. M¨ulle Muheim52, M. Mulder27, C.H. Murphy57, D. Murray56, D. M¨uller42, J. M¨uller10, K. M¨uller44 V. M¨uller10, P. Naik48, T. Nakada43, R. Nandakumar51, A. Nandi57, T. Nanut43, I. Nasteva2 M¨uller10, P. Naik48, T. Nakada43, R. Nandakumar51, A. Nandi57, T. Nanut43, I. Nasteva2, Needham52, N. Neri21, S. Neubert12, N. Neufeld42, M. Neuner12, T.D. Nguyen43, M. Needham52, N. Neri21, S. Neubert12, N. Neufeld42, M. Neuner12, T.D. Nguyen43, C. Nguyen-Mau43,n, S. Nieswand9, R. Niet10, N. Nikitin35, A. Nogay69, D.P. The LHCb collaboration Serra44, J. Serrano6, Sestini23, P. Seyfert42, M. Shapkin39, Y. Shcheglov33,†, T. Shears54, L. Shekhtman38,w, L. Sestini23, P. Seyfert42, M. Shapkin39, Y. Shcheglov33,†, T. Shears54, L. Shekhtman38,w, V. Shevchenko69, E. Shmanin70, B.G. Siddi16, R. Silva Coutinho44, L. Silva de Oliveira2, Shevchenko69, E. Shmanin70, B.G. Siddi16, R. Silva Coutinho44, L. Silva de Oliveira2, G. Simi23,o, S. Simone14,d, N. Skidmore12, T. Skwarnicki61, J.G. Smeaton49, E. Smith9, G. Simi23,o, S. Simone14,d, N. Skidmore12, T. Skwarnicki61, J.G. Smeaton49, E. Smith9, T. Smith52, M. Smith55, M. Soares15, l. Soares Lavra1, M.D. Sokoloﬀ59, F.J.P. Soler53, I.T. Smith52, M. Smith55, M. Soares15, l. Soares Lavra1, M.D. Sokoloﬀ59, F.J.P. Soler53, Souza De Paula2, B. Spaan10, P. Spradlin53, F. Stagni42, M. Stahl12, S. Stahl42, P. Stefko43, B. Souza De Paula2, B. Spaan10, P. Spradlin53, F. Stagni42, M. Stahl12, S. Stahl42, P. Ste S. Stefkova55, O. Steinkamp44, S. Stemmle12, O. Stenyakin39, M. Stepanova33, H. Stevens10, Stefkova55, O. Steinkamp44, S. Stemmle12, O. Stenyakin39, M. Stepanova33, H. Stevens10, S. Stone61, B. Storaci44, S. Stracka24,p, M.E. Stramaglia43, M. Straticiuc32, U. Straumann44 Stone61, B. Storaci44, S. Stracka24,p, M.E. Stramaglia43, M. Straticiuc32, U. Straumann44, S. Strokov71, J. Sun3, L. Sun64, K. Swientek30, V. Syropoulos28, T. Szumlak30, M. Szymanski63 S. Strokov71, J. Sun3, L. Sun64, K. Swientek30, V. Syropoulos28, T. Szumlak30, M. Szymanski63 S. T’Jampens4, Z. Tang3, A. Tayduganov6, T. Tekampe10, G. Tellarini16, F. Teubert42, E. Thomas42, J. van Tilburg27, M.J. Tilley55, V. Tisserand5, S. Tolk42, L. Tomasset Wang61, M. Wang3, Y. Wang65, Z. Wang44, D.R. Ward49, H.M. Wark54, N.K. Watson47, Websdale55, A. Weiden44, C. Weisser58, M. Whitehead9, J. Wicht50, G. Wilkinson57, Wilkinson61, I. Williams49, M.R.J. Williams56, M. Williams58, T. Williams47, F.F. Wilson51,42, J. Wimberley60, M. Winn7, J. Wishahi10, W. Wislicki31, M. Witek29, G. Wormser7, S.A. Wotton49, K. Wyllie42, D. Xiao65, Y. Xie65, A. Xu3, M. Xu65, Q. Xu63, Z. Xu3, Z. Xu4, Z. Yang3, Z. Yang60, Y. Yao61, L.E. Yeomans54, H. Yin65, J. Yu65,ab, X. Yuan61, O. Yushchenko39, K.A. Zarebski47, M. Zavertyaev11,c, D. Zhang65, L. Zhang3, W.C. Zhang3,aa, Y. Zhang7, A. Zhelezov12, Y. Zheng63, X. Zhu3, V. Zhukov9,35, J.B. Zonneveld52, S. Zucchelli15 Z. Xu3, Z. Xu4, Z. Yang3, Z. Yang60, Y. Yao61, L.E. Yeomans54, H. Yin65, J. Yu65,ab, X. Yuan61, O. Yushchenko39, K.A. Zarebski47, M. Zavertyaev11,c, D. Zhang65, L. Zhang3, W.C. 1 Centro Brasileiro de Pesquisas F´ısicas (CBPF), Rio de Janeiro, Brazil 2 Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 3 Center for High Energy Physics, Tsinghua University, Beijing, China 4 Univ. Grenoble Alpes, Univ. Savoie Mont Blanc, CNRS, IN2P3-LAPP, Annecy, France 5 Clermont Universit´e, Universit´e Blaise Pascal, CNRS/IN2P3, LPC, Clermont-Ferrand, France 6 Aix Marseille Univ, CNRS/IN2P3, CPPM, Marseille, France 7 LAL, Univ. Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France 8 LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France The LHCb collaboration O’Hanlon15, A. Oblakowska-Mucha30, V. Obraztsov39, S. Ogilvy18, R. Oldeman22,f, C.J.G. Onderwater68, A. Oblakowska-Mucha30, V. Obraztsov39, S. Ogilvy18, R. Oldeman22,f, C.J.G. Onderwater68, M. Palutan18,42, G. Panshin71, A. Papanestis51, M. Pappagallo52, L.L. Pappalardo16,g, M. Palutan18,42, G. Panshin71, A. Papanestis51, M. Pappagallo52, L.L. Pappalardo16,g, Perret5, L. Pescatore43, K. Petridis48, A. Petrolini19,h, A. Petrov69, S. Petrucci52, Petruzzo21,q, B. Pietrzyk4, G. Pietrzyk43, M. Pikies29, M. Pili57, D. Pinci26, J. Pinzino42, Pisani42, A. Piucci12, V. Placinta32, S. Playfer52, J. Plews47, M. Plo Casasus41, F. Polci8, M. Poli Lener18, A. Poluektov50, N. Polukhina70,c, I. Polyakov61, E. Polycarpo2, G.J. Pomery48, M. Poli Lener18, A. Poluektov50, N. Polukhina70,c, I. Polyakov61, E. Polycarpo2, G.J. Pomery48, – 4 – S. Ponce42, A. Popov39, D. Popov47,11, S. Poslavskii39, C. Potterat2, E. Price48, J. Prisciandaro4 nce42, A. Popov39, D. Popov47,11, S. Poslavskii39, C. Potterat2, E. Price48, J. Prisciandaro41, , p , p , , , , , Prouve48, V. Pugatch46, A. Puig Navarro44, H. Pullen57, G. Punzi24,p, W. Qian63, J. Qin63, C. Prouve48, V. Pugatch46, A. Puig Navarro44, H. Pullen57, G. Punzi24,p, W. Qian63, J. Qin6 , g , g , , , Q , Q , Quagliani8, B. Quintana5, B. Rachwal30, J.H. Rademacker48, M. Rama24, M. Ramos Pernas41, S. Rangel2, F. Ratnikov37,x, G. Raven28, M. Ravonel Salzgeber42, M. Reboud4, F. Redi43, M.S. Rangel2, F. Ratnikov37,x, G. Raven28, M. Ravonel Salzgeber42, M. Reboud4, F. Redi43, Reichert10, A.C. dos Reis1, F. Reiss8, C. Remon Alepuz72, Z. Ren3, V. Renaudin7, S. Reichert10, A.C. dos Reis1, F. Reiss8, C. Remon Alepuz72, Z. Ren3, V. Renaudin7, S. Ricciardi51, S. Richards48, K. Rinnert54, P. Robbe7, A. Robert8, A.B. Rodrigues43, E. Rodrigues59, J.A. Rodriguez Lopez66, M. Roehrken42, A. Rogozhnikov37, S. Roiser42, A. Rollings57, V. Romanovskiy39, A. Romero Vidal41, M. Rotondo18, M.S. Rudolph61, T. Ruf42, A. Rollings57, V. Romanovskiy39, A. Romero Vidal41, M. Rotondo18, M.S. Rudolph61, T. Ru C. Sanchez Gras27, C. Sanchez Mayordomo72, B. Sanmartin Sedes41, R. Santacesaria26, C. Sanchez Gras27, C. Sanchez Mayordomo72, B. Sanmartin Sedes41, R. Santacesaria26, C. Santamarina Rios41, M. Santimaria18, E. Santovetti25,j, G. Sarpis56, A. Sarti18,k, JHEP10(2018)107 JHEP10(2018)107 C. Satriano26,s, A. Satta25, M. Saur63, D. Savrina34,35, S. Schael9, M. Schellenberg10, Schiller53, H. Schindler42, M. Schmelling11, T. Schmelzer10, B. Schmidt42, O. Schneider43, A. Schopper42, H.F. Schreiner59, M. Schubiger43, M.H. Schune7, R. Schwemmer42, B. Sciascia Schopper42, H.F. Schreiner59, M. Schubiger43, M.H. Schune7, R. Schwemmer42, B. Sciascia18, A. Sciubba26,k, A. Semennikov34, E.S. Sepulveda8, A. Sergi47,42, N. Serra44, J. Serrano6, Sciubba26,k, A. Semennikov34, E.S. Sepulveda8, A. Sergi47,42, N. 8 LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France The LHCb collaboration Zhang3,aa, Y Zhang7 A Zhelezov12 Y Zheng63 X Zhu3 V Zhukov9,35 J B Zonneveld52 S Zucchelli15 5 Clermont Universit´e, Universit´e Blaise Pascal, CNRS/IN2P3, LPC, Clermont-Ferrand, France 6 Aix Marseille Univ, CNRS/IN2P3, CPPM, Marseille, France / 7 LAL, Univ. Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France – 5 – 9 I. Physikalisches Institut, RWTH Aachen University, Aachen, Germany 9 I. The LHCb collaboration Physikalisches Institut, RWTH Aachen University, Aachen, Germany 10 Fakult¨at Physik, Technische Universit¨at Dortmund, Dortmund, Germany 10 Fakult¨at Physik, Technische Universit¨at Dortmund, Dortmund, Germany 11 Max-Planck-Institut f¨ur Kernphysik (MPIK), Heidelberg, Germany 11 Max-Planck-Institut f¨ur Kernphysik (MPIK), Heidelberg, Germany 12 Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany 12 Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany Physikalisches Institut, Ruprecht-Karls-Universit¨at H 12 Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, H School of Physics, University College Dublin, Dublin, 14 INFN Sezione di Bari, Bari, Italy 15 INFN Sezione di Bologna, Bologna, Italy 16 INFN Sezione di Ferrara, Ferrara, Italy 17 INFN Sezione di Firenze, Firenze, Italy 18 INFN Laboratori Nazionali di Frascati, Frascati, Italy 19 INFN Sezione di Genova, Genova, Italy 20 INFN Sezione di Milano-Bicocca, Milano, Italy JHEP10(2018)107 21 INFN Sezione di Milano, Milano, Italy 22 INFN Sezione di Cagliari, Monserrato, Italy 23 INFN Sezione di Padova, Padova, Italy 24 INFN Sezione di Pisa, Pisa, Italy 25 INFN Sezione di Roma Tor Vergata, Roma, Italy 26 INFN Sezione di Roma La Sapienza, Roma, Italy 27 Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands Nikhef National Institute for Subatomic Physics, A 28 Nikhef National Institute for Subatomic Physics and VU University Amsterdam, Amsterdam, Netherlands 29 Henryk Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Krak´ow, Poland 29 Henryk Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Krak´ow, Poland 30 AGH — University of Science and Technology, Faculty of Physics and Applied Computer Science, Krak´ow, Poland 29 Henryk Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Krak´ow, Poland 30 AGH — University of Science and Technology, Faculty of Physics and Applied Computer Science, 30 AGH — University of Science and Technology, Faculty of Physics and Applied Computer Science, Krak´ow, Poland 31 National Center for Nuclear Research (NCBJ), Warsaw, Poland 32 Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest-Magurele, Romania 33 Petersburg Nuclear Physics Institute (PNPI), Gatchina, Russia 34 Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia 35 Institute of Nuclear Physics, Moscow State University (SINP MSU), Moscow, Russia 36 Institute for Nuclear Research of the Russian Academy of Sciences (INR RAS), Moscow, Russia 36 Institute for Nuclear Research of the Russian Academy of Sciences (INR RAS), Moscow, Russia 37 Yandex School of Data Analysis, Moscow, Russia 37 Yandex School of Data Analysis, Moscow, Russia 37 Yandex School of Data Analysis, Moscow, Russia 38 Budker Institute of Nuclear Physics (SB RAS), N 39 Institute for High Energy Physics (IHEP), Protvino, Russia stitute for High Energy Physics (IHEP), Protvino, R 40 ICCUB, Universitat de Barcelona, Barcelona, Spain 41 Instituto Galego de F´ısica de Altas Enerx´ıas (IGFAE), Universidade de Santiago de Compostela, Santiago de Compostela, Spain 41 Instituto Galego de F´ısica de Altas Enerx´ıas (IGFAE), Universidade de Santiago de Compostela, Santiago de Compostela, Spain 42 European Organization for Nuclear Research (CERN), Geneva, Switzerland 42 European Organization for Nuclear Research (CERN), Geneva, Switzerland 43 Institute of Physics, Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Lausanne, Switzerland 44 Ph ik I tit t U i it¨t Z¨ i h Z¨ i h S it l d 43 Institute of Physics, Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Lausanne, Switzerland 44 Physik-Institut Universit¨at Z¨urich Z¨urich Switzerland 43 Institute of Physics, Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Lausann Physik-Institut, Universit¨at Z¨urich, Z¨urich, Switzer 45 NSC Kharkiv Institute of Physics and Technology (NSC KIPT), Kharkiv, Ukr 45 NSC Kharkiv Institute of Physics and Technology (NSC KIPT), Kharkiv, Ukrai SC Kharkiv Institute of Physics and Technology (NS 46 Institute for Nuclear Research of the National Academy of Sciences (KINR), Kyiv 46 Institute for Nuclear Research of the National Academy of Sciences (KINR), Kyiv, Ukraine Institute for Nuclear Research of the National Academy of Sciences (KINR), Kyiv, Ukraine 47 University of Birmingham, Birmingham, United Kingdom 47 University of Birmingham, Birmingham, United Kingdom 48 H.H. The LHCb collaboration Wills Physics Laboratory, University of Bristol, Bristol, United Kingdom 48 H.H. The LHCb collaboration Wills Physics Laboratory, University of Bristol, Bristol, United Kingdom 49 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 49 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom University of Cambridge, Cambridge, United Kingdo 50 Department of Physics, University of Warwick, Coventry, United Kingdom 50 Department of Physics, University of Warwick, Coventry, United Kingdom 51 STFC Rutherford Appleton Laboratory, Didcot, United Kingdom 52 School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 52 School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom Physics and Astronomy, University of Edinburgh, E School of Physics and Astronomy, University of Edin School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 53 School of Physics and Astronomy, University of Glasgow, Glasgow, United King Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 54 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 55 Imperial College London, London, United Kingdom 56 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 56 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom – 6 – 57 Department of Physics, University of Oxford, Oxford, United Kingdom 58 Massachusetts Institute of Technology, Cambridge, MA, United States 59 University of Cincinnati, Cincinnati, OH, United States 60 University of Maryland, College Park, MD, United States 61 Syracuse University, Syracuse, NY, United States 62 Pontif´ıcia Universidade Cat´olica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil, associated to2 63 University of Chinese Academy of Sciences, Beijing, China, associated to3 64 School of Physics and Technology, Wuhan University, Wuhan, China, associated to3 65 65 Institute of Particle Physics, Central China Normal University, Wuhan, Hubei, China, associated to3 Departamento de Fisica, Universidad Nacional de Co JHEP10(2018)107 JHEP10(2018)107 Departamento de Fisica, Universidad Nacional de Colombia, Bogota, Colombia, associated to 67 Institut f¨ur Physik, Universit¨at Rostock, Rostock, Germany, associated to12 67 Institut f¨ur Physik, Universit¨at Rostock, Rostock, Germany, associated to12 68 Van Swinderen Institute, University of Groningen, Groningen, Netherlands, associated to27 69 National Research Centre Kurchatov Institute, Moscow, Russia, associated to34 70 National University of Science and Technology “MISIS”, Moscow, Russia, associated to34 National Research Tomsk Polytechnic University, Tomsk, Russia, associated to34 71 National Research Tomsk Polytechnic University, Tomsk, Russia, associated to34 72 Instituto de Fisica Corpuscular, Centro Mixto Universidad de Valencia — CSIC, Valencia, Spain associated to40 73 University of Michigan, Ann Arbor, United States, associated to61 74 74 Los Alamos National Laboratory (LANL), Los Alamos, United States, associated to61 74 Los Alamos National Laboratory (LANL), Los Alamos, United States, associated to61 a Universidade Federal do Triˆangulo Mineiro (UFTM), Uberaba-MG, Brazil Universidade Federal do Triˆangulo Mineiro (UFTM b Laboratoire Leprince-Ringuet, Palaiseau, France c P.N. The LHCb collaboration Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russia d U i it` di B i B i It l c P.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS) d Universit`a di Bari, Bari, Italy e Universit`a di Bologna, Bologna, Italy e Universit`a di Bologna, Bologna, Italy f Universit`a di Cagliari, Cagliari, Italy g Universit`a di Ferrara, Ferrara, Italy h Universit`a di Genova, Genova, Italy i Universit`a di Milano Bicocca, Milano, Italy j Universit`a di Roma Tor Vergata, Roma, Italy j Universit`a di Roma Tor Vergata, Roma, Italy k Universit`a di Roma La Sapienza, Roma, Italy l AGH — University of Science and Technology, Faculty of Computer Science, Electronics and l AGH — University of Science and Technology, Faculty of Computer Science, Electronics and l AGH — University of Science and Technology, Telecommunications, Krak´ow, Poland m LIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain m LIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain n Hanoi University of Science, Hanoi, Vietnam n Hanoi University of Science, Hanoi, Vietnam o Universit`a di Padova, Padova, Italy o Universit`a di Padova, Padova, Italy p Universit`a di Pisa, Pisa, Italy p Universit`a di Pisa, Pisa, Italy q Universit`a degli Studi di Milano, Milano, Italy r Universit`a di Urbino, Urbino, Italy r Universit`a di Urbino, Urbino, Italy s Universit`a della Basilicata, Potenza, Italy s Universit`a della Basilicata, Potenza, Italy t Scuola Normale Superiore, Pisa, Italy t Scuola Normale Superiore, Pisa, Italy u Universit`a di Modena e Reggio Emilia, Modena, Italy u Universit`a di Modena e Reggio Emilia, Modena, Italy v MSU — Iligan Institute of Technology (MSU-IIT), Iligan, Philippines v MSU — Iligan Institute of Technology (MSU-IIT), Iligan, Philippines w Novosibirsk State University, Novosibirsk, Russia w Novosibirsk State University, Novosibirsk, Russia x National Research University Higher School of Economics, Moscow, Russia x National Research University Higher School x National Research University Higher School of Economics, Moscow, Ru y Sezione INFN di Trieste, Trieste, Italy y Sezione INFN di Trieste, Trieste, Italy z Escuela Agr´ıcola Panamericana, San Antonio de Oriente, Honduras z Escuela Agr´ıcola Panamericana, San Antonio de Oriente, Honduras aa School of Physics and Information Technology, Shaanxi Normal University (SNNU), Xi’an, China aa School of Physics and Information Technology, Shaanxi Normal Univer ab Physics and Micro Electronic College, Hunan University, Changsha City, China ab Physics and Micro Electronic College, Hunan University, Changsha City, China † – 7 –
https://openalex.org/W4386949943	https://periodicos.ufba.br/index.php/revistagerminal/article/download/54706/29945	Portuguese	null	Via Campesina, práticas educativas e a construção do internacionalismo	Germinal	2,023	cc-by	11,380	Debate DOI: http://doi.org/10.9771/gmed.v15i2.54706 Ellen Cristine dos Santos Ribeiro1 Betânea Moreira de Moraes2 Josefa Jackline Rabelo3 Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 Introdução Diversos autores (Borras, 2004; Vieira, 2011; Desmarais, 2013; Patel, 2004), ao analisarem o cenário que culminaria com o surgimento da Via Campesina Internacional, consideram a mercantilização massiva e internacionalização da agricultura ocorrida na década de 1980 como um fator crucial. O sistema de produção alimentar concentrado nas mãos de multinacionais, acabou por subordinar os agricultores a uma engrenagem que os enfraquecia não apenas em relação à produção, mas também na circulação e na homogeneização do consumo de alimentos. Como consequência houve aumento da concentração de terras, empobrecimento dos agricultores, êxodo rural e agravamento da situação de dependência. O desenvolvimento do capitalismo na América Latina se deu de forma desigual, não-linear. Essa peculiaridade foi responsável pela combinação de formas de acumulação primitivas e desenvolvidas, permitindo que as economias centrais concentrassem riquezas a partir da superexploração das economias latino-americanas e da transferência de capital. A interferência desses interesses externos acabou por moldar os processos de desenvolvimento econômico e integração, deformando-os e limitando-os. Campos (2014) constata que a dependência externa da região é inversamente proporcional às possibilidades de integração que, por sua vez, representa uma importante ferramenta de enfrentamento dessa dependência. Aos poucos, com agudização ao final do século XX, essa lógica passou a englobar novos processos, potencializando a privatização de recursos até então considerados públicos, como a terra, a água e até o patrimônio genético. Em reposta a essa especulação massiva da atividade agrícola e ao avanço das táticas de desarticulação do campesinato estouraram movimentos e organizações populares em vários países. Para Moyo e Yeros (2005), no último quarto de século, ocorreram intensas mudanças socioeconômicas e políticas nos países da periferia. Sob a influência dos programas de ajuste estrutural, camponeses e trabalhadores tiveram suas condições de reprodução social deterioradas, atropeladas pela busca desesperada de alternativas econômicas e políticas que se mostraram, muitas vezes, precipitadas ou desalinhadas aos interesses da classe trabalhadora. Nessas circunstâncias de aumento das possibilidades de articulação e intercâmbio das organizações camponesas em nível internacional, surge a Via Campesina. Desponta, assim, a partir das relações dialéticas produzidas pelo próprio capitalismo, expressas através de conflitos intrínsecos a este modelo. O cenário político-social em questão destruía os saberes e cultura locais, além de arruinar o controle dos pequenos agricultores e camponeses sobre suas terras. Debate Josefa Jackline Rabelo3 Resumo: O artigo, de natureza teórico-bibliográfica, analisa a contribuição educacional engendrada pela Via Campesina na construção do internacionalismo. Para tanto, apresentou-se o processo histórico que culminou na organização da maior organização camponesa em escala mundial, bem como seus princípios de luta e formas de atuação, a fim de examinar os pressupostos educacionais que regem suas práticas educativas. Foi possível constatar que, ao educar os sujeitos campesinos em contraposição ao modelo hegemônico de educação, por meio de práticas solidárias e cooperativas, o movimento soma forças e avança em direção da construção de um internacionalismo potencialmente revolucionário, especialmente nas lutas sociais do campo na América Latina, a exemplo do Movimento dos Trabalhadores Rurais Sem Terra (MST). Palavras-chave: Via Campesina. Práticas Educativas. Internacionalismo. Resumen: El artículo, de carácter teórico-bibliográfico, analiza el aporte educativo que engendra la Vía Campesina en la construcción del internacionalismo. Para ello, se presentó el proceso histórico que culminó con la organización de la mayor organización campesina a escala mundial, así como sus principios de lucha y formas de acción, a fin de examinar los supuestos educativos que rigen sus prácticas educativas. Se pudo constatar que, al educar sujetos campesinos en oposición al modelo hegemónico de educación, a través de prácticas solidarias y cooperativas, el movimiento suma fuerzas y avanza hacia la construcción de un internacionalismo potencialmente revolucionario, especialmente en las luchas sociales del campo en América Latina, como el Movimiento de los Trabajadores Rurales Sin Tierra (MST). Palabras clave: La Vía Campesina. Prácticas Educativas. Internacionalismo. Abstract: The article, of a theoretical-bibliographical nature, analyze the educational contribution engendered by Via Campesina in the construction of internationalism. To this end, the historical process that culminated in the organization of the largest peasant organization on a world scale was presented, as well as its principles of struggle and forms of action, in order to examine the educational assumptions that govern its educational practices. It was possible to verify that, by educating peasant subjects in opposition to the hegemonic model of education, through solidary and cooperative practices, the movement joins forces and advances towards the construction of a potentially revolutionary internationalism, especially in the social struggles of the countryside in America Latin America, such as the Landless Workers Movement (MST). Keywords: Via Campesina. Educational Practices. Internationalism. Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 320 Debate Debate nacional e internacional” (FERNANDES, 2010, p. 187), trouxe um novo componente à ampliação da luta camponesa internacional. As práticas educativas engendradas pelos movimentos sociais populares do campo contemplam tanto a educação em sentido lato quanto em sentido stricto. Junto às políticas de formação da Via Campesina, se somam inúmeras ações pedagógicas – em termos gramscianos – executadas pelos movimentos camponeses que se conectam através dela, e que apontam para a formação de quadros dirigentes e militantes dentro desses movimentos, intelectuais forjados no seio da própria classe, atentos às particularidades e necessidades de seu país e da América Latina, representando, em suma, mais um passo em direção à construção do internacionalismo. O Brasil, seguindo a mesma organização dos outros países vinculados à VC, promove, principalmente por meio do MST, cursos livres, seminários e cursos de escolarização formal. Também são coordenadas inúmeras atividades de cunho formativo, que se articulam através do internacionalismo dessas organizações, ou seja, experiências político-pedagógicas construídas pela articulação internacional de movimentos sociais do campo. Em escala mundial, a Via Campesina opera a partir do que é consenso entre as grandes regiões, buscando alinhar lutas e estratégias comuns diante do vasto número de organizações e movimentos sociais do campo. O tema do internacionalismo, ainda pouco explorado4, sobretudo nas pesquisas em educação, é considerado um elemento-chave para compreender os processos sociais latino-americanos contemporâneos (BRINGEL; FALERO, 2008). Optamos, assim, por enfrentar a temática, localizando o protagonismo internacional das práticas educativas da Via Campesina, considerando a centralidade da educação popular e potencialmente revolucionária que promove e da possibilidade de articular essa educação como manifestação política, carregada de experiências solidárias e estratégias de organização, buscando novos percursos para as práticas sociais. Introdução A VC pode ser definida como uma organização mundial que articula movimentos e povos do campo, empunhando, principalmente, as bandeiras da agricultura familiar e da agroecologia como principais matrizes de referência para a conquista da soberania alimentar e da produção de alimentos saudáveis (VIA CAMPESINA, 2005). Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 321 Entre os objetivos que alicerçam a atuação da Via Campesina, destacam-se: construir relações de solidariedade, mediante a compreensão da diversidade do campesinato no mundo; vivificar a preservação do meio ambiente, protegendo a biodiversidade do planeta; e conceber um modelo de agricultura que desenvolva e garanta a soberania alimentar, entendida como um direito dos povos, assim como a autonomia para definir políticas agrícolas alinhadas às distintas realidades camponesas. A filiação do MST à Via Campesina, “principal interlocutora dos movimentos camponeses nas negociações de políticas em escala Os Estados Unidos gestavam uma nova condição de inserção internacional dos Estados na era contemporânea das relações internacionais. A superioridade econômica, associada à capacidade e à vontade para sobrepujar as potências europeias tradicionais, elevava os Estados Unidos ao cerne das decisões internacionais (SARAIVA, 2007, p. 181). Debate Debate O contato com as fontes primárias do Banco Mundial permitiu a Gonçalves (2014) comprovar que mesmo as organizações multilaterais reconhecem as consequências veladas que a superexploração do capitalismo dependente causa ao meio rural, bem como a profundidade desses impactos. Os programas agrários do Banco Mundial são instrumentos poderosos da hegemonia financeira e política norte-americana, que sobrevalorizam o direito à propriedade privada como fundamento de construção de regimes e projetos de desenvolvimento social para países periféricos. A abordagem mercadológica conferida pelo Banco Mundial às políticas agrárias e direitos fundiários coletivos agudizam as contradições sociais na medida em que assentem um padrão hegemônico de produção agropecuária, materializado através do modelo do agronegócio, que homogeneíza os territórios e se apropria deles sem qualquer constrangimento legal ou política efetiva de reforma agrária ou demarcação de terras. Para Cândido (1979), a expansão do mercado capitalista não apenas obriga o camponês a intensificar o esforço físico, como também atrofia as possibilidades coletivas de organização do trabalho e ajuda mútua. Individualizado, entregue a si mesmo, o trabalhador é projetado de um meio comunitário para uma esfera influenciada pela economia regional. Ao galgar condições de sobrevivência, precisa renunciar aos antigos padrões de trabalho integral. Gonçalves (2014, p. 41), complementa: A expansão do agronegócio nas áreas rurais brasileiras, cuja produção mecanizada em larga escala possui baixos índices de geração de emprego em comparação com a agricultura familiar, enseja o desalojamento das populações rurais (e de comunidades indígenas, quilombolas e tradicionais) orientadas à migração para as periferias dos centros urbanos em busca de trabalho, promovendo o desmantelando das culturas camponesa, caipira, indígena, quilombola, tradicionais, populares, entre outras. A expansão do agronegócio nas áreas rurais brasileiras, cuja produção mecanizada em larga escala possui baixos índices de geração de emprego em comparação com a agricultura familiar, enseja o desalojamento das populações rurais (e de comunidades indígenas, quilombolas e tradicionais) orientadas à migração para as periferias dos centros urbanos em busca de trabalho, promovendo o desmantelando das culturas camponesa, caipira, indígena, quilombola, tradicionais, populares, entre outras. Contexto de surgimento e critérios de organização da Via Campesina Alguns historiadores internacionalistas, a exemplo de Saraiva (2007), indicam que o mundo liberal se ergueu a partir de uma nova ordem internacional, gestada nos moldes de um liberalismo central, vinculada às metas estratégicas das políticas externas dos países mais desenvolvidos. Esse bloco hegemônico, por sua vez, tem se orientado pelo Estado-nação que consolidou maior influência e poder no século XX: os Estados Unidos. Se, às vésperas da mundialização da Segunda Guerra, a cooperação econômica entre EUA e Grã- Bretanha pareceu fundamental para o futuro econômico pós-guerra, as bases do Plano Marshall, gestadas antes mesmo do ingresso norte-americano no conflito, juntamente com ‘diplomacia do dólar’ e a ampliação do domínio sobre a América Latina. Assim, os anos 1940 viram emergir um novo conceito: o de superpotência. Os Estados Unidos gestavam uma nova condição de inserção internacional dos Estados na era contemporânea das relações internacionais. A superioridade econômica, associada à capacidade e à vontade para sobrepujar as potências europeias tradicionais, elevava os Estados Unidos ao cerne das decisões internacionais (SARAIVA, 2007, p. 181). Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 322 Debate contava com 61 movimentos camponeses agregados e 125 pedidos de filiação (FERNANDES, 2008). Com crescimento exponencial consolidado, em 2014 já articulava mais de 100 movimentos. No hoje, é considerada um dos principais agentes na discussão de questões agrárias, agrícolas e alimentares, sendo ouvida por seguimentos da ONU como a FAO e o Conselho de Direitos Humanos, além da grande representatividade entre movimentos sociais de todo o globo. Segundo dados atualizados em 20187, a Via campesina reúne por volta de 164 organizações em 73 países. Estima-se que cerca de 200 milhões de pessoas estão envolvidas em suas causas. [...] Via Campesina se revelou como um ator principal nas lutas populares internacionais contra o neoliberalismo que, entre outras coisas, exigem responsabilidades das agências intergovernamentais, enfrentam e se opõem ao controle corporativo sobre os recursos naturais e a tecnologia, e defendem a soberania alimentar. Além disso, desempenhou um papel destacado em campanhas de grande polêmica como, por exemplo, as dirigidas contra a OMC, contra os gigantes corporativos mundiais como McDonalds, e contra os organismos geneticamente modificados (OGM) e as multinacionais que os fomentam, como a Monsanto (BORRAS, 2004, p.3)8. Sob forte influência das lutas revolucionárias da América Latina, a La Via Campesina9 traz, já no nome, o cerne de uma proposta contrária aos modelos vigentes, conforme enfatiza Francisca Rodriguez, líder de uma das organizações integrantes, a Asociación Nacional de Mujeres Rurales e Indígenas (Anamuri), no Chile: E é por isso que se chama Via Campesina, não é a confederação, não é a união internacional, é esse processo em que nós estamos levando a cabo a construção de uma via alternativa, a partir dos camponeses, frente às políticas neoliberais (Entrevista com Francisca Rodriguez In VIEIRA, 2011, p. 181). Na América Latina, segundo aponta Batista (2013), houve um significativo investimento na organização dos camponeses como classe social nos anos 1940, deliberação dos Partidos Comunistas latinos. No caso brasileiro, especialmente após 1945, foram articuladas diversas associações, como as Ligas Camponesas10, que contaram com importantes membros do Partido Comunista Brasileiro (PCB) em sua composição. O êxito da Revolução Cubana, em 1959, assim como outras revoluções proletárias do século XX, contou com expressivo apoio do campesinato. Embora tenha contornos próprios, a reforma agrária impulsionada pela revolução contou com a solidariedade internacional de diversas organizações de esquerda, em escala mundial. Rosset (2006) assinala que os governos nacionais conseguiram acomodar características do capitalismo neoliberal na agricultura através de políticas de ajuste estrutural influenciadas por agências multilaterais, como o Banco Mundial, a OMC5 e o FMI6: Essas políticas incluíram a liberalização do comércio e a subsequente inundação de mercados locais com importação de alimentos baratos subfaturados, com os quais os agricultores locais dificilmente conseguem competir; o corte da sustentação de preços e dos subsídios para produtores de alimentos; a privatização do crédito, da comercialização e da assistência técnica; a promoção excessiva da exportação; patenteamento de recursos genéticos de cultivares; e um favorecimento da pesquisa agrícola em prol de tecnologias caras como a engenharia genética (ROSSET, 2006, P. 316-317). A formação da Via Campesina Internacional surgiu a partir da reunião de diversos líderes de movimentos camponeses em ocasião do II Congresso da UNAG – Unión Nacional de Agricultores y Granaderos de Nicarágua, em 1992, na cidade de Manágua. Tanto as pesquisas de Fernandes (2012) quanto as de Vieira (2011) apontam a disposição comum de criar um projeto coletivo de alternativas ao neoliberalismo. A partir deste evento, foi produzida a Declaração de Manágua, documento que sublinha a urgência de unificação entre os movimentos americanos e europeus, a fim de que suas propostas reverberassem na consolidação e reafirmação dos espaços de resistência. Segundo Desmarais (2013), no entanto, o movimento só foi oficializado um ano depois, quando 46 líderes de diversos países se reuniram em Mons, na Bélgica. A Via Campesina Internacional emerge, em 1992, a partir de uma miríade de coletivos camponeses organizados na Europa, Ásia, África e América, o que lhe confere engajamento mundial. Em 2008, já Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 323 Debate Debate do Brasil (CNBB) saiu em defesa dos direitos humanos, das CEBs e das pastorais (da Terra, da Juventude, Operária, Indigenista). Durante toda a década e início dos anos 1980, as CEBs deram cobertura à atuação política de muitos setores sociais, ainda que conservassem um caráter predominantemente religioso. As CEBs chegavam aonde o padre não alcançava. Era comum, tanto nas periferias quanto em comunidades rurais, a existência de líderes religiosos que exerciam também grande influência política. A atuação de membros das CEBs impulsionou a articulação de cooperativas e associações de moradores e, aos poucos, também foi alcançando sindicatos, movimentos sociais, partidos políticos – a exemplo do Partido dos Trabalhadores (PT) –, e coletivos como a Central Única das Favelas (CUT), a Central de Movimentos Populares (CMP) e o próprio Movimento dos Trabalhadores Rurais Sem Terra (MST)12. Sobre a conjuntura que precedeu o surgimento da Via Campesina, Batista (2013) destaca o triunfo da Revolução Nicaraguense, em 1979. A Frente Sandinista de Libertação Nacional (FSLN) derrubou o governo ditatorial de Anastasio Somoza, estabeleceu um governo revolucionário e realizou a reforma agrária. A efetivação do processo revolucionário abrangeu outras questões sociais como, por exemplo, as brigadas de alfabetização que irromperam pelo país, através da Cruzada Nacional de Alfabetização. Além da atuação dos combatentes sandinistas, brigadas solidárias internacionais encorparam o movimento, enviando membros de organizações camponesas latino-americanas para auxiliarem na produção de alimentos. Percebemos assim, que a articulação internacional de camponeses não é recente e faz parte da história das lutas dos trabalhadores. No caso específico da conformação da Via Campesina, destacamos a importante influência do processo revolucionário cubano, da ação das CEBs e da teologia da libertação, assim como do processo revolucionário nicaraguense. Nestes, a solidariedade e a articulação internacional foram bases para a conformação da articulação internacional de organizações do campo, a Via Campesina. Pois diversos militantes e dirigentes que participaram de processos acima citados, são hoje militantes e dirigentes de organizações sociais partícipes da VCI (BATISTA, 2013, p. 78). O contexto de surgimento da Via Campesina é determinado também, além dos fatores já expostos, pelo incentivo à privatização massiva das terras por meio da titularidade individual. Vieira (2011) aponta que o fenômeno ocorreu principalmente na África e nas terras indígenas da América Latina e Ásia, historicamente comunais. Como retribuição a esta solidariedade, mas também como princípio revolucionário Cuba possibilitou a formação de profissionais em medicina para militantes dos diferentes países, através da ELAM (Escuela Latino-Americana de Medicina). Também possibilitou a formação de muitos militantes de movimentos sociais emergentes na América Latina desde a escola de mulheres, e a escola de formação de pequenos agricultores da ANAP11 (BATISTA, 2013, p. 77). Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 324 Os impactos da Teologia da Libertação na América Latina também precisam ser mencionados. A corrente teológica emergiu na década de 1970, proclamando sua opção pelos mais pobres. Sob essa orientação, o movimento passou a concentrar energias na assistência às comunidades vulneráveis e na luta anticolonialista, o que o aproximou dos coletivos de base e de luta pela terra. As Comunidades Eclesiais de Base (CEBs), surgiram nos anos 1970, período marcado pelo autoritarismo da ditadura militar e pela consequente obstrução dos canais de participação política. Como reação, a Conferência Nacional dos Bispos Debate monopólio na medida em que facilita a determinação dos preços de toda a cadeia produtiva mundial pelas das multinacionais. Pode-se definir a Via Campesina como um movimento camponês internacional central no fortalecimento do conceito de classe e de ações práticas de cooperação e solidariedade internacional que reverberam, consequentemente, em ações de integração latino-americana (MARTINS, 2014). Outro pilar impulsionado pelo movimento, conforme já mencionado, é a agroecologia13, objeto medular das experiências desencadeadas pela articulação da Via. Enfoca-se, pois, que a baliza empírica da atuação da Via Campesina desemboca em categorias que questionam o caráter hegemônico dos modelos e concepções de campo. A Via Campesina é, portanto, uma articulação internacional de trabalhadores que constrói sua identidade em contraposição ao modelo dominante de agricultura, afirmando a economia, a cultura, os valores, os modos de vida do campesinato. Ao mesmo tempo, busca estabelecer relações com outros movimentos e organizações, pois entende o modelo agrícola como parte do formato mais geral do capitalismo mundial na contemporaneidade (VIEIRA, 2011, p. 251). A Via Campesina é, portanto, uma articulação internacional de trabalhadores que constrói sua identidade em contraposição ao modelo dominante de agricultura, afirmando a economia, a cultura, os valores, os modos de vida do campesinato. Ao mesmo tempo, busca estabelecer relações com outros movimentos e organizações, pois entende o modelo agrícola como parte do formato mais geral do capitalismo mundial na contemporaneidade (VIEIRA, 2011, p. 251). A rede de movimentos sociais que integram a Via Campesina aponta para o acirramento de formas violentas de apropriação do meio rural pelo capital transnacional, fato que impulsionou o surgimento de organizações camponesas vinculadas internacionalmente. Contudo, a Via Camponesa não é apenas uma articulação de enfrentamento do modelo neoliberal. Empenha-se, também, em recuperar a agenda camponesa de esquerda. Na América Latina, seus dirigentes identificam uma continuidade das lutas empreendidas nos anos 1960, 1970 e 1980, que culminaram em sua criação. Relacionam, por exemplo, pautas como o combate às ditaduras, o papel da igreja progressista, os embates contra a concretização do capitalismo na agricultura e a experiência adquirida pelos militantes nesses processos de confronto (VIEIRA, 2011). Claramente, a Via Campesina está preenchendo uma lacuna importante. Também esquadrinha a incorporação de pequenos agricultores à agroindústria por meio de programas privados de financiamento e a pressão sobre os Estados para executarem a chamada reforma agrária de mercado, defendida pelas agências multilaterais como parâmetro de aquisição de terras para a reforma agrária segundo valores de mercado. Sobre tais políticas, Borras (2004) afirma que procuram homogeneizar os direitos de propriedade por todo o mundo com o intuito de estimular a acumulação de capital privado na economia rural. O fortalecimento da industrialização na agricultura foi outro aspecto relevante para o surgimento da Via Campesina Internacional. Para Vieira (2011), a questão se conecta com a produção em larga escala e com a padronização em escala mundial dos produtos alimentícios concentrados, conforme já dito, nas mãos do grande empresariado. Essa concentração se deu tanto na esfera dos insumos (fertilizantes, agrotóxicos, sementes) quanto no campo dos produtos da agricultura (comércio mundial de frutas, grãos, matérias primas para a produção industrial). Há, ainda, o apontamento de que essa concentração exerce uma função de Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 325 Debate Debate Camponesas) e MPA (Movimento dos Pequenos Agricultores), além de consultar outras entidades sul- americanas. Desmarais (2013) destaca que, apesar de sua estrutura global, a VC dá muita importância aos movimentos de base, compreendendo que a articulação internacional só é possível a partir da consolidação dos coletivos locais e regionais. Na estrutura organizativa da Via Campesina, as Conferências, com periodicidade de quatro anos, representam o órgão máximo de decisão política. A sede do evento é itinerante, variando a cada encontro, visando contemplar todas as regiões do mundo, ratificando seu caráter internacionalista. Desmarais (2013) protocola que nas Conferências também é eleito o Comitê Coordenador Internacional (CCI), que também se comporta como um organismo-chave nas decisões políticas. Em cada uma das oito regionais da VC há um coordenador e uma coordenadora, membros de coletivos diferentes, responsáveis pela articulação, organização, comunicação e decisão política. Nesse sentido, desde os primeiros momentos afirmou-se que era preciso construir uma articulação que estivesse enraizada nos movimentos de base e evitasse a burocratização de lideranças ou mesmo a consolidação de grandes estruturas materiais. No entendimento da Via Campesina, as experiências concretas dos movimentos é que devem servir de base para a construção da articulação internacional (VIEIRA, 2013, p. 202). A II Conferência da Via Campesina ocorreu em 1996, na cidade de Tlaxcala (México). Contou com a presença de 69 organizações, representando 37 países. Também houve participação de delegados oriundos de coletivos ainda em processo de vinculação à Via Campesina, principalmente dos continentes africano e asiático. Edelman (2003) registra a impossibilidade de dezenas de militantes em comparecer ao evento por terem seus vistos negados pelo governo mexicano. Outra dificuldade apontada pelo autor foi a forma apressada com que o evento foi preparado. Estava previsto para ser sediado pelas Filipinas, meses depois, mas conflitos locais impediram a realização naquele país. Vieira (2011) considera que, apesar das dificuldades para que a II Conferência fosse realizada, seus desdobramentos foram concretos e expressivos. Registra que, pela primeira vez, se discutiu os pilares do conceito de soberania alimentar, além de discutir de forma mais madura um modelo camponês de agricultura. O Encontro também debateu o Massacre de Eldorado do Carajás14, decorrido em 17 de abril daquele ano, no estado do Pará, responsável por chacinar 19 militantes do MST. O massacre repercutiu em todo o mundo, chamando atenção, inclusive, pela crueldade das execuções. A sua própria existência é uma evidência de novas estruturas de ação coletiva no campo; suas estratégias desafiam padrões tradicionais de organização no setor agrícola e a ampla magnitude de sua presença internacional – sua natureza dinâmica, diversidade cultural e ampla distribuição geográfica – expressa o seu potencial transformador (DESMARAIS, 2013, p. 7). Ao detalhar os objetivos da Via Campesina, Vieira (2011) registra que visa influenciar os núcleos de poder e tomada de decisões dos governos e órgãos multilaterais a fim de orientar políticas econômicas e agrárias voltadas à produção de pequeno e médio porte. Elabora, assim, propostas desafiadoras englobando os seguintes temas: reforma agrária, soberania alimentar, agricultura camponesa sustentável, biodiversidade, direitos humanos, recursos genéticos, migração de trabalhadores rurais e questões de gênero. Cada tópico compõe uma Comissão Temática da qual as organizações-membro devem participar de ao menos uma. Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 326 O principal critério de ingresso das organizações na Via Campesina é o engajamento nas lutas sociais concretas que envolvem a questão agrária e no enfrentamento do modelo vigente. Também há a exigência de aprovação por parte das entidades locais e regionais que compõem a articulação. No caso brasileiro, por exemplo, a admissão de uma nova organização passa pelo crivo do MST (Movimento dos Trabalhadores Rurais Sem Terra), MAB (Movimento dos Atingidos por Barragens), MMC (Movimento de Mulheres 326 [...] Portanto, o que se propõe como metodologia é que todos os militantes das organizações camponesas, mesmo que não participem diretamente da conferência, possam debater os temas e acompanhar o seu processo de construção e, ao mesmo tempo, que se estimule a realização de encontros regionais e nacionais (VIA CAMPESINA, 2003, p. 5). O processo de preparação dessa IV Conferência foi iniciado em maio de 2003 [...] no qual se discutiu que tão importante quanto garantir a presença das organizações membros [...] seria realizarmos um grande mutirão para que a notícia da conferência, seus temas, sua metodologia fossem debatidos nas bases, em cada país e região. [...] Portanto, o que se propõe como metodologia é que todos os militantes das organizações camponesas, mesmo que não participem diretamente da conferência, possam debater os temas e acompanhar o seu processo de construção e, ao mesmo tempo, que se estimule a realização de encontros regionais e nacionais (VIA CAMPESINA, 2003, p. 5). Diante do compromisso com a igualdade de gênero, às vésperas da Conferência, realizaram-se a I Assembleia Mundial de Jovens e a II Assembleia Mundial de Mulheres Camponesas da Via Campesina. Vieira (2011) relata a incorporação de 40 novas organizações camponesas à VC, além da inclusão da África como uma oitava região articulada, organizadas em: América do Norte, América do sul, América Central, Caribe, Europa, Sudeste da Ásia, Sul da Ásia e África. Após a inclusão do território africano, o CCI passou a ter 16 membros. O comitê organizador conseguiu inserir no evento linguagens e processos diversos de debate. Vieira (2001) destaca, por exemplo, a utilização de místicas, manifestações culturais espontâneas, palavras de ordem em diferentes línguas, festas, músicas e cerimônias diversas que garantiram o tom plural do evento. Por meio de um compromisso de reativar e fortalecer as suas comissões temáticas internacionais, a conferência da Via Campesina consolidou posições e futuros planos de ação sobre sete questões fundamentais: soberania alimentar e liberalização do comércio; biodiversidade e recursos genéticos; reforma agrária, gênero, agricultura camponesa sustentável, direitos humanos, e migração e trabalhadores agrícolas (DESMARAIS, 2013, p. 283). A V Conferência se deu em Matola, município moçambicano, em 2008, reflexo da atuação crescente da Via campesina no continente africano. Após o encontro, a regionalização da África precisou ser dividida em duas: África 1 e África 2. A posição da Via Campesina em relação ao evento foi de imediato repúdio e denúncia dos brutais assassinatos. Estabeleceu o 17 de abril como o Dia Internacional da Luta Campesina, data destinada a rememorar as vítimas da luta pela terra e chamar atenção para o debate da soberania alimentar. A III Conferência foi realizada em 2000, em Bangalore, Índia. Participaram mais de 100 delegados discutindo a construção da identidade da Via Campesina, já que o debate em torno da soberania alimentar já estava avançado. As discussões em torno das questões de gênero também tiveram lugar de destaque, reflexo do engajamento das mulheres dentro da organização, iniciado em 1996. O conhecido lema “globalizemos a luta, globalizemos a esperança” foi firmado durante o evento e se mantém vivo até os dias atuais. Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 327 327 Debate Entre 2000 e 2004, as lutas em escala internacional tomaram novas dimensões. A quantidade de protestos contra as organizações multilaterais, a concepção e realização de várias edições do Fórum Social Mundial, tudo isso colocou a Via Campesina em destaque. Em maio de 2003, dez anos após a Conferência de fundação, foi realizado um encontro para pensar as estratégias da Via Campesina. Membros do CCI discutiram em Natoye (Bélgica) tanto as questões internas como as externas, e produziram um documento que serviria de base para os debates preparatórios para a IV Conferência (VIEIRA, 2011, p. 196). O documento suprarreferido apresentou uma análise da situação agrária internacional, além de expor textos que discutem a política de alianças, comunicação, necessidade de formação política, gênero e juventude. Vieira (2011) acrescenta que trouxe, ao final, um roteiro de perguntas acerca do funcionamento interno e sobre a plataforma política da Via Campesina. Com o lema “organizar a luta: terra, alimento, dignidade e vida” a IV Conferência, ocorreu em 200415, na cidade paulista Itaici, no Brasil, com a presença de 400 delegados, representantes de 76 países. O evento ficou sob a responsabilidade do MST e de outros movimentos brasileiros integrantes da Via Campesina. O processo de preparação dessa IV Conferência foi iniciado em maio de 2003 [...] no qual se discutiu que tão importante quanto garantir a presença das organizações membros [...] seria realizarmos um grande mutirão para que a notícia da conferência, seus temas, sua metodologia fossem debatidos nas bases, em cada país e região. Debate Debate O documento final resultante da V Conferência reconheceu o papel central das mulheres na agricultura e repudiou todas as formas de violência de gênero. As discussões sobre soberania alimentar e agroecologia foram intensificadas, o que suscitou a necessidade de formação específica para empreender a transição do modelo de agricultura convencional para o modelo agroecológico. É nesse contexto que surgem experiências formativas de cunho político e agroecológico no seio da Via Campesina. Assim, a trajetória da VCI tem sido marcada por conquistas e desafios. Destes podemos mencionar como: solidariedade internacional; a articulação da luta internacional dos sujeitos do campo; mobilizações constantes contra ação dos organismos do capital no campo; construção de estratégias fundadas numa nova forma de agricultura baseada na transformação da sociedade; construção de uma forma organizativa horizontal e integradora; e a preocupação constante na formação de sua militância e quadros (BATISTA, 2013, p. 87). Desmarais (2013) aponta que a identidade da VC é coletiva. As estratégias e posições que sustenta foram forjadas a partir da oposição à OMC e do diálogo com outras organizações da sociedade civil, como, por exemplo, a International Federation of Agricultural Producers (IFAP) e ONGs empenhadas no desenvolvimento internacional. Pollack (2001) faz uma distinção entre os movimentos “de dentro” e os “de fora”, ou seja, entre os movimentos que estão diretamente ligados ou dependentes de estruturas maiores e aqueles cuja existência se vê ameaçada pela globalização. Essa diferenciação nos ajuda a compreender melhor os limites e possibilidades das mudanças sociais. A IFAP está bem preparada e situada para dialogar com instituições econômicas multilaterais; essa é uma das estratégias principais no esforço para reformar acordos de comércio e abordagens de desenvolvimento para servir melhor aos interesses dos agricultores. Isso nos ajuda a entender por que a oposição da Via Campesina à OMC é tão implacável: a IFAP e Via Campesina não falam a mesma língua – elas professam visões de futuro diametralmente opostas. As organizações de camponeses e agricultores que formaram a Via Campesina estão convencidas de que estratégias e posições mais radicais são necessárias com urgência para tratar da crise no campo. Ao formarem a Via Campesina, elas criaram efetivamente uma alternativa progressista em relação à IFAP (DESMARAIS, 2013, p. 30, grifo nosso). Assim sendo, destaca-se o objetivo primordial da Via Campesina: construir um modelo de agricultura inédito até então, radicalmente diferente, assentado no conceito de soberania alimentar. Rosset e Torres (2010) consideram que o evento foi o marco de uma nova fase (2008-2010) para a Via Campesina. Com uma crise mundial de alimentos como pano de fundo, foi a primeira vez em que o capitalismo foi apontado como fonte dos problemas que assolam o campo, e as empresas transnacionais como as maiores antagonistas dos camponeses de todo o mundo. erminal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 328 Debate da Via Campesina somado às mudanças agrícolas ocorridas no final do século XX, levaram a IFAP a se voltar também aos pequenos produtores. Desmarais (2013), Vieira (2011) e Edelman (2003) evidenciam o fato de que a IFAP faz o intermédio de recursos entre os agricultores e as multinacionais, o que capitaliza um enorme poder para si diante dos camponeses. Este e outros aspectos afastam, de forma irreconciliável, Via Camponesa e IFAP, tornando-as opostas frente aos interesses que representam. Apesar de sua existência em países da África e da Ásia, a IFAP ainda tem sua base muito concentrada na Europa e nos Estados Unidos, em especial nas organizações de médios e grandes produtores. Analistas e dirigentes da Via Campesina afirmam que nos últimos anos tem havido uma deliberada estratégia de avançar sobre as bases da Via Campesina. A maior dificuldade causada para a Via Campesina pela existência da IFAP é, contudo, o espaço de representação em organismos multilaterais. Nos encontros da FAO, por exemplo, a IFAP tem lugar assegurado e compete com a Via Campesina para falar em nome dos camponeses do mundo (VIEIRA, 2011, p. 221). Apesar de alguns teóricos ainda abordarem a capacidade de organização dos camponeses com olhos de dúvida, enfocando sempre sua diversidade, Desmarais (2013) aponta que a Via Campesina conseguiu converter esse caráter heterogêneo em seu maior trunfo. Tal diversidade confere ao movimento transnacional uma maior abrangência, oportunizando ações tanto em meios rurais remotos quanto no extremo de meios urbanos. O próprio MST, por exemplo, além de organizar os trabalhadores sem-terra no campo, também coordena assentamentos na periferia, alocando famílias urbanas em pequenas porções de terra. Outro exemplo apontado pela autora é o caso da Unión Nacional de Organizaciones Regionales Campesinas Autónomas (UNORCA), em Puebla, no México. O coletivo dá suporte a vendedores ambulantes urbanos. A Union Paysanne agrega, no Quebec, agricultores, pesquisadores, estudantes, consumidores e empresários do ecoturismo, todos comprometidos com o desenvolvimento de alternativas à alimentação artificial e à agricultura industrial. Ao formar a Via Campesina, as organizações camponesas e agrícolas efetivamente transnacionalizaram e conquistaram um espaço na arena internacional. A Via Campesina está preenchendo aquele espaço com vozes camponesas, articulando as demandas camponesas e as alternativas em esforços para resistir à imposição de um modelo corporativo de agricultura. A solidariedade e a unidade experimentadas com a Via Campesina geram talvez a mais preciosa de todas as conquistas, a esperança. De acordo com Desmarais (2013), para o movimento camponês, essa tarefa pode se concretizar por meio da solidariedade e unidade entre as diversas e complexas organizações camponesas do mundo. A tática já garantiu à Via Campesina a consolidação da identidade de “povo da terra”, oposição ferrenha às instituições multilaterais, definição de políticas alternativas para problemas relativos às comunidades rurais e engajamento nas ações coletivas de construção da soberania alimentar. Assim, torna-se importante diferenciar – e mesmo afastar – a Via Campesina da IFAP16. Autores como Borras (2004) e Vieira (2011), a quem muito recorremos a fim de melhor compreender os processos de gênese e consolidação da VC, apontam que a IFAP, ao ser criada em 1946, agregou médios e grandes agricultores, em sua maioria, oriundos de países desenvolvidos. Alegam, ademais, que a hegemonia dos grandes produtores no interior da organização acabou por consolidá-la como representante de seus interesses e de suas negociações com o agronegócio e com entidades intergovernamentais. O surgimento Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 329 Debate Debate A diversidade da Via Campesina também se reflete na maneira como as organizações de agricultores e de camponeses estão estruturadas. Há coletivos organizados apenas em nível estadual, como no caso da Associação de Agricultores do Estado de Kamataka (KRRS). O National Farmers Union of Canada (NFU) se organiza por todo o território canadense; a já mencionada UNORCA se constitui como uma federação nacional, atuando em 23 estados mexicanos. Temos, ainda, a Coordenation Paysanne Européenne (CPE) e a Asociación de Organizaciones Campesinas Centroamericanas para la Cooperación y el Desarrollo (ASOCODE), entidades regionais que agregam organizações nacionais. Desmarais (2013) destaca que uma das principais capacidades da VC consiste em fazer confluir as organizações que a integram. Apesar das distintas particularidades e dos contextos sociais, econômicos, políticos e culturais diversos, consegue estabelecer uma unidade. Nascida no cume das consequências neoliberais para a agricultura, a Via Campesina se estabeleceu, ao longo dos anos, como o principal meio de articulação dos sujeitos do campo, enfrentando não apenas o momento histórico de seu surgimento, de grande ofensiva neoliberal, mas também o desenvolvimento do capital no campo, que se desdobra principalmente através da ação de multinacionais na agricultura, recursos hídricos e mineração, que geram impacto e desterritorializam os povos do campo. A luta contra o poder hegemônico do capital no campo é, pois, reafirmada constantemente através dos diversos movimentos camponeses que a integram, a exemplo do MST. A esperança de que “outra” agricultura é possível. De fato, a Via Campesina permite-nos imaginar que a mudança é possível e que um projeto alternativo está sendo criado. Isso foi claramente capturado no lema da Via Campesina: “globalizar a luta, globalizar a esperança” (DESMARAIS, 2013, p. 303). O processo de formação da Via Campesina ocorreu em paralelo à crescente mercantilização e internacionalização da agricultura. Concretizou-se, em 1993, a partir das fronteiras abertas da América Latina, em resistência à ofensiva neoliberal, fortalecendo movimentos de lutas camponesas. Organiza-os em espaços independentes, dando-lhes visibilidade local, regional e internacional, conduzindo-os entre os diferentes setores da sociedade civil. Em sua dinâmica, permite ações de protesto descentralizadas ou coordenadas de maneira simultânea, em diferentes partes do mundo, o que comprova seu poder encorajador e mobilizador (DESMARAIS, 2007). Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 330 Debate Debate A partir de 1920, ocorrem profundas alterações na estrutura econômica do país, impossíveis de serem anotadas, dada sua complexidade e impactos no processo de industrialização. A fim de não fugir ao escopo da temática, importa saber que, a partir da ascensão do neoliberalismo, as medidas econômicas implementadas pelos governos da década de 199017 representaram a aliança firmada entre as classes dominantes brasileiras e o capital internacional: de absoluta subordinação à financeirização do capital. Esse marco representou um modelo de agricultura centralizado nas fazendas com grandes extensões de terra; no uso de agroquímicos e agrotóxicos; na monocultura de produtos para exportação (MAZZEO, 1995). Tal modelo se generalizou em proporções avassaladoras. A proposta neoliberal continuou avançando nos governos dos anos 200018, tomando conta do setor agrícola e selando uma aliança de subordinação em relação às empresas transnacionais, controladoras do comércio agrícola internacional, sementes, agrotóxicos e agroindústria, seguindo hegemônicas e com políticas econômicas de atendimento aos seus interesses e necessidades. A fórmula do agronegócio brasileiro estava dada: diminuição da mão-de- obra do campo em troca maquinários de alta produtividade e superexploração do campesinato com salários muito abaixo da média, ou seja: escala aumentada em grandes extensões de terra ajustada aos salários da agricultura entre os mais baixos do país. A Via Campesina Brasil tem se voltado a temas relevantes para a atual conjuntura, tais como: soberania alimentar, reforma agrária e agricultura camponesa sustentável, organização política e econômica de estrutura coletiva e horizontal, articulação internacional com formação de novos quadros, além de outros assuntos cujo debate se faz urgente na sociedade, a exemplo das questões de gênero e direitos humanos. Também articula 15 importantes movimentos camponeses, dentre os quais: Movimento dos Trabalhadores Rurais Sem Terra (MST); Movimento dos Pequenos Agricultores (MPA); Movimento dos Atingidos por Barragens (MAB); Movimento de Mulheres Camponesas (MMC); Conselho Indigenista Missionário (CIMI); Movimento dos Pescadores e Pescadoras Artesanais (MPP). Há, ainda, outros movimentos articulados à Via Campesina Internacional, que não possuem uma matriz exclusivamente campesina, mas também estudantil e religiosa: Federação dos Estudantes de Agronomia do Brasil (FEAB); Federação dos Estudantes de Engenharia Florestal (ABEEF); Comissão Pastoral da Terra (CPT); Pastoral da Juventude Rural (PJR). A particularidade brasileira e a possibilidade de cooperar com a construção do internacionalismo pelas vias da educação A particularidade brasileira exibe nuances que merecem destaque. A ausência de conflitos no seio da classe dominante, ou entre estas e as classes subalternas, não litigou a distribuição de terras. A partir dessa constatação, pode-se comparar o processo brasileiro ao alemão, levando em consideração que as antigas classes dominantes e a grande propriedade fundiária permaneceram. A concepção da questão agrária, como uma tarefa democrática dentro do processo revolucionário burguês clássico, mesmo no exemplo russo, teve como principal característica o choque entre as classes de ordem feudal. Não pesou, portanto, a contradição entre capital e trabalho, assim como não sofreu a ação do proletariado. Para Paulino e Almeida (2010), o Brasil se constituiu sob guarida do capitalismo comercial, com parte significativa de suas riquezas controladas por agraristas comprometidos com a economia agroexportadora, responsáveis por direcioná-la para atividades urbano-industriais, em primeiro plano. A estratégia fomentava os lucros na seara agrícola. Dessa forma, foram estruturadas duas situações de classe: proprietários fundiários e empreendedores urbano-industriais. Mazzeo (1995) explica que a burguesia brasileira não rompeu com a ordem latifundiário-escravista, caracterizando uma economia agroexportadora e importadora de bens industrializados. Paulatinamente, a burguesia agrária transformou-se em burguesia industrial. A produção cafeeira do século XIX possibilitou a acumulação de capital através do campo, ainda que por meio de uma industrialização secundária em relação à atividade agroexportadora. Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 331 Debate Debate econômicas e políticas, livres das imposições de organizações internacionais como o Fundo Monetário Internacional (FMI), o Banco Mundial, a Organização Mundial do Comércio (OMC), entre outras. Sua palavra de ordem “Globalizemos a luta, globalizemos a esperança” representa esta característica (GONÇALVES, 2010, p. 45). Nos últimos 30 anos, os movimentos camponeses têm tido um expressivo destaque na agenda de lutas latino-americana. Destacam-se: o movimento camponês indígena bolivariano, representado principalmente pela Central Sindical Única dos Trabalhadores Camponeses da Bolívia (CSUTCB); o Exército Zapatista de Libertação Nacional (EZLN), no México; e, claro, o Movimento dos Trabalhadores Rurais Sem Terra (MST), no Brasil. Cada um a seu modo, encabeçou, a partir da década de 1980, a luta contra as políticas neoliberais em seus respectivos países. Cumprem, desde meados dos anos 1980, um papel de fortalecimento da figura do camponês como um sujeito revolucionário, reacendendo a chama da luta social. Na trilha de Marx e Engels (2010), o modelo de educação praticado no campo vai ao encontro da premissa de que ideias revolucionárias podem surgir no seio da velha sociedade, formando elementos para uma nova. A dissolução das antigas ideias marcha juntamente com a dissolução das antigas condições de existência de vida. Ainda que submetidos a relações sociais capitalistas, os movimentos sociais do campo seguem em luta, resistindo às explorações e expropriações impostas pelo capital. Nessa jornada, engendram práticas sociais que reverberam em práticas educativas que apontam para uma nova forma de organização da sociedade: [...] o campesinato é um grupo social – parte da classe trabalhadora – que historicamente tem resistido à desterritorialização. Mas é um grupo social singular, porque sua subordinação ao capital não é total, como é a do assalariado [...]. No caso do campesinato, a terra de trabalho é um território de resistência. Na luta pela terra, na ocupação do território do latifúndio, o acampamento é um espaço de resistência (MARTIN; FERNANDES, 2004, p. 178). De acordo com Vieira (2011) a Via Campesina não se organiza, ainda, em busca de mudanças estruturais. Está firmada na luta anti-imperialista e antineoliberal, mas não alcança a luta anticapitalista efetivamente, ainda que suas principais organizações reclamem tais rupturas e levantem a bandeira da luta pelo socialismo. Em escala mundial, a VC opera a partir do que é consenso entre as grandes regiões, buscando alinhar lutas e estratégias comuns diante do vasto número de organizações e movimentos sociais do campo. A luta política encabeçada pela Via Campesina, que desemboca em movimentos sociais como o Movimento dos Trabalhadores Rurais Sem Terra (MST), se localiza, historicamente, na organização da sociedade civil para a construção de uma nova hegemonia, capaz de superar a trajetória de exploração forjada a partir da exploração. A análise de Fernandes (2001) atesta que na ausência de reforma agrária, a ocupação tem representado uma considerável forma de acesso à terra. Ademais, este processo é uma forma de intervenção dos trabalhadores em meio ao sistema político e econômico da expropriação. Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 332 Em 1996, o MST filia-se à Via Campesina, contribuindo para a articulação transnacional dos movimentos sociais contra-hegemônicos, bem como para a mundialização da luta pela emancipação humana. A Via Campesina configura-se como importante espaço para troca de experiências entre os movimentos sociais, diálogo, apoio, articulações de ações conjuntas, além de formulação de alternativas à ordem mundial vigente, defendendo a autodeterminação dos povos e a autonomia das sociedades em suas decisões A variedade de bases sociais das organizações-membro estabelece contradições, ou, no mínimo, ambivalências, pois articula grupos que possuem concepções diferentes sobre determinados temas comuns. Os grupos e organizações que compõem a Via Campesina representam, na totalidade, as particularidades nas quais se inserem, assim como refletem as contradições e trajetórias do capital nos territórios onde atuam. Vieira (2011) e Batista (2013), ao discutirem o processo de consolidação da VC destacam, por exemplo, que os coletivos latino-americanos não entendem a luta política e o papel da Via Campesina da mesma que os europeus, o que se justifica diante de processos históricos e trajetórias diferentes. Esses contrastes, segundo afirmam, não significam meras disputas nacionalistas, mas a existência de processos mais complexos da articulação internacional. Esses embates, no entanto, não as impedem de atuarem juntas e consolidarem lutas comuns. Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 333 Debate O internacionalismo, assim como o método de construir a organização política, são espaços onde se possibilita a unidade e coerência no território imaterial, na consciência individual e coletiva das organizações que fazem parte desta articulação. Aí reside também o papel da formação política de militantes e quadros, que se dá a partir das escolas, cursos, e atividades formativas, mas essencialmente na atuação cotidiana na organização política. Esta última tem um papel chave para abrir as inquietações e possibilidades de avanço na consciência organizativa e do sistema capitalista em sua totalidade. Somente com estas inquietações à flor da pele, ou com a necessidade da busca de respostas, é que as atividades de formação político-profissional realizadas pela VC tem um caráter realmente pedagógico (BATISTA, 2013, p. 259). Nesse sentido, é importante esclarecer que a categoria prática educativa, central nesse estudo, abarca ações que vão além dos espaços formais de estudo. Caldart (2011) salienta que a educação do campo não está restrita à escola física, embora a luta por ela seja um ponto essencial no histórico de lutas e organização coletiva dos sujeitos do campo. A busca pelo acesso à escolarização enfrenta a negação do conhecimento imposta a estes povos, uma das faces mais emblemáticas e sórdidas de um projeto de educação que prioriza a degradação e dominação das condições de vida no campo. A compreensão da expropriação provocada pelo capital segue integrada nas estratégias de resistência, não podendo ser dissociada. Debate Debate e economia promovidos no interior do MST. Afinado à VC, concentra esforços e canaliza visibilidade para três importantes pautas: luta pela terra, luta por sustentabilidade socioambiental e luta pela por educação. Mais do que colocar essa agenda em evidência, MST e VC consumam processos efetivos de formação e de conscientização política. Nesse caminho, conforme indicam Stédile e Fernandes (2005), o MST, à frente da luta pela reforma agrária como fundamento de um novo projeto de sociedade, reclama a necessidade de amarrar essa luta à democratização da educação, reforçando que a disputa pela terra requer uma correlação de forças que se alternam de acordo com o avanço da capacidade de organização e mobilização dos trabalhadores. A fim de melhor ilustrar as experiências educativas realizadas através da Via Campesina e dos movimentos que a compõem, é importante destacar as ações dos Institutos de Agroecologia Latino Americanos (IALAs) como parte de processos formativos mais amplos que ocorrem no interior desses coletivos de forma orgânica. Tais instituições de ensino, são resultado da articulação internacional de experiências formativas da Coordenadoria Latino-Americana de Organizações do Campo (CLOCVC), representação importante no processo de (re)construção dos territórios camponeses e indígenas através da consolidação de projetos político-educativos de experiências agroecológicas locais, regionais, nacionais e continentais. As experiências formativas da rede de IALAs tomam a agroecologia como o princípio político- educativo basilar. Embora o conceito de agroecologia não seja exatamente o mesmo para todos os movimentos que constituem a Via Campesina, sofrendo variações e diferenciações, há o consenso e convergência quanto ao reconhecimento de ser o ponto comum em torno da defesa da soberania alimentar, a premissa a partir da qual se articula a base epistêmica camponesa. Ainda sobre as práticas educativas da VC/MST, faz-se importante mencionar outras experiências pulverizadas na América Latina e Caribe por meio da Via Campesina e MST, tais como: Curso para militantes de base da região Cone Sul, itinerante; Escola de formação de militantes de base da região dos Andes, itinerante; Escola Nacional Florestan Fernandes – ENFF, Brasil; Escola Latino Americana de Comunicação Popular da CLOC-VC, itinerante; Escola de Agroecologia Raul Balbuena, Colômbia; Escola Nacional de Agroecologia do Equador – ENA; Escola de Formação de Dirigentes Egídio Brunetto, itinerante. Ao mencionar tais práticas, é importante enfatizar o protagonismo que os Movimentos Sociais do Campo desempenharam na construção dessas experiências. Também são pedagógicos os congressos, encontros, conferências, reuniões, espaços de formação política e toda a pluralidade de atividades sistemáticas que se estruturam a fim de garantir a aquisição de conhecimentos fundamentais à organização e emancipação dos camponeses: leitura de conjuntura, planejamento de ações, organização da luta, intercâmbios de experiências. São pedagógicas todas as ações capazes de forjar mecanismos de mobilização, avanço da consciência e ampliação de tudo o que se reputa necessário à edificação de uma hegemonia camponesa. Caldart (2012) reforça que a educação do campo não nasce como uma teoria educacional. Ao contrário, suas primeiras inquietações foram práticas. E que os desafios atuais continuam sendo práticos, ao passo que uma mera disputa teórica não daria conta dessa categoria. As práticas educativas promovidas pela Via Campesina/MST em áreas de assentamento compreendem esses territórios como fontes de apropriação dos conhecimentos científicos acumulados historicamente pelo conjunto da humanidade, bem como oportunidades de produção de conhecimentos e relações entre a vida cotidiana e a ciência. Os sujeitos do campo esperam/necessitam que esses espaços formativos possam ampliar suas possibilidades de conhecimento, a partir de processos pedagógicos que tenham como ponto de partida as particularidades de sua realidade. As práticas educativas empreendidas pelos movimentos sociais do campo estão inseridas na realidade concreta. A decisão sobre o que deve ser aprendido emerge dos conflitos e necessidades protagonizados pelos trabalhadores do campo, a partir de ações coletivas que priorizam seus interesses políticos, econômicos e sociais. Tais práticas, ultrapassam os limites da escola ou universidade, pois alcançam sujeitos em movimento, que resistem a partir da organização de seus coletivos, atrelando conhecimentos à garantia da própria existência. Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 334 A título de exemplificação, convém elencar, de forma concreta, algumas práticas educativas, como as que se estabelecem nos processos de formação política, centrais diante da perspectiva de transformação da realidade. Dentre diversos formatos, estão os estudos sistemáticos e coletivos sobre análise de conjuntura 334 Considerações finais A organização internacional da classe trabalhadora, nos diversos momentos históricos, procurou dar respostas aos desafios de seu tempo, englobando pautas ainda inconclusas diante da complexidade da luta pela emancipação humana. Nessa trajetória, a questão agrária e a questão do sujeito revolucionário foram temas de intenso debate, manifestando-se em diferentes expressões. Mais recentemente, a necessidade de organizar um Movimento Camponês Internacional – Via Campesina, aflorou em um contexto de enfrentamento da globalização neoliberal, acomodando novos desafios que ultrapassam a particularidade do campo, incorporando problemas da classe trabalhadora como um todo (VIEIRA, 2011; DESMARAIS, 2013). A influência do Banco Mundial no meio agrário brasileiro a partir da segunda metade do século XX, buscou imprimir essa política globalizada neoliberal através da narrativa de construção de políticas fundiárias pensadas a partir de necessidades observadas no país. Foi possível estabelecer dois elementos primordiais para a compreensão do contexto de surgimento da Via Campesina: a globalização do modelo de agricultura industrial moderno e, na contracorrente, a tentativa de buscar uma abordagem alternativa pelos setores mais impactados pelo modelo dominante, isto é, o próprio campesinato. Problematizamos o cenário em que despontaram protestos e pautas de reivindicações da Via Campesina no país, contestando os novos contornos do capitalismo mundial. Os movimentos sociais de luta pela terra e pela reforma agrária têm se articulado em resistência à ordem, política, ideologia e economia pautadas no agronegócio e latifúndio. No Brasil, mais precisamente a partir da década de 1980, esse enfrentamento tem ocorrido também pelas vias da educação popular – formal e não formal, em sentido lato e sentido stricto. As práticas educativas no/do campo priorizam a produção coletiva do conhecimento, por meio de experiências concretas validadas a partir das práticas sociais em perspectiva histórica. Organizando diferentes sujeitos, realidades, espaços e processos educativos, sistematizam, de forma dialética e dialógica, processos pedagógicos com potencial de contribuir com a edificação de um projeto de sociedade emancipatório, socialmente sustentável, fornecendo as bases de uma solidariedade internacionalista. Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 336 É seguro afirmar que as práticas educativas da Via Campesina na América Latina têm somado forças para a construção de uma hegemonia camponesa, forjando novos quadros políticos e intelectuais no/do campo. Debate Debate mundial, estabelecendo uma correlação na qual se constituem práticas educativas populares que diferem das convencionais, englobando um conjunto de organismos também formativos e potencialmente revolucionários. Fornecem, portanto, elementos categoriais que permitem alargar a perspectiva de educação dentro dos limites da escola burguesa, mantendo a percepção crítica de tal projeto. O maior desafio, assim como em outras experiências de educação formal no/do campo, consiste em concretizar um projeto educativo que forme seus próprios intelectuais, dotados de conhecimentos técnicos, mas também de consciência de classe, conhecimentos políticos, históricos, culturais e, principalmente, com vontade de atuar de forma diferente da convencional, de forma a superar o modelo agrário comprometido com o agronegócio, que historicamente tem empobrecido o campo de todas as formas (RIBEIRO, 2023). Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 335 O MST, em parceria com a Via Campesina, ao desempenhar o papel de formar lideranças e multiplicadores de uma hegemonia camponesa ao mesmo tempo em que luta pela terra e pela reforma agrária, cumpre a tarefa de semear práticas revolucionárias. A elevação político-intelectual dos povos do campo é um elemento-chave na linha de atuação dos movimentos sociais camponeses articulados em escala Debate Debate construção de uma nova sociedade, cuja efetivação só é possível diante de uma ampla reforma agrária popular e de rupturas estruturais. As práticas educativas populares da Via Campesina não dizem respeito apenas aos territórios20 em que são praticadas, mas comtemplam o desenvolvimento do campo e de seus povos. Reforçam, assim, o paradigma de uma educação que ocorra no espaço físico do campo, mas que pertença ao campo, pensada pelos camponeses, que leve em conta suas necessidades e realidades e que forje seus próprios intelectuais. A resistência camponesa tem demonstrado a capacidade de enfrentar as imposições das relações capitalistas no campo. As lutas engendradas pelos movimentos sociais do campo expressam não apenas potência e resistência, mas se comportam como um lugar social no qual é possível realizar e interpretar essas batalhas, erguendo elementos importantes para a construção de uma nova hegemonia. O caráter classista das relações corrompidas pelo capital é denunciado ao mesmo tempo em que é confrontado pela existência de experiências campesinas orientadas pela solidariedade, coletividade, colaboração e internacionalismo. Considerações finais A perspectiva de educar os povos do campo em contraposição a uma educação hegemônica caminha, lado a lado, com a luta pelo acesso à terra e reforma agrária, o que caracteriza uma concepção revolucionária de educação. Diante dos limites de que a educação, sozinha19, não possa dar conta de mobilizar as estruturas sociais, tais práticas educativas seguem formando novos homens e mulheres, alargando não apenas suas capacidades intelectuais, mas compondo um projeto maior que vislumbra a Debate MARTIN, Jean-Yves; FERNANDES, Bernardo Mançano. Movimento socioterritorial e “globalização”: algumas reflexões a partir do caso do MST. Revista Lutas Sociais, n. 11/12, 2004, p. 173-185. MARTINS, Fernando José. MST, Via Campesina e educação: integração e o Instituto de Agroecologia Latino-Americano (IALA) Guarani. In: MARTINS, Fernando José (Org.). Práticas educativas da Via Campesina. Curitiba: CRV, 2014, p. 37-51. MARX, Karl; ENGELS, Friedrich. Manifesto comunista. São Paulo: Boitempo, 2010. MAZZEO, Antonio Carlos. Burguesia e Capitalismo no Brasil. 2. ed. São Paulo: Ática, 1995. MAZZEO, Antonio Carlos. Burguesia e Capitalismo no Brasil. 2. ed. São Paulo: Ática, 1995. MOYO Sam; YEROS Paris Reclaiming the land: the resurgence of rural movements in Africa Asia MOYO, Sam; YEROS, Paris. Reclaiming the land: the resurgence of rural movements in Africa and Latin America. Londres: Zed Books, 2005. PAULINO, Eliane Tomiasi; ALMEIDA, Rosemeire Aparecida de. Terra e Território: a questão camponesa no capitalismo. São Paulo: Expressão Popular, 2010. PATEL, Raj. Agricultural imperialism and new peasant solidarities. Presentation at the Seminar Series of the Centre for Civil Society. University of KwaZulu-Natal, South Africa, 2004. RIBEIRO, Ellen Cristine dos Santos. Questão agrária, práticas educativas e internacionalismo: uma análise a partir do Instituto Agroecológico Latino Americano (IALA) Amazônico. Orientadora: Betânea Moreira de Moraes. Tese (Doutorado em Educação). Universidade Estadual do Ceará (UECE), 2023, 206p. ROSSET, Peter. Alternativa à política fundiária de mercado: reforma agrária e soberania alimentar. In: SAUER, Sérgio; PEREIRA, João Marcio Mendes (Orgs.). Capturando a terra: Banco Mundial, políticas fundiárias neoliberais e reforma agrária de mercado. São Paulo: Expressão Popular, 2006. ROSSET, Peter; TORRES, Maria Elena Martínez. Del conflicto de modelos para el mundo rural emerge la vía campesina como movimiento social transnacional. Journal of Peasant Studies, v. 37, n.1, p. 149-175, 2010. SARAIVA, José Flávio Sombra. História das relações internacionais contemporâneas: da sociedade internacional do século XIX à era da globalização. 2 ed. São Paulo: Saraiva, 2007. VIA CAMPESINA. IV Conferência Interacional da Via Campesina: documentos preparatórios. 2003 VIA CAMPESINA. Protocolo de intenciones que hacen entre sí: la Via Campesina, el gobierno de la República Bolivariana de Venezuela, el gobierno del Estado de Paraná e instituciones de enseñanza del Brasil e de Venezuela. Tapes, 2005 (mimeo). VIEIRA, Flávia Braga. Dos proletários unidos à globalização da esperança: um estudo sobre internacionalismos e a Via Campesina. 1ª edição. São Paulo: Alameda, 2011. Referências: BATISTA, Ândrea Francine. Consciência e territorialização contra-hegemônica: uma análise das políticas de formação da Via Campesina América do Sul. Orientador: Eduardo Girardi. Dissertação (Mestrado em Geografia). Universidade Estadual Paulista (UNESP), 2013, 276p. BORRAS, Saturnino. La Via Campesina: un movimiento en movimiento. Amsterdã: Transnational Institute, 2004. CALDART, Roseli Salete. Educação do Campo. In: CALDART, Roseli Salete, PEREIRA, Isabel Brasil, ALENTEJANO, Paulo, FRIGOTTO, Gaudêncio. (Orgs.). Dicionário da Educação do Campo. São Paulo: Expressão Popular, 2012. CAMPOS, João Carlos de. A integração latino-americana nas Escolas Latino-Americanas de Agroecologia da CLOC-Via Campesina no Brasil e Venezuela. Orientador: Francis Mary Nogueira. Dissertação (Mestrado em Educação). Universidade Estadual do Oeste do Paraná (UNIOESTE), 2014, 110p. CÂNDIDO, A. Os parceiros do Rio Bonito: estudo sobre o caipira paulista e a transformação dos seus meios de vida. São Paulo: Livraria Duas Cidades, 1979. DESMARAIS, Annette Aurélie. A Via Campesina: a globalização e o poder do campesinato. São Paulo: Cultura Acadêmica; Expressão Popular, 2013. EDELMAN, Marc. Transnational peasant and farmer movements and networks. In: GLASIUS, H; KALDOR, M. (Orgs.). Global civil Society Yearbook. Londres: Oxford University Press, 2003. FERNANDES, Bernardo Mançano. A ocupação como forma de acesso à terra. In: Congresso Internacional da Associação de Estudos Latino-Americanos, 23., 2001, Washington, D.C., 2001. FERNANDES, Bernardo Mançano. 27 anos do MST em luta pela terra. In: FERRANTE, V.; WHITAKER, D. (Orgs.). Reforma Agrária e desenvolvimento: desafios e rumos da política de assentamentos rurais. Brasília: MDA, 2008, p. 27-52. GONÇALVES, Laura. Movimentos sociais e Relações Internacionais: a luta pela emancipação humana. Orientador: Estevão Martins. Trabalho de Conclusão de Curso (Bacharelado em Relações Internacionais). Universidade Estadual Paulista Júlio de Mesquita Filho, 2010. GONÇALVES, Laura. O agrário na contemporaneidade: Banco Mundial, Via Campesina e o Estado brasileiro. Orientador: Estevão Martins. Dissertação (Mestrado em Relações Internacionais) – Universidade de Brasília (UnB), Brasília, 2014, 322p. erminal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 337 7 Dados atualizados em outubro de 2018, através do sítio eletrônico da Federação de Estudantes de Agronomia do Brasil (FEAB). Disponível em: https://feab.wordpress.com/via-campesina-e-msps/. 7 Dados atualizados em outubro de 2018, através do sítio eletrônico da Federação de Estudantes de Agronomia do Brasil (FEAB). Disponível em: https://feab.wordpress.com/via-campesina-e-msps/. 8 Tradução nossa. Texto original: “[...] Vía Campesina se ha revelado como un actor principal en las actuales luchas populares internacionales contra el neoliberalismo que, entre otras cosas, exigen responsabilidades a las agencias intergubernamentales, se enfrentan y se oponen al control corporativo sobre los recursos naturales y la tecnología, y defienden la soberanía alimentaria. Además, ha contado con un papel destacado en campañas de gran polémica política como, por ejemplo, las dirigidas contra la OMC, los gigantes corporativos mundiales como McDonalds, y los organismos modificados genéticamente (OMG) y las multinacionales que los fomentan, como Monsanto”. 9 Frequentemente, o termo é utilizado em castelhano como forma de salientar seu caráter latino, em clara referência à experiência de Mons, na Bélgica, remetendo às origens e à articulação dos povos latino-americanos e europeus do sul. 10 Emergem no Brasil em 1955, na região Nordeste, e foram violentamente reprimidas depois do golpe militar de 1964. Admitia como sujeito histórico de luta tanto o camponês (pequeno possuidor de terra e de meios para viver daquilo que planta e colhe) quanto o trabalhador rural (assalariado; necessita vender sua força de trabalho para sobreviver). As Ligas Camponesas entendiam o campesinato como uma força revolucionária hegemônica capaz de efetivar o fim do latifúndio e do imperialismo. 11 Asociación Nacional de Agricultores Pequeños. Fundada em 1961, em Cuba, como umas das resoluções do I Congresso Nacional de Pequenos Agricultores. 12 Apesar da vinculação inicial com a Igreja Católica, através da Comissão Pastoral da Terra (CPT), a atuação do MST ocorre de forma independente e será abordada no próximo subitem dessa exposição. 13 De acordo com Leite e Medeiros (2012, p. 85), o modelo agroecológico se contrapõe ao modelo do agronegócio, priorizando, para além do uso e posse da terra, práticas agrícolas alinhadas à preservação da biodiversidade, equilíbrio e saúde do solo, a partir de construções coletivas de conhecimento agregados pelos povos do campo, agricultores e cientistas. Trata-se de uma perspectiva pautada “na valorização da agricultura camponesa e nos princípios da policultura, nos cuidados ambientais e no controle dos agricultores sobre a produção de suas sementes”. Debate 4 Destacamos os trabalhos de Bringel (2015); Bringel e Cairo (2010); Bringel e Falero (2008); Landaluze e Barrera (2008); Rubbo (2014) e Bringel e Vieira (2015), fundamentais para a construção deste estudo. 4 Destacamos os trabalhos de Bringel (2015); Bringel e Cairo (2010); Bringel e Falero (2008); Landaluze e Barrera (2008); Rubbo (2014) e Bringel e Vieira (2015), fundamentais para a construção deste estudo. 5 Organização Mundial do Comércio. 5 Organização Mundial do Comércio. 6 Fundo Monetário Internacional. 6 Fundo Monetário Internacional. 6 Fundo Monetário Internacional. Notas 1 Doutora em Educação (UECE). Professora da rede municipal de Fortaleza/CE. Pesquisadora do Instituto de Estudos e Pesquisas do Movimento Operário (IMO/UECE). Currículo Lattes: http://lattes.cnpq.br/3336841935789898. Orcid: https://orcid.org/0000-0002-7558-5547. E-mail: ellencristineribeiro@hotmail.com. Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 338 1 Doutora em Educação (UECE). Professora da rede municipal de Fortaleza/CE. Pesquisadora do Instituto de Estudos e Pesquisas do Movimento Operário (IMO/UECE). Currículo Lattes: http://lattes.cnpq.br/3336841935789898. Orcid: https://orcid.org/0000-0002-7558-5547. E-mail: ellencristineribeiro@hotmail.com. 2 Doutora em Educação (UFC). Professora Adjunta da Universidade Estadual do Ceará (UECE). Pesquisadora do Instituto de Estudos e Pesquisas do Movimento Operário (IMO/UECE). Currículo Lattes: http://lattes.cnpq.br/0834231585359453. Orcid: https://orcid.org/0000-0001-8760-0380. E-mail: betaneamoraes@gmail.com. 3 Doutora em Educação (UFC). Professora Titular da Universidade Federal do Ceará (UFC). Pesquisadora do Instituto de Estudos e Pesquisa do Movimento Operário (IMO/UECE). Currículo Lattes: http://lattes.cnpq.br/8231954289757480. Orcid: https://orcid.org/0000-0002-4933-631X. E-mail: jacklinerabelo@gmail.com. 2 Doutora em Educação (UFC). Professora Adjunta da Universidade Estadual do Ceará (UECE). Pesquisadora do Instituto de Estudos e Pesquisas do Movimento Operário (IMO/UECE). Currículo Lattes: http://lattes.cnpq.br/0834231585359453. Orcid: https://orcid.org/0000-0001-8760-0380. E-mail: betaneamoraes@gmail.com. 2 Doutora em Educação (UFC). Professora Adjunta da Universidade Estadual do Ceará (UECE). Pesquisadora do Instituto de Estudos e Pesquisas do Movimento Operário (IMO/UECE). Currículo Lattes: http://lattes.cnpq.br/0834231585359453. Orcid: https://orcid.org/0000-0001-8760-0380. E-mail: betaneamoraes@gmail.com. 3 Doutora em Educação (UFC). Professora Titular da Universidade Federal do Ceará (UFC). Pesquisadora do Instituto de Estudos e Pesquisa do Movimento Operário (IMO/UECE). Currículo Lattes: http://lattes.cnpq.br/8231954289757480. Orcid: https://orcid.org/0000-0002-4933-631X. E-mail: jacklinerabelo@gmail.com. rminal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 7 Dados atualizados em outubro de 2018, através do sítio eletrônico da Federação de Estudantes de Agronomia do Brasil (FEAB). Disponível em: https://feab.wordpress.com/via-campesina-e-msps/. 14 “Em 17 de abril de 1996, de acordo com informações fornecidas pelo MST, a Polícia Militar abriu fogo contra um grupo de 1.500 pessoas que estava em uma marcha para Belém, capital do Pará, para exigir uma solução legal para uma ocupação de terras que mais de 4 mil pessoas haviam realizado na fazenda Macaxeira (território de Curionópolis) – a cerca de 650nquilômetros do destino planejado. Sob o pretexto de garantir que os manifestantes não interrompessem o trânsito, o governador do Pará enviou um batalhão de 2 mil soldados bem armados. Quando os manifestantes se recusaram a sair da estrada a polícia abriu fogo, matando dezenove camponeses e ferindo trinta mais” (DESMARAIS, 2013, P. 233). 15 Vieira (2011) relembra que a II Assembleia Mundial de Mulheres Camponesas e a I Assembleia Mundial de Jovens da Via Campesina ocorreram no mesmo ano, às vésperas da IV Conferência. 15 Vieira (2011) relembra que a II Assembleia Mundial de Mulheres Camponesas e a I Assembleia Mundial d da Via Campesina ocorreram no mesmo ano, às vésperas da IV Conferência. 16 International Federation of Agricultural Producers. 16 International Federation of Agricultural Producers. ndo Collor de Melo (1990-1992); Itamar Franco (1992-1994); Fernando Henrique Cardoso (1995-1998 e 1999- 18 Luís Inácio Lula da Silva (2003-2006 e 2007-2010); Dilma Rousseff (2011-2014 e 2015-até o golpe jurídico- parlamentar de 2016). 18 Luís Inácio Lula da Silva (2003-2006 e 2007-2010); Dilma Rousseff (2011-2014 e 2015-até o golpe jurídico- parlamentar de 2016). 19 Resguarda-se, aqui, a premissa lukacsiana segundo a qual a relação que se estabelece entre a educação e o complexo fundante – o trabalho – é de dependência ontológica, autonomia relativa e reciprocidade dialética. 20 Abre-se, aqui, um parêntese para expor a concepção de território na perspectiva dos assentamentos do MST, indissociável ao projeto de reforma agrária que reivindica. A educação ensejada em áreas de luta pela reforma agrária integra-se ao desenvolvimento político-social dos sujeitos do campo, evidenciando a territorialidade dos assentados, refletida através da perspectiva da multidimensionalidade vivenciada por esses membros, de organização social coletiva. Logo, tal concepção de territorialidade, advém da consciência integrante desse território, da participação de sujeitos que se sabem fundamentais para o desenvolvimento sócio-econômico-cultural do assentamento, desde sua formação histórica, de forma subjetiva e prática (FOLMER; MEURER, 2019). Recebido em: 29 de maio 2023 Aprovado em: 23 de jul. 2023 Recebido em: 29 de maio 2023 Aprovado em: 23 de jul. 7 Dados atualizados em outubro de 2018, através do sítio eletrônico da Federação de Estudantes de Agronomia do Brasil (FEAB). Disponível em: https://feab.wordpress.com/via-campesina-e-msps/. 2023 Recebido em: 29 de maio 2023 Aprovado em: 23 de jul. 2023 Germinal: marxismo e educação em debate, Salvador, v.15, n.2, p.320-339, ago. 2023. ISSN: 2175-5604 339
https://openalex.org/W4313830867	https://www.zora.uzh.ch/id/eprint/226643/1/ZORA_s00701_022_05477_3.pdf	English	null	Ex vivo and in vivo evaluation of transsphenoidal Liqoseal application to prevent cerebrospinal fluid leakage	Acta neurochirurgica	2,023	cc-by	6,989	Zurich Open Repository and Archive Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2023 Ex vivo and in vivo evaluation of transsphenoidal Liqoseal application to prevent cerebrospinal fluid leakage Slot, Emma M H ; Colmer, Nadia ; Serra, Carlo ; Holzmann, David ; Regli, Luca ; van Doormaal, Tristan P DOI: https://doi.org/10.1007/s00701-022-05477-3 DOI: https://doi.org/10.1007/s00701-022-05477-3 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-226643 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-226643 Journal Article Published Version ollowing work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Slot, Emma M H; Colmer, Nadia; Serra, Carlo; Holzmann, David; Regli, Luca; van Doormaal, Tristan P C (2023). Ex vivo and in vivo evaluation of transsphenoidal Liqoseal application to prevent cerebrospinal fluid leakage. Acta Neurochirurgica, 165(6):1511-1521. DOI: https://doi.org/10.1007/s00701-022-05477-3 Acta Neurochirurgica https://doi.org/10.1007/s00701-022-05477-3 ORIGINAL ARTICLE - NEUROSURGICAL TECHNIQUE EVALUATION Ex vivo and in vivo evaluation of transsphenoidal Liqoseal application to prevent cerebrospinal fluid leakage Received: 8 September 2022 / Accepted: 20 December 2022 © The Author(s) 2023 Abstract Background Despite improvements in closure techniques by using a vital nasoseptal flap, the use of sealing materials, and improved neurosurgical techniques, cerebrospinal fluid (CSF) leak after transsphenoidal surgery still is a clinically relevant problem. Liqoseal® (Polyganics bv, Groningen, The Netherlands) is a CE-approved bioresorbable sealant patch for use as an adjunct to standard methods of cranial dural closure to prevent CSF leakage. This study aims to evaluate the application of Liqoseal in transsphenoidal surgery ex vivo and in vivo. q p g y Methods 1. We created an ex vivo setup simulating the sphenoidal anatomy, using a fluid pump and porcine dura positioned on a conus with the anatomical dimensions of the sella to evaluate whether the burst pressure of Liqoseal applied to a bulging surface was above physiological intracranial pressure. Burst pressure was measured with a probe connected to dedicated computer software. Because of the challenging transsphenoidal environment, we tested in 4 groups with varying compression weight and time for the application of Liqoseal. 2. We subsequently describe the application of Liqoseal® in 3 patients during transsphenoidal procedures with intraoperative CSF leakage to prevent postoperative CSF leakage. Results 1. Ex vivo: The overall mean burst pressure in the transsphenoidal setup was 231 (± 103) mmHg. There was no significant difference in mean burst pressure between groups based on application weight and time (p = 0.227). 2. In Vivo: None of the patients had a postoperative CSF leak. No nose passage problems were observed. One patient had a postoperative meningitis and ventriculitis, most likely related to preoperative extensive CSF leakage. Postoperative imaging did not show any local infection, swelling, or other device-related adverse effects. Conclusions We assess the use of Liqoseal® to seal a dural defect during an endoscopic transsphenoidal procedure as to be likely safe and potentially effective. g g y g Conclusions We assess the use of Liqoseal® to seal a dural defect during an endoscopic transsphenoidal procedure as to be likely safe and potentially effective. Abstract Keywords Cerebrospinal fluid leakage · Case report · Device · Transsphenoidal surgery Keywords Cerebrospinal fluid leakage · Case report · Device · Transsphenoidal surgery Keywords Cerebrospinal fluid leakage · Case report · Device · Transsphenoidal surgery Abbreviations ANOVA Analysis of variance CE Conformité Européenne CSF Cerebrospinal fluid ELD External lumbar drain EVD External ventricular drain MRI Magnetic resonance imaging mm Millimeters cm Centimeters mmHg Millimeters of mercury N Number NSF Nasoseptal flap PEEP Positive end-expiratory pressure TSS Transsphenoidal surgery USA United States of America This article is part of the Topical Collection on Neurosurgical technique evaluation * Tristan P. C. van Doormaal Theodorus.vandoormaal@usz.ch 1 Department of Neurology and Neurosurgery, University Medical Center Utrecht, Utrecht, The Netherlands 2 Department of Translational Neuroscience, University Medical Center, Utrecht Brain Center, Utrecht University, Utrecht, The Netherlands 3 Department of Neurosurgery, Clinical Neuroscience Center, University Hospital Zurich, Zurich, Switzerland 4 Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Zurich, Zurich, Switzerland :(0123 1 3456789) 3 Acta Neurochirurgica Fig. 1 Liqoseal. Length 8 cm, width 8 cm, weight 1600 to 2000 mg. Reproduced with permission from the copyright owner [10]: Van Doormaal TPC, Germans MR, Sie M, Brouwers B, Fierstra J, Dep- auw PRAM, Robe PA, Regli L. Single-arm, open-label, multicenter study to evaluate the safety and performance of dura sealant patch in reducing cerebrospinal fluid leakage following elective cranial sur- gery: the ENCASE Trial Study Protocol. Neurosurgery. 2020 Feb 1;86(2):E203-E208. Website URL: https:// journ als. lww. com/ neuro surge ry/ Fullt ext/ 2020/ 02000/ Single_ Arm,_ Open_ Label ,_ Multi center_ Study_ to. 36. aspx. Neurosurgery is the official journal of the congress of neurological surgeons. The creative commons license does not apply to this content. Use of the material in any format is prohibited without written permission from the publisher, Wolters Kluwer Health, Inc. Please contact permissions@lww.com for further information Introduction Cerebrospinal fluid (CSF) leak is a frequent complica- tion after transsphenoidal surgery (TSS), with an overall prevalence of 3.4% [7]. The prevalence of CSF leak for indications other than pituitary adenomas (i.e., crani- opharyngioma, meningioma, Rathke’s cleft cysts) is 7.1%, which is similar to that found for craniotomies [7]. CSF leak is associated with various complications such as meningitis, CSF hypotension syndrome, and intrac- erebral hemorrhage causing increased morbidity and mortality [4, 8]. Furthermore, hospital costs for patients with CSF leak after TSS are significantly higher than for patients without [4, 11]. Despite improvements in closure techniques by using a vital nasoseptal flap (NSF), the use of sealing materi- als, and improved neurosurgical techniques, CSF leak after TSS still is a clinically relevant problem, for intra- dural and invasive lesions, such as craniopharyngiomas or tuberculum sellae meningiomas, especially. Retro- spective analyses of the use of a patch sealant, TachoSil (Takeda Pharmaceuticals, Tokyo, Japan), in TSS show variable postoperative CSF leak results ranging from 0.8 to 7.8% [2, 3, 12]. For liquid sealants, Tisseel (Baxter, Deerfield, USA) and DuraSeal (Integra Lifesciences, Princeton, USA), similar results have been reported in retrospective analyses with postoperative CSF leak rang- ing from 1 to 12.5% [13, 14]. Pereira et al. [14] did not find a statistically significant difference in postoperative CSF leak for the use of Tisseel® or DuraSeal®. To fur- ther improve the advancement of TSS effective solutions to prevent postoperative CSF leaks are warranted. Fig. 1 Liqoseal. Length 8 cm, width 8 cm, weight 1600 to 2000 mg. Reproduced with permission from the copyright owner [10]: Van Doormaal TPC, Germans MR, Sie M, Brouwers B, Fierstra J, Dep- auw PRAM, Robe PA, Regli L. Single-arm, open-label, multicenter study to evaluate the safety and performance of dura sealant patch in reducing cerebrospinal fluid leakage following elective cranial sur- gery: the ENCASE Trial Study Protocol. Neurosurgery. 2020 Feb 1;86(2):E203-E208. Website URL: https:// journ als. lww. com/ neuro surge ry/ Fullt ext/ 2020/ 02000/ Single_ Arm,_ Open_ Label ,_ Multi center_ Study_ to. 36. aspx. Neurosurgery is the official journal of the congress of neurological surgeons. The creative commons license does not apply to this content. Use of the material in any format is prohibited without written permission from the publisher, Wolters Kluwer Health, Inc. Please contact permissions@lww.com for further information Ex vivo Liqoseal (Polyganics bv, Groningen, The Nether- lands) is a CE (Conformité Européenne) approved biore- sorbable sealant patch for use as an adjunct to standard methods of cranial dural closure. The patch is composed of a white foam layer containing Polyethylene glycol- N-hydroxysuccinimide, the adhesive component, and buffer salt [10]. The blue layer is made of polyurethane and provides the watertight seal (Fig. 1) [10]. The first in the human study (ENCASE) has shown that the patch is safe and potentially efficacious for reducing CSF leak- age after intracranial surgery [10, 15]. Model We created an ex-vivo transsphenoidal burst pressure model by adapting an earlier published dural sealing model [16, 9] with a conus in the shape of the sella to mimic the application area (Fig. 2A). The dimensions of the conus (17 × 7.5 mm) were based on measurements of the pituitary gland and sella turcica on 23 anonymized MRI scans of patients with pituitary adenomas. Cranial porcine dura was harvested at an abattoir and cut into circles with a 30 mm diameter. A circular gap of 3 mm was punched out in the center. Liqoseal® was cut into circles of 15 mm in diameter. The dura was clamped above the open pressure chamber and the Liqoseal® was applied manually to cover the gap from the outside with a 5 mm overlap. TSS is regarded as a form of cranial surgery, and thus Liqoseal® application is not off-label [6]. However, the surrounding tissue and dimensions in this approach are different compared to a craniotomy. Therefore, this study evaluates the application of Liqoseal® in TSS in preclinical (ex vivo) settings and 3 endoscopic trans- sphenoidal cases. Liqoseal® was compressed by equally and continu- ously applying a standardized weight on a moist gauze 1 3 Acta Neurochirurgica A Set up for burst pres- easurement. B Example of burst pressure soft- LabChart, AD Instru- Fig. 2 A Set up for burst pres- sure measurement. B Example output of burst pressure soft- ware (LabChart, AD Instru- ments) for a specified time period. For the cranial application of Liqoseal® a compression time of 2 min with a compres- sion weight of 1 kg was used, to allow optimal adhesion by the formation of amide bonds between the foam layer of the patch and the dura mater [16]. However, the difficult corridor in TSS could, in practice, result in the prescribed application pressure not being met. Therefore, the acute burst pressure was evaluated with a compression weight of 1 kg and 0,25 kg. Furthermore, a shorter compression time would be clinically advantageous. Hence, we compared acute burst pressure for compression times of 2 min and 1 min, respectively. for a specified time period. For the cranial application of Liqoseal® a compression time of 2 min with a compres- sion weight of 1 kg was used, to allow optimal adhesion by the formation of amide bonds between the foam layer of the patch and the dura mater [16]. Model However, the difficult corridor in TSS could, in practice, result in the prescribed application pressure not being met. Therefore, the acute burst pressure was evaluated with a compression weight of 1 kg and 0,25 kg. Furthermore, a shorter compression time would be clinically advantageous. Hence, we compared acute burst pressure for compression times of 2 min and 1 min, respectively. set-ups was to determine if Liqoseal would adhere to the dura with mean burst pressure above the higher end of the physiological intracranial pressure range (> 30 mmHg) [1] on a surface resembling the shape of the sella with varying compression weight and time during application. Fig. 2 A Set up for burst pres- sure measurement. B Example output of burst pressure soft- ware (LabChart, AD Instru- ments) In vivo We performed a retrospective evaluation of all transsphenoi- dal surgeries in which Liqoseal® was used in the University Hospital of Zurich, Switzerland, between the 3rd of Janu- ary 2020 (when Liqoseal® was approved) and the 1st of March 2022. Three Liqoseal applications were performed in these procedures. Liqoseal® was applied on the outside of the defect in all cases. All 3 patients provided a general informed consent for the use of all clinical data and imaging for research. Spearman’s rank-order correlation showed no significant correlation between mean burst pressure and the interval between experiment and harvesting (rs = 0.031, p = 0.759). Statistics The required sample size to detect statistically significant differences between groups with an alpha of 0.05 and power of 90% was determined at 23 measurements per subgroup, using the power analysis for One-way Analysis of Variance (ANOVA). Input for sample size calculation was based on the results of the previous cranial and spinal measurements [16]. A total of 3 additional measurements were planned per subgroup to allow for loss of measurements due to experimental failure, so in total 104 measurements were performed. The four groups varying in compression weight (1 kg vs. 0.25 kg) and time (1 min vs. 2 min) were compared using ANOVA. Post hoc Bonferroni correction was applied to adjust for multiple comparisons. Spearman’s rank-order correlation was used to evaluate the association between A fluid pump with a constant flow of 2.0 mL/min of artificial CSF (EcoCyte Bioscience, Germany) was used to increase the pressure in the chamber. The pressure was con- tinuously measured using a blood pressure probe (AD instru- ments MLT0670 Disposable BP transducer) connected to a computer using LabChart v8.1.14 software (ADInstruments, Australia). Burst pressure was defined as the maximum pressure in millimeters of mercury (mmHg) determined on the continuous measurement in LabChart (Fig. 2B) at the moment of fluid leakage. The aim of these experimental 1 3 Acta Neurochirurgica burst pressure and the interval between measurement and harvesting of the dura. All analyses were performed in SPSS version 27 (IBM). leakage in the experimental setup prevented adequate pres- sure built-up. The overall mean burst pressure in the trans- sphenoidal setup was 231 (± 103) mmHg (Fig. 3, Table 1). There was no significant difference in mean burst pres- sure between groups based on application time and weight (p = 0.227). In Vivo Liqoseal® was applied in 3 endoscopic transsphenoidal sur- geries until March 1, 2022. Results Patient 1 (63 years old male) was diagnosed with a hor- mone-inactive growing gonadotrophic macroadenoma (Fig. 4, Table 2, Supplementary Information 1). Intra- operatively an evident CSF leak occurred (Fig. 5A). The patient was operated using the mononostril “chopstick” approach with the aim to preserve healthy mucosal tissue Patient 1 (63 years old male) was diagnosed with a hor- mone-inactive growing gonadotrophic macroadenoma (Fig. 4, Table 2, Supplementary Information 1). Intra- operatively an evident CSF leak occurred (Fig. 5A). The patient was operated using the mononostril “chopstick” approach with the aim to preserve healthy mucosal tissue Table 1 Burst pressure in 4 groups; 1 kg/2 min, 1 kg/1 min, 0.25 kg/2 min, and 0.25 kg/1mi Ex vivo A total of 100 measurements were included in the analysis. Four measurements were excluded from the analysis because Fig. 3 Boxplot (minimum, Q1, median, Q3, and maxi- mum) of burst pressure in 4 groups varying compression weight and time: 1 kg/2 min, 1 kg/1 min, 0.25 kg/2 min, and 0.25 kg/1 min Table 1 Burst pressure in 4 groups; 1 kg/2 min, 1 kg/1 min, 0.25 kg/2 min, and 0.25 kg/1mi SD, standard deviation; N, number Group Mean Burst Pressure (mmHg) SD Lowest value Highest value N included N performed 1 kg, 2 min 241.4 135.0 69.4 459.0 25 26 1 kg, 1 min 257.3 102.0 70.1 426.4 24 26 0.25 kg, 2 min 229.5 77.5 53.4 352.6 25 26 0.25 kg, 1 min 199.0 85.1 62.9 397.4 26 26 Fig. 3 Boxplot (minimum, Q1, median, Q3, and maxi- mum) of burst pressure in 4 groups varying compression weight and time: 1 kg/2 min, 1 kg/1 min, 0.25 kg/2 min, and 0.25 kg/1 min 1 3 Acta Neurochirurgica Fig. 4 Preoperative MRI patient 1 showing a macroadenoma in A sagittal view and B coronal view Fig. 4 Preoperative MRI patient 1 showing a macroadenoma in A sagittal view and B coronal view [5]. Considering the small size of the defect, preparing an NSF resulting in damage to the nasal mucosa was not considered favorable. Therefore, it was decided to seal with Liqoseal® combined with external lumbar drain- age (ELD). A piece of plastic was used to assess the size of the bony defect in the sella. A circular piece of Liqoseal® was cut with 10 mm margin at all sides. After trying several folding options, the piece was folded in 2 with the white side out and parachuted in holding the patch at the front tip to pull the patch forward instead of pushing it. After positioning, a series of small cottonoids was positioned over the Liqoseal® before compressing for 2 min with a 90-degree ring curette. This led to a good adherence over bone and sella region. However, a small bottom part of the sealant was hampered by loose mucosa. The Liqoseal® could be removed with a gentle pulling force via the forceps. The basal bone was cleaned, mucosa removed and a second circular piece of the same patch of Liqoseal® was applied that covered the whole sella defect with a margin of 10 mm (Fig. 5B). Case 2 PEEP test (20 cm H2O for 20 s) showed no intraoperative leakage. The patch was covered with Tisseel® and Spongostan®. A nasal packing was put in place. No rhi- noliquorrhea was observed after this surgery. Patient developed a combined meningitis and ventriculitis at day 4 after the 3rd surgery, which was treated with intravenous antibiotics. The ELD was exchanged for an external ventricular drain (EVD) at this day to treat the infection and resulting hydrocephalus. The treating neurosurgeon did not consider Liqoseal® as the source of the infection, hence the nose was not surgically revised. At day 12 an MRI was made (Fig. 9). Individual Liqoseal® patch recognition was not possible and there were no signs of infec- tion or swelling of the patch. Temporary closure of the EVD resulted repeatedly in hydrocephalus (still without leakage). Therefore, a ventriculoperitoneal shunt was placed at day 40. Patient was discharged at day 44. Final follow-up was 6 months after the surgery in which Liqoseal® was applied. Visual dis- turbances persisted. Patient-reported no nasal complaints and good olfactory function. She refused further follow-up. Case 2 Patient 2 (54 years old female) was diagnosed with a giant mac- roadenoma causing bitemporal hemianopsia (Fig. 7, Table 2, Supplementary Information 1). First surgery (day -17) was complicated by postoperative rhinorliquorrhea. A revision sur- gery was performed using a vascularized NSF to seal the defect and decreasing CSF pressure with ELD (day -7). The leakage continued postoperatively despite increasing CSF drainage vol- ume. During the second revision surgery (day 0) a defect just above the vital NSF was observed (Fig. 8A). As salvage treat- ment a fat plug was placed in the small defect. Subsequently, Liqoseal was inserted with the same method as described in patient 1 (Fig. 8B–C). PEEP test (20 cm H2O for 20 s) showed no intraoperative leakage. The patch was covered with Tisseel® and Spongostan®. A nasal packing was put in place. No rhi- noliquorrhea was observed after this surgery. Patient developed a combined meningitis and ventriculitis at day 4 after the 3rd surgery, which was treated with intravenous antibiotics. The ELD was exchanged for an external ventricular drain (EVD) at this day to treat the infection and resulting hydrocephalus. The treating neurosurgeon did not consider Liqoseal® as the source of the infection, hence the nose was not surgically revised. At day 12 an MRI was made (Fig. 9). Individual Liqoseal® patch recognition was not possible and there were no signs of infec- tion or swelling of the patch. Temporary closure of the EVD resulted repeatedly in hydrocephalus (still without leakage). Therefore, a ventriculoperitoneal shunt was placed at day 40. Patient was discharged at day 44. Final follow-up was 6 months after the surgery in which Liqoseal® was applied. Visual dis- turbances persisted. Patient-reported no nasal complaints and good olfactory function. She refused further follow-up. Patient 2 (54 years old female) was diagnosed with a giant mac- roadenoma causing bitemporal hemianopsia (Fig. 7, Table 2, Supplementary Information 1). First surgery (day -17) was complicated by postoperative rhinorliquorrhea. A revision sur- gery was performed using a vascularized NSF to seal the defect and decreasing CSF pressure with ELD (day -7). The leakage continued postoperatively despite increasing CSF drainage vol- ume. During the second revision surgery (day 0) a defect just above the vital NSF was observed (Fig. 8A). As salvage treat- ment a fat plug was placed in the small defect. Subsequently, Liqoseal was inserted with the same method as described in patient 1 (Fig. 8B–C). Ex vivo Posi- tive end-expiratory pressure (PEEP) test was performed (20 cm H2O for 20 s) showing no leakage. The patch was covered with Tisseel® and to prevent the patch from being exposed to air and Spongostan (Ethicon, Raritan, USA) to further cover the patch and mucosa, to fill up the cavity and provide additional tissue support (Fig. 5C). A nasal packing was put in place to further provide support to the surrounding tissues and to tamponade any small bleeding afterwards. Postoperatively, no rhinoliquorrhea was observed. The ELD was removed at day 6. Patient was discharged day 8 after surgery without complica- tions. Three-month endoscopic control showed complete re-endothelialization (Fig. 5D). At further MRI follow- up (Fig. 6) individual patch recognition was not pos- sible, but no signs of infection or swelling of the patch were observed. During the entire follow-up period of 15 months, there were no nasal complaints and good olfactory function Case 3 Age Sex BMI Smoking Relevant medical history Indication Intraoperative complications Other closure techniques External CSF drainage Postoperative complications Discharge (d) Postoperative treatment Neurological deficit Final follow-up (m) 1 63 M 26 Former (20 PY) None Macro adenoma CSF leakage Tisseel, Spongostan, Fat, Nose tampon ELD day 0–6 None 7 None None 15 2 54 F 23 No 2 times TSS Revision CSF leakage None Tisseel, Spongostan, Fat, Nose tampon ELD day 0–4 EVD day 4–40 VPS at day 40 Meningitis and ven- triculitis, Hydrocepha- lus 44 None Bitemporal hemiano- psia 6 3 7 F 15 No None Clival chor- doma CSF leakage Tisseel, Spon- gostan, Fat ELD day 0–8 None 12 Proton beam therapy Abducens nerve palsy 7 Age Sex BMI Smoking Relevant medical history Indication Intraoperative complications Other closure techniques External CSF drainage Postoperative complications Discharge (d) Postoperative treatment Neurological deficit Final follow-up (m) 1 63 M 26 Former (20 PY) None Macro adenoma CSF leakage Tisseel, Spongostan, Fat, Nose tampon ELD day 0–6 None 7 None None 15 2 54 F 23 No 2 times TSS Revision CSF leakage None Tisseel, Spongostan, Fat, Nose tampon ELD day 0–4 EVD day 4–40 VPS at day 40 Meningitis and ven- triculitis, Hydrocepha- lus 44 None Bitemporal hemiano- psia 6 3 7 F 15 No None Clival chor- doma CSF leakage Tisseel, Spon- gostan, Fat ELD day 0–8 None 12 Proton beam therapy Abducens nerve palsy 7 Case 3 Patiënt 3 (7 years old female) presented with an abducens nerve palsy caused by a clivus chordoma (Fig. 10, Table 2, Supplementary Information 1). After resection a large defect 1 3 Acta Neurochirurgica in the clivus resulted with a central dural defect (Fig. 11A). A NSF was not prepared and it was considered by the oper- ating surgeon that it would be difficult in this case to make it large enough to cover the total defect appropriately. How- ever, no dural sealants have been CE approved for use in children. So on the discretion of the operating surgeons Liqoseal® was chosen to be used off-label. This application area was deeper and flatter than in the previous 2 patients. This caused the Liqoseal® application to be more difficult and a re-application was necessary. The final positioning showed wrinkles and internal Liqoseal® folds (Fig. 11B). The operating surgeon however decided to leave the patch in place because the dural defect was covered. The Liqo- seal® was covered with a fat plug harvested from the peri- umbilical region (Fig. 11C). Tisseel® and fat were thereafter alternately applied. Finally, the construct was covered with Spongostan® to further fill the cavity and deliver additional tissue support (Fig. 11D). No PEEP test was performed. Because of the high risk of postoperative leakage associated with the dural defect an ELD was placed intraoperatively as well. Postoperatively, no rhinoliquorrhea was observed. The ELD was removed at day 8. No postoperative complications occurred and patient was discharged at day 12 after surgery. Intraoperative and postoperative MRI showed a small chor- doma rest at the cavernous sinus which was considered inop- erable. The patient was radiated with proton beam 7 weeks after surgery. Latest follow-up was at 7 months after surgery. The abducens paresis persisted. Patient showed good nasal passage and olfactory function up until this time. MRI con- trol at this timepoint showed no swelling of the Liqoseal® patch and slow resolving of the fat plug (Fig. 12). Fig. 6 MRI follow-up patient 1 showing smoothening of the sellar wall over time. No signs of infection, swelling, or other pathological reactions were observed. A Intraoperative MRI (no Liqoseal), B day 6 postop- eratively, C 3 months post- operatively, and D 15 months postoperatively Fig. 7 Preoperative CT patient 2 showing pneumocephalus due to CSF leakage after previous surgery in A sagittal view and B axial view Fig. 5 Endoscopic images of patient 1 showing A intra- operative CSF leakage, B final Liqoseal positioning, C intraoperative end situation, and D 3-month follow-up with full re-endothelisation in patient 1 Fig. 8 Endoscopic images patient 2 showing A intraopera- tive CSF leakage, B folding of Liqoseal during application, C final Liqoseal positioning, and D intraoperative end situation in patient 2 Discussion This is the first study that evaluates the application of Liqo- seal® during TSS. We report excellent ex vivo and in vivo results. The overall mean burst pressure of Liqoseal® in this transsphenoidal model (231 ± 103 mmHg) and mean burst pressures in individual groups based on compression weight and time were all well above physiological intracranial pres- sure [1]. Mean burst pressure in this model was shown to be similar to those found in our cranial and spinal model (145 ± 39 mmHg and 233 ± 81 mmHg, respectively) [16]. Liqoseal® was successfully applied during endoscopic endonasal surgery in 3 patients. Given their clinical history, each of these patients can be considered as high risk for post- operative CSF leakage. None of these patients’ postoperative CSF leakage required revision surgery or had nasal passage problems. There was one infectious complication in patient 2 that occurred 4 days after the implantation of the device. This patient was at increased risk for infection because of 1 3 Acta Neurochirurgica Fig. 5 Endoscopic images of patient 1 showing A intra- operative CSF leakage, B final Liqoseal positioning, C intraoperative end situation, and D 3-month follow-up with full re-endothelisation in patient 1 1 3 Fig. 6 MRI follow-up patient 1 showing smoothening of the sellar wall over time. No signs of infection, swelling, or other pathological reactions were observed. A Intraoperative MRI (no Liqoseal), B day 6 postop- eratively, C 3 months post- operatively, and D 15 months postoperatively Fig. 7 Preoperative CT patient 2 showing pneumocephalus due to CSF leakage after previous surgery in A sagittal view and B axial view Fig. 6 MRI follow-up patient 1 showing smoothening of the sellar wall over time. No signs of infection, swelling, or other pathological reactions were observed. A Intraoperative MRI (no Liqoseal), B day 6 postop- eratively, C 3 months post- operatively, and D 15 months postoperatively Fig. 7 Preoperative CT patient 2 showing pneumocephalus due to CSF leakage after previous surgery in A sagittal view and B axial view 1 3 Acta Neurochirurgica 1 3 Fig. 8 Endoscopic images patient 2 showing A intraopera- tive CSF leakage, B folding of Liqoseal during application, C final Liqoseal positioning, and D intraoperative end situation in patient 2 Fig. Fig. 10 Preoperative MRI showing a clivus chordoma in A and B sagittal view and C axial view Discussion 9 MRI follow-up patient 2 A 16 days before 3rd surgery, B day 6 after 3rd surgery, C day 12 postoperatively (sagittal), and D day 12 postoperatively (transversal) showing no swell- ing of the patch or signs of infection Fig. 10 Preoperative MRI showing a clivus chordoma in A and B sagittal view and C axial view Fig. 9 MRI follow-up patient 2 A 16 days before 3rd surgery, B day 6 after 3rd surgery, C day 12 postoperatively (sagittal), and D day 12 postoperatively (transversal) showing no swell- ing of the patch or signs of infection B Fig. 10 Preoperative MRI showing a clivus chordoma in A and B sagittal view and C axial view 1 3 Acta Neurochirurgica continuous CSF leakage prior to the surgery in which Liqo- seal® was applied, and the infection was treatable with antibi- otics [4]. We deem the infection unlikely to be device-related. We found no indications of safety issues for the transsphenoi- dal application of Liqoseal® based on these 3 patients. Limitations The most important limitation of the current study is the applied which does not allow for any conclusions about effi- cacy. Moreover, all patients in this study received an intra- operative ELD to decrease the CSF pressure and support the healing of the dura which may have positively contrib- uted to the prevention of CSF leakage and the functioning of the patch. In addition, fibrin glue (Tisseel®) and gelatin sponge (Spongostan®) were used as a coverage. Further- more, endoscopic inspection of the nasal mucosa (not stand- ard of care) was performed in one patient only, showing Fig. 11 Endoscopic images showing patient 3 A intraop- erative CSF leakage, B final Liqoseal positioning, C fat plug fixated with Tisseel on top of Liqoseal, and D intraoperative end situation in patient 3 Fig. 12 MRI results in patient 3 A intraoperatively, B 1 month postoperatively. C 5 months postoperatively, and D 8 months postoperatively, showing no swelling of the patch or signs of infection. Slow resolving of fat patch can be observed Fig. 11 Endoscopic images showing patient 3 A intraop- erative CSF leakage, B final Liqoseal positioning, C fat plug fixated with Tisseel on top of Liqoseal, and D intraoperative end situation in patient 3 Fig. 12 MRI results in patient 3 A intraoperatively, B 1 month postoperatively. Discussion C 5 months postoperatively, and D 8 months postoperatively, showing no swelling of the patch or signs of infection. Slow resolving of fat patch can be observed applied which does not allow for any conclusions about effi- cacy. Moreover, all patients in this study received an intra- operative ELD to decrease the CSF pressure and support the healing of the dura which may have positively contrib- uted to the prevention of CSF leakage and the functioning of the patch. In addition, fibrin glue (Tisseel®) and gelatin sponge (Spongostan®) were used as a coverage. Further- more, endoscopic inspection of the nasal mucosa (not stand- ard of care) was performed in one patient only, showing re-endothelization. applied which does not allow for any conclusions about effi- cacy. Moreover, all patients in this study received an intra- operative ELD to decrease the CSF pressure and support the healing of the dura which may have positively contrib- uted to the prevention of CSF leakage and the functioning of the patch. In addition, fibrin glue (Tisseel®) and gelatin sponge (Spongostan®) were used as a coverage. Further- more, endoscopic inspection of the nasal mucosa (not stand- ard of care) was performed in one patient only, showing re-endothelization. continuous CSF leakage prior to the surgery in which Liqo- seal® was applied, and the infection was treatable with antibi- otics [4]. We deem the infection unlikely to be device-related. We found no indications of safety issues for the transsphenoi- dal application of Liqoseal® based on these 3 patients. Recommendations Based on our experience in these first 3 cases, we think that there are a number of technical aspects to take into consid- eration when applying Liqoseal® in TSS. Firstly, we recom- mend patch sizing to allow for margins of minimally 5 mm, taking into consideration that a larger sized patch is more difficult to introduce. When fat tissue is placed under Liqo- seal®, we recommend a margin of 10 mm as Liqoseal® does not adhere to fat. Secondly, we recommend to fold the patch with the white side (PEG-NHS side) outwards. This has the advantage of easier unfolding, yet does expose the foam layer to possible absorption of blood and damage. Thirdly, the patch should be held at the most distal point with a small rongeur while being introduced in the nose to exert a pulling force on the patch instead of a pushing force. Fourthly, in these 3 cases, compression for 2 min using moistened cot- tonoids and a patty was performed with a 90-degree bended ring curette. Despite the results of the ex vivo experiments showing that 1 min compression appears to be sufficient, we still recommend to compress for a minimum of 2 min as stated in the instructions for use for security and consistency reasons. Finally, the dural defects in the cases presented in this article were relatively small. Liqoseal® is intended for use on defects with a maximum size of 3 mm. Use over larger defects is thus off-label. We recommend to use Liqo- seal® in cases with larger defects with caution and only in combination with a construct allowing endothelization and formation of new dura (i.e., covering the mucosal tissue with muscle tissue or fat). It is important to note that Liqoseal does not adhere to fat tissue and that fat tissue will resorb over time. Considering the relatively fast endothelization we have observed, the primary goal of using Liqoseal® in such cases is to overcome the time until endothelization without CSF leakage. Data Availability Data is available from the corresponding author upon reasonable request. Limitations The most important limitation of the current study is the small number of TSS cases in which Liqoseal® has been 1 3 Acta Neurochirurgica indicate that the use of Liqoseal® in the sphenoid sinus to seal a dural defect in TSS is likely safe and potentially effective. Finally, the experimental model was designed based on the sella region. This is representative for the majority of transsphenoidal cases, but not all of them. For example, patient 3 had a clivus tumor that grew under the sella and the surface of this region does not resemble the surface of the ex vivo model. Furthermore, the gap size in the dura in the experimental setup was 3 mm in diameter. In clinical practice, the gap size in the dura, especially in cases leading to CSF leak postoperatively, may in fact be larger. Supplementary Information The online version contains supplemen- tary material available at https:// doi. org/ 10. 1007/ s00701- 022- 05477-3. Author contribution Conceptualization: Tristan van Doormaal; Meth- odology: Tristan van Doormaal, Nadia Colmer, Emma Slot, Formal analysis and investigation: Emma Slot, Tristan van Doormaal; Surger- ies: Tristan van Doormaal, Carlo Serra, David Holzmann, Luca Regli; Writing—original draft preparation: Emma Slot, Tristan van Door- maal; Writing—review and editing: all authors; Supervision: Tristan van Doormaal. Conflict of interest E.M.H.S. received a research grant through Po- lyganics b.v. Conflict of interest E.M.H.S. received a research grant through Po- lyganics b.v. T.P.C.vD is a consultant for Polyganics b.v. 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/. References 1. Albeck MJ, Børgesen SE, Gjerris F, Schmidt JF, Sørensen PS (1991) Intracranial pressure and cerebrospinal fluid outflow con- ductance in healthy subjects. J Neurosurg. https:// doi. org/ 10. 3171/ jns. 1991. 74.4. 0597 j 2. Hong CK, Kim YB, Hong JB, Lee KS (2015) Sealing of cerebro- spinal fluid leakage during conventional transsphenoidal surgery using a fibrin-coated collagen fleece. J Clin Neurosci. https:// doi. org/ 10. 1016/j. jocn. 2014. 10. 019 2. 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Conclusion Kinaci A, van Thoor S, Redegeld S, Tooren M, van Doormaal TPC (2021) Ex vivo evaluation of a multilayered sealant patch for watertight dural closure: cranial and spinal models. J Mater Sci: Mater Med, https:// doi. org/ 10. 1007/ s10856- 021- 06552-4 p g 8. Van Aken MO, Feelders RA, de Marie S et al (2004) Cerebrospi- nal fluid leakage during transsphenoidal surgery: postoperative external lumbar drainage reduces the risk for meningitis. Pituitary. https:// doi. org/ 10. 1007/ s11102- 005- 5351-3 17. Kinaci A, Algra A, Heuts S, O’Donnell D, van der Zwan A, van Doormaal T (2018) Effectiveness of dural sealants in prevention of cerebrospinal fluid leakage after craniotomy: a systematic review world neurosurgery. https:// doi. org/ 10. 1016/j. wneu. 2018. 06. 196 9. Van Doormaal T, Kinaci A, van Thoor S, Redegeld S, Bergmann W, van der Zwan A (2018) Usefulness of sealants for dural clo- sure: evaluation in an in vitro model. 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https://openalex.org/W3108677007	https://www.researchsquare.com/article/rs-27275/v1.pdf	English	null	Myoinositol to total choline ratio in IDH wild-type gliomas as a prognostic factor on preoperative magnetic resonance spectroscopy	Research Square (Research Square)	2,020	cc-by	6,680	Myoinositol to total choline ratio in IDH wild-type gliomas as a prognostic factor on preoperative magnetic resonance spectroscopy gliomas as a prognostic f magnetic resonance spec Masanobu Kumon Department of Neurosurgery, Fujita Health Univers Shunsuke Nakae (  snakae.1977@gmail.com ) Kazuhiro Murayama Fujita Health University Takema Kato Fujita Health University Shigeo Ohba Fujita Health University Joji Inamasu Fujita Health University Masato Abe Fujita Health University Seiji Yamada Fujita Health University Hikaru Sasaki Department of Neurosurgery, Keio University Yoshiharu Ohno Fujita Health University Mitsuhiro Hasegawa Fujita Health University Hiroki Kurahashi Fujita Health University Yuichi Hirose Fujita Health University Research article Keywords: glioma, IDH wild-type, magnetic resonance spectroscopy, myoinositol Posted Date: May 11th, 2020 Keywords: glioma, IDH wild-type, magnetic resonance spectroscopy, myoinositol Posted Date: May 11th, 2020 Page 1/18 DOI: https://doi.org/10.21203/rs.3.rs-27275/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License DOI: https://doi.org/10.21203/rs.3.rs-27275/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/18 Abstract Background: Isocitrate dehydrogenase (IDH) wild-type gliomas tend to be pathologically defined as glioblastomas. We previously reported that, unlike IDH -mutant gliomas, IDH wild-type gliomas showed significantly lower ratios of myoinositol to total choline (i.e., the Ins/Cho ratio) on magnetic resonance (MR) spectroscopy. Given that IDH -mutant gliomas also have much better prognoses than IDH wild-type gliomas, we hypothesized that this lower Ins/Cho ratio is associated with malignancy in adults with supratentorial gliomas. Therefore, we calculated the Ins/Cho ratios of patients with supratentorial IDH wild-type gliomas and investigated their progression free survival (PFS) and overall survival (OS) to determine its utility as a prognostic marker. Methods: We classified IDH wild-type gliomas (n = 30) into two groups based on the Ins/Cho ratios, and compared patient backgrounds, pathological findings, PFS, OS, and copy number aberrations. Methods: We classified IDH wild-type gliomas (n = 30) into two groups based on the Ins/Cho ratios, and compared patient backgrounds, pathological findings, PFS, OS, and copy number aberrations. Results: Compared with the group with high Ins/Cho ratios, the group with low Ins/Cho ratios had shorter PFS (P = 0.020) and OS (P = 0.037) durations. Multivariate analysis demonstrated that the Ins/Cho ratio corelated significantly with PFS (hazard ratio 0.34, P = 0.027). Results: Compared with the group with high Ins/Cho ratios, the group with low Ins/Cho ratios had shorter PFS (P = 0.020) and OS (P = 0.037) durations. Multivariate analysis demonstrated that the Ins/Cho ratio corelated significantly with PFS (hazard ratio 0.34, P = 0.027). Conclusion: We conclude that the preoperative Ins/Cho ratio can be used as a novel prognostic factor for IDH wild-type gliomas. Conclusion: We conclude that the preoperative Ins/Cho ratio can be used as a novel prognostic factor for IDH wild-type gliomas. Study design We retrospectively investigated 30 cases of newly diagnosed IDH wild-type glioma in patients analyzed by MR spectroscopy between 2013 and 2018 at Fujita Health University hospital. Patients who had already received treatment before MR spectroscopy or who were younger than 20 years old were excluded. The primary outcomes were the progression free survival (PFS) and the overall survival (OS). PFS was calculated from the date of first operation to the date of confirmed recurrence. OS was calculated from the date of first resection to the date of death. Patient information was last updated in April 2019 for follow-up purposes. All resected tissues were assessed by neuropathologists according to the WHO classification [1]. Background Gliomas, which are diagnosed based on pathological and genetic findings, are among the most common brain tumors [1–4]. Extensive, surgical resection is the preferred initial therapeutic strategy to improve prognosis [5, 6]; however, such an approach also increases the risk of brain dysfunction because either the border between the tumor and normal brain tissue is unclear or because tumors infiltrate invasively into adjacent healthy brain tissue. Therefore, the ability to predict tumor malignancy preoperatively could help surgeons to plan optimal surgical strategies. Magnetic resonance (MR) spectroscopy enables us to quantify tumor metabolites noninvasively by analyzing their spectra, and thus, it is widely used for preoperative diagnosis of brain tumors [7]. We previously examined tumor metabolites in adult supratentorial gliomas and reported that the myoinositol to total choline (Ins/Cho) ratios in isocitrate dehydrogenase (IDH) wild-type gliomas were significantly lower than in IDH-mutant gliomas [8]. Given that total choline is thought to represent cell density [9], we presumed that lower ratios indicate myoinositol consumption in a glioma cell. Myoinositols are sugar alcohol in phospholipids of the intracellular membrane [10], and they are produced through food digestion or by hydrolysis or dephosphorylation of the intracellular membrane [10–12]. In astrocytes, myoinositols contribute to adjusting osmotic pressure in response to changes in intracranial pressure [13]. Page 3/18 Most supratentorial gliomas harboring wild-type IDH are pathologically classified as glioblastomas and tend to have dismal prognoses [1–3]. Molecular factors that contribute to this poor prognosis have been identified based on genetic subtype, including TERT promoter mutations, copy number aberrations (e.g., + 7, 10q and 9p21), and epigenetic changes (e.g., histone H3) [14–18]. However, such information is only available after surgical resection, and we ideally need a method for predicting outcomes in patients suspected of having glioblastomas before surgery. Based on the observation that supratentorial gliomas with IDH mutations have better outcomes than those with wild-type IDH [1, 2, 3], we hypothesized that low Ins/Cho ratios in IDH wild-type gliomas can be used as a prognostic marker for this genetic subtype of glioma. − − In the present study, we calculated Ins/Cho ratios by MR spectroscopy for adults with supratentorial IDH wild-type gliomas to investigate the ratio’s association with prognosis, its correlation with previously reported prognostic factors, and whether it could be used as a preoperative prognostic factor. Next generation sequencing analysis and comparison of Copy number aberration We analyzed samples that met our inclusion criteria and classified them by Ins/Cho ratio into high and low and a low ratio groups. We prepared diluted genomic DNA for subsequent experiments. After whole genome amplification using a SurePlex DNA Amplification System (Illumina, San Diego, CA, USA), library preparation was performed with a Nextera XT DNA Library Preparation Kit (Illumina). Sequencing analysis was conducted with a VeriSeq PGS Kit-MiSeq (Illumina), and results were analyzed with BlueFuse Multi Software (Illumina). In all cases, chromosomes were divided into 2500 windows of approximately 1 Mb in size. Evaluation of IDH mutation status We evaluated IDH mutations using the Sanger method, with codon 132 in IDH1 and codon 172 in IDH 2 analyzed by polymerase chain reaction (PCR). Briefly, DNA was extracted from resected frozen tissue and formalin-fixed and paraffin-embedded tissue (FFPE), using DNeasy blood and tissue kits (QIAGEN, Hulsterweg, Netherland) and an REPLI-g FFPE Kit (QIAGEN).The reaction mixtures for PCR comprised DNA, primers, 10 PCR buffer, 10 mM dNTP mix (Thermo Fisher Scientific, Waltham, MA, USA), 50 mM MGCL₂, and PLATINUM TagDNA polymerase (Thermo Fisher Scientific). After we confirmed the DNA bands of the PCR products in electrophoresis, we added BigDye Sequencing Buffer (Thermo Fisher Scientific), Ready Reaction Mix (Thermo Fisher Scientific), and the same primer to the PCR products, and repeated the PCR. Sequencing was performed with a BigDye Terminator version 3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and results were analyzed on an ABI 3100 (Applied Biosystems, Waltham, MA, USA). × MR spectroscopy A single-voxel 1H-MR spectroscopy with point-resolved spectroscopy sequence was performed with a 3 T (T) scanner (Ingenia 3T; Philips Healthcare, Best, The Netherlands) using a dS Head coil and Vantage Titan 3T (Canon Medical Systems Corporation, Otawara, Japan) using a 16 or a 32-channel coil. In each patient, 1H-MR spectroscopy was performed using the following parameters: repetition time (TR)/echo time (TE), 2000/144 and 35 ms; number of excitation (NEX), 128; bandwidth, 1.61 HZ/point; voxel of interest (VOI) size for metabolic measurements, 15 × 15 × 15 mm. T2 weighted images in 3 directions for setting the VOI were determined for each patient by means of the following parameters: repetition time (TR)/echo time (TE), 4250/82 ms; acquisition matrix size, 416 × 344; reconstruction matrix, 640 × 640; field of view (FOV), 230 × 230 mm; slice thickness, 4.0 mm; slice gap, 0.8 mm; number of excitation (NEX), 1; reduction factor 1.9. The VOI was selected by a board certified neuroradiologist (K.M) with an experience of 10–15 years’ to include the VOIs inside the lesion based on T2 weighted images in 3 Page 4/18 Page 4/18 directions. When the tumor contains necrotic and cystic components, these components were included within VOI to accurately evaluate tumor characteristics. In 1H-MR spectroscopy, myoinositol and total choline were measured with short TE (TE = 35 ms) and long TE (TE = 144 ms) respectively [19, 20, 21] because of their relaxation time in T2, and mean concentration of MR spectroscopy values were analyzed by automatic quantification program (LCModel; Stephen Provencher, Oakville, Ontario, Canada) [22]. PFS and OS analysis We compared prognosis by the Ins/Cho ratio. During follow-up, tumor recurrence occurred in 94% of patients with low Ins/Cho ratio (i.e., 16/17) and in 77% with high Ins/Cho ratio (i.e., 10/13). Moreover, 82% (14/17) of patients with low ratios and 38% (5/13) with high ratios died. Figures 1a and 1b show that the PFS and OS in those with high ratios were significantly shorter than in those with low ratios, indicating higher recurrence and mortality rates in the patients with low Ins/Cho ratios (P = 0.020 and 0.037, respectively). In the multivariate analysis, Cox proportional hazards models revealed that the Ins/Cho ratio was significantly associated with PFS (hazard ratio 0.34, P = 0.027), which suggested that the Ins/Cho ratio was useful outcome predictor, especially for PFS. These data are summarized in Tables 3a and 3b. Relation of patient and pathology characteristics with the Ins/Cho ratio The mean and median values of the Ins/Cho ratios for the 30 patients included in this cohort were 0.75 and 0.67, respectively. We opted for 0.7 as the cutoff value and classified those patients into high (≥0.7) and low (<0.7) Ins/Cho ratio groups (n = 13 and 17, respectively). Tables 1 and 2 show the patient characteristics, pathologies, Ins/Cho ratios, and postoperative therapies. Case comprised 25 glioblastoma, two gliosarcomas, three astrocytomas. Two of three astrocytomas gained chromosome 7 and/or loss of chromosome 10q, but the other was not associated with them. There were no statistically significant differences in sex, age at onset, laterality, MIB-1 index, or grade Ⅳ WHO classification between the two groups. Overall, eight patients underwent reoperation, six patients underwent additional chemotherapy, and seven patients had no treatment after recurrence. Analysis of copy number aberration by next generation sequencing We included 20 cases in the next generation sequencing, analyzing nine samples in the group with high Ins/Cho ratios (≥0.7) and 11 samples in the group with low Ins/Cho ratios (<0.7). The overall noise value was <0.3. Analysis focused on regions where specific genes exist that are associated with poor prognosis in glioblastoma, including 7p11.2 (EGFR), 9p21.3 (p16), 10q23.3 (PTEN) [15, 16]. Gain of 7p11.2 was detected in seven cases with high ratios and in six cases with low ratios, loss of 9p21.3 was detected in four cases with high ratios and in three cases with low ratios, and loss of 10q23.3 was detected in four cases with high ratios and in three cases with low ratios (Table 4). There were no significant differences between the groups in any other window. Statistical analysis We used Fisher's exact test and the Mann Whitney U test to compare age at onset, sex, laterality, MIB-1 index, and pathology between the two groups. PFS and OS were analyzed using the Kaplan Meier method and compared with the log-rank test. Cox proportional hazards models were used to determine the relationship between the Ins/Cho ratio and prognosis. Given that most patients were treated with surgery, temozolomide and radiotherapy, and bevacizumab, we selected the following as explanatory factors in the multivariate analysis: gross total resection; subtotal resection, which was defined as > 90% resection; temozolomide and radiotherapy; bevacizumab; and the Ins/Cho ratio. All statistical analyses were conducted using EZR [23]. – – Page 5/18 Page 5/18 Discussion Page 6/18 In this study, we demonstrated that a high Ins/Cho ratio on MR spectroscopy was associated with a better prognosis than a low Ins/Cho ratio according to Kaplan Meier curves. The Ins/Cho ratio was significantly associated with PFS by Kaplan Meier and multivariate analysis. Of the three cases in which the Ins/Cho ratios exceeded 1.0 and that started the Stupp regimen soon after maximum safe resection, the PFS durations were 26, 17, and ≥ 39 months (Cases 7, 8, and 26, respectively). Given that total choline in MR spectroscopy reveals cell density [9], low myoinositol levels are considered associated with poor prognosis. Indeed, a recent cohort study of recurrent glioblastoma treated with bevacizumab reported that a high myoinositol value on MR spectroscopy was associated with a good prognosis [24]. Given all these results, we considered that myoinositol affects as a second messenger and has antitumor effects. Myoinositols are located in glial cells, especially in astrocytes [25], and regulate intracranial osmotic pressure [13]. Phosphatidylinositol 3-phosphate, which contains myoinositols in its structure, is produced by intracellular membrane metabolism [10] and activates the PI3K-Akt pathway as a second messenger [26]. Activation of this pathway may cause a poorer prognosis in patients with a low Ins/Cho ratio by prompting cell proliferation and myoinositol consumption. As shown in a previous study, oral administration of myoinositols can inhibit malignant transformation of tumor cells in patients suffering non-small-cell lung cancer, in which the PI3K-Akt pathway is important [27]. Other researchers also reported that myoinositols have antitumor effects that result from their phosphorylated metabolites, such as inositol 1,3,4,5,6-pentaphosphate and inositol hexaphosphate, which can induce tumor cell apoptosis [28, 29]. These previous studies suggest that malignancy in IDH wild-type gliomas with low Ins/Cho ratios is associated with a simple reduction in antitumor effects. – – However, multivariate analysis only showed that the Ins/Cho ratio was significantly associated with PFS and not OS (Fig. 1, Table 3). We assumed that the therapeutic strategies used after recurrence affected these results in the multivariate analyses. Indeed, some patients chose both surgical resection and additional chemotherapy (13%), whereas other patients opted for no additional therapy after recurrence (23%), potentially affecting outcomes. Notably, all patients younger than 50 years at diagnosis were in the group with a low Ins/Cho ratio, and 60% of these chose surgical resection and chemotherapy (Table 1). Discussion Two of these patients (cases 1 and 3) survived for approximately 2 years despite having low Ins/Cho ratios (0.57 and 0.51, respectively). By contrast, 20% of patients older than 60 years at diagnosis underwent surgical resection, and only 4% of these chose additional chemotherapy. Patients who opted for no therapy after recurrence had short survival time (e.g., cases 15 and 27) despite having relatively high Ins/Cho ratios (0.85 and 0.69, respectively). Copy number analysis by next generation sequencing revealed no significant correlations between the Ins/Cho ratio and chromosomal areas, despite the association of specific copy number aberrations with glioblastoma (Table 4). Previous studies have shown that such copy number aberrations are an early stage of tumorigenesis in glioblastoma [15, 30, 31], and our results suggest that low myoinositol levels are less likely to be caused by decreased myoinositol synthesis due to chromosomal change and gene expression. Rather, the low myoinositol levels can be considered to result from consumption due to tumor growth. Page 7/18 Page 7/18 This study is limited by its retrospective cohort design and lack of control of therapeutic strategies (e.g., resection extent by tumor location, postoperative bevacizumab use, and radiological dosages) or postoperative complications (e.g., delayed wound healing or high-fever affected the start of adjuvant therapy). These factors could have significantly affected the clinical courses and outcomes. We must now investigate more cases to clarify the putative correlations between the Ins/Cho ratio and patient prognosis. Conclusions The noninvasive Ins/Cho ratio can serve as a novel prognostic marker for adults with supratentorial IDH wild-type gliomas, providing useful preoperative information in patients with suspected glioblastomas. However, we can only speculate on why myoinositols are associated with patient outcomes, and questions around this will be targeted in future research. Abbreviations FFPE formalin-fixed and paraffin-embedded t IDH isocitrate dehydrogenase Ins/Cho ratio ratio of myoinositol to total choline MR Magnetic resonance spectroscopy OS overall survival PCR polymerase chain reaction PFS progression free survival VOI voxel of interest Consent for publication All authors read and approved the final manuscript. Authors' contributions MK is the author and who conducted this study. TK, HK conducted the copy number analysis and drafted the manuscript. KM and YO analyzed metabolites of glioma in this study and drafted the manuscript. MA and SY diagnosed tissue pathologically according to the WHO classification. SN, SO, JI, HS, MH, and YH revised and edited the manuscript. Declarations Ethical approval and consent to participate: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the local ethical review board of Fujita Health University (HG19-017). Page 8/18 Page 8/18 Acknowledgements We would like to thank Ms. Fujiko Sueishi for technical support. Availability of data and materials We already provided adequate information of patients enrolled in the present study in manuscript and tables. For this reason, we will not deposit further information as dataset. Funding This study was funded by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology in Japan (# 16K20029 to S.N.). Competing interests The authors declare that they have no conflict of interest. the Central Nervous System: a summary. Acta Neuropathol. 2016, 131:803-820. https://doi.org/1007/s00401-016-1545-1. 2. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, Kos I, Batinic-Haberle I, Jones S, Riggins GJ, Friedman H, Friedman A, Reardon D, Herndon J, Kinzler KW, Velculescu VE, Vogelstein B, Bigner DD. IDH1 and IDH2 mutations in gliomas. N Engl J Med. 2009, 360:765- 773. https://doi.org/10.1056/NEJMoa0808710. 2. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, Kos I, Batinic-Haberle I, Jones S, Riggins GJ, Friedman H, Friedman A, Reardon D, Herndon J, Kinzler KW, Velculescu VE, Vogelstein B, Bigner DD. IDH1 and IDH2 mutations in gliomas. 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Clin Cancer Res. 2014, 20:5601-5611. https://doi.org/1158/1078-0432.CCR-14-0831. 31. Huse JT, Aldape KD. The evolving role of molecular markers in the diagnosis and management of diffuse glioma. Clin Cancer Res. 2014, 20:5601-5611. https://doi.org/1158/1078-0432.CCR-14-0831. 2187. https://doi.org/10.3390/ijms18102187. Methodological consensus on Page 12/18 Page 12/18 clinical proton MRS of the brain: Review and recommendations. 2019 82:527-550. doi: 10.1002/mrm.27742. Epub 2019 Mar 28. clinical proton MRS of the brain: Review and recommendations. 2019 82:527-550. doi: 10.1002/mrm.27742. Epub 2019 Mar 28. 22. Provencher SW. Automatic quantitation of localized in vivo 1H spectra with LCModel. NMR Biomed. 2001, 14:260-264. https://doi.org/10.1002/nbm.698. 22. Provencher SW. Automatic quantitation of localized in vivo 1H spectra with LCModel. NMR Biomed. 2001, 14:260-264. https://doi.org/10.1002/nbm.698. 23. Kanda Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone Marrow Transplant. 2013, 48:452-458. https://www.nature.com/articles/bmt2012244. 23. Kanda Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone Marrow Transplant. 2013, 48:452-458. https://www.nature.com/articles/bmt2012244. 24. Steidl E, Pilatus U, Hattingen E, Steinbach JP, Zanella F, Ronellenfitsch MW, Bähr O. Myoinositol as a Biomarker in Recurrent Glioblastoma Treated with Bevacizumab: A 1H-Magnetic Resonance Spectroscopy Study. PLoS One. 2016, 11:e0168113. https://doi.org/10.1371/journal.pone.0168113. 24. Steidl E, Pilatus U, Hattingen E, Steinbach JP, Zanella F, Ronellenfitsch MW, Bähr O. Myoinositol as a Biomarker in Recurrent Glioblastoma Treated with Bevacizumab: A 1H-Magnetic Resonance Spectroscopy Study. PLoS One. 2016, 11:e0168113. https://doi.org/10.1371/journal.pone.0168113. 25. Brand A, Richter-Landsberg C, Leibfritz D. Multinuclear NMR studies on the energy metabolism of glial and neuronal cells. Dev Neurosci. 1993, 15:289-298. https://doi.org/10.1159/000111347. 25. Brand A, Richter-Landsberg C, Leibfritz D. Multinuclear NMR studies on the energy metabolism of glial and neuronal cells. Dev Neurosci. 1993, 15:289-298. https://doi.org/10.1159/000111347. 26. Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1998, 273:13375- 13378.https://doi.org/10.1074/jbc.273.22.13375. 26. Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1998, 273:13375- 13378.https://doi.org/10.1074/jbc.273.22.13375. 27. Han W, Gills JJ, Memmott RM, Lam S, Dennis PA. The chemopreventive agent myoinositol inhibits Akt and extracellular signal-regulated kinase in bronchial lesions from heavy smokers. Cancer Prev Res (Phila). 2009, 2:370-376. https://doi.org/1158/1940-6207.CAPR-08-0209. 27. Han W, Gills JJ, Memmott RM, Lam S, Dennis PA. The chemopreventive agent myoinositol inhibits Akt and extracellular signal-regulated kinase in bronchial lesions from heavy smokers. Cancer Prev Res (Phila). 2009, 2:370-376. https://doi.org/1158/1940-6207.CAPR-08-0209. 28. Piccolo E, Vignati S, Maffucci T, Innominato PF, Riley AM, Potter BV, Pandolfi PP, Broggini M, Iacobelli S, Innocenti P, Falasca M. Inositol pentakisphosphate promotes apoptosis through the PI 3- K/Akt pathway. Oncogene. 2004, 23:1754-1765. https://doi.org/10.1038/sj.onc.1207296. 28. Tables Table 1. Characteristics and treatments of patients Table 1. Table 3a. Multivariate analysis of PFS Tables Characteristics and treatments of patients Page 13/18 Case Age, sex Pathology Recurrence PFS Follow-up terms Outcome Ins/Cho First operation / Postoperative therapies / therapies after recurrence 1 42F GBM, Gr Ⅳ Yes 9 22 Dead 0.573 GTR / TMZ + RT / TMZ + RT + BEV+ other chemotherapies + operation 2 43M GBM, Gr Ⅳ Yes 5 12 Dead 0.475 PR / TMZ + RT / BEV + another chemotherapy + operation 3 43F GBM, Gr Ⅳ Yes 1 31 Dead 0.507 GTR / No treatment / TMZ + RT + BEV + other chemotherapies + operations 4 44M GBM, Gr Ⅳ Yes 10 14 Dead 0.167 GTR / TMZ + RT + BEV / other chemotherapy 5 46F GBM, Gr Ⅳ Yes 3 12 Dead 0.450 GTR / TMZ + RT / TMZ + RT + BEV + other chemotherapy 6 62M GBM, Gr Ⅳ Yes 14 49 Dead 0.516 Biopsy / TMZ + RT / TMZ + RT + BEV + operation 7 62M GS, Gr Ⅳ Yes 26 58 Alive 1.149 STR / TMZ + RT / TMZ + RT + operation 8 63F GBM, Gr Ⅳ Yes 17 36 Alive 1.149 GTR / TMZ + RT / TMZ + RT + BEV 9 67M GBM, Gr Ⅳ Yes 2 2 Alive 0.044 PR / TMZ + RT / BEV 10 67M GBM, Gr Ⅳ Yes 9 10 Alive 0.816 PR / TMZ + RT + BEV / TMZ 11 68F GBM, Gr Ⅳ Yes 8 8 Dead 0.227 GTR / TMZ + RT / No treatment 12 68M GBM, Gr Ⅳ NO 6 10 Alive 1.182 STR / TMZ + RT / No treatment 13 69F GBM G Ⅳ Y 1 12 D d 2 054 14 69F GBM, Gr Ⅳ Yes 12 20 Dead 0.655 GTR / TMZ + RT / BEV 15 70M GBM, Gr Ⅳ NO 3 11 Dead 0.854 GTR / TMZ + RT / No treatment 16 71M DA, Gr Ⅱ Yes 9 39 Dead 1.702 Biopsy / TMZ + RT / operation 17 72M GBM, Gr Ⅳ Yes 1 22 Dead 0.451 GTR / TMZ + RT / TMZ + RT + BEV 18 72F DA, Gr Ⅱ Yes 4 7 Alive 0.952 PR / No treatment / No treatment 19 73F GBM, Gr Ⅳ Yes 1 23 Dead 0.405 STR / No treatment / TMZ + RT + BEV + operation 20 74M GBM, Gr Ⅳ Yes 2 20 Dead 0.758 GTR / TMZ + RT / TMZ + BEV 21 74M GBM, Gr Ⅳ Yes 1 20 Dead 0.240 GTR / TMZ + RT / TMZ + BEV + another chemotherapy + Operation 22 74M GBM, Gr Ⅳ Yes 10 19 Alive 0.893 STR / TMZ + RT + BEV / TMZ + RT + BEV 23 75F GBM, Gr Ⅳ NO 6 6 Alive 0.405 GTR / TMZ + RT 24 76F AA, Gr Ⅲ Yes 1 24 Dead 0.635 Biopsy / TMZ + RT / TMZ + BEV 25 77M GS, Gr Ⅳ Yes 13 14 Dead 0.695 GTR / TMZ + BEV / No treatment 26 80F GBM, Gr Ⅳ NO 39 39 Alive 1.176 PR / TMZ + RT 27 81M GBM, Gr Ⅳ Yes 2 3 Dead 0.692 Biopsy / TMZ + BEV / No treatment 28 81M GBM Gr Ⅳ Yes 4 4 Alive 1 397 GTR / No treatment 29 85M GBM, Gr Ⅳ Yes 2 5 Alive 0.279 Biopsy / TMZ + BEV +RT / TMZ 30 86F GBM, Gr Ⅳ Yes 21 24 Dead 1.111 STR / TMZ / BEV Data show the patient and treatment details after the first operation and after recurrence for supratentorial IDH wild-type glioma (n=30). Tables Pathology was diagnosed according to the WHO classification 2016. Abbreviations: AA, anaplastic astrocytoma; BEV, bevacizumab; DA, diffuse astrocytoma; F, female; GBM, glioblastoma; Gr, grade; GS, gliosarcoma; GTR, Gross total resection; Ins/Cho, ratio of myoinositol to total choline; IDH, isocitrate dehydrogenase; M, male; PFS, progression free survival; PR, Partial resection; RT, radiotherapy; STR, Subtotal resection; TMZ, temozolomide. Table 2. Comparison of patient characteristics by Ins/Cho ratio threshold Ins/Cho ratio≥0.7 (n = 13) Ins/Cho ratio<0.7 (n = 17) P value Male, n 8 9 0.72 Age at onset, median (years) 71 69 0.38 Left side, n 8 7 0.46 MIB-1 index, median 38 46 0.41 Grade Ⅳ, n 11 16 0.57 Abbreviations: Grade Ⅳ, grade Ⅳ in WHO Classification; Ins/Cho, ratio of myoinositol to total choline; n, number of patients. Ins/Cho ratio≥0.7 Ins/Cho ratio<0.7 P value Abbreviations: Grade Ⅳ, grade Ⅳ in WHO Classification; Ins/Cho, ratio of myoinositol to total choline; n, number of patients. de Ⅳ, grade Ⅳ in WHO Classification; Ins/Cho, ratio of myoinositol to total choline; n, number of patients. Abbreviations: Grade Ⅳ, grade Ⅳ in WHO Classification; Ins/Cho, ratio of myoinositol to total choline; n, nu Abbreviations: Grade Ⅳ, grade Ⅳ in WHO Classification; Ins/Cho, ratio of myoinositol to total choline; n, number of patients. Table 3a. Multivariate analysis of PFS Page 16/18 Page 16/18 Explanatory factor Hazard ratio (95% CI) P value GTR or STR 0.84 (0.36-2.0) 0.68 Use of BEV 0.76 (0.26-2.2) 0.62 Both use of TMZ and RT 0.52 (0.20-1.4) 0.18 Ins/Cho ratio 0.34 (0.13-0.88) 0.027 Explanatory factor Hazard ratio (95% CI) P value Table 3b. Multivariate analysis of OS Table 3b. Multivariate analysis of OS Explanatory factor Hazard ratio (95% CI) P value GTR or STR 1.6 (0.44-5.6) 0.48 Use of BEV 0.91 (0.28-3.0) 0.88 Both use of TMZ and RT 0.70 (0.22-2.2) 0.55 Ins/Cho ratio 0.33 (0.10-1.1) 0.067 Tables 3a and 3b show the results of multivariate analysis: we chose GTR or STR, BEV, TMZ and RT, and the Ins/Cho ratio as explanatory factors and analyzed their relevance to PFS and OS. Abbreviations: BEV, bevacizumab; GTR, Gross total resection; Ins/Cho, ratio of myoinositol to total choline; OS, overall survival; PFS, progression free survival; RT, radiotherapy; STR, subtotal resection; TMZ, temozolomide. Table 3b. Multivariate analysis of OS Tables Tables 3a and 3b show the results of multivariate analysis: we chose GTR or STR, BEV, TMZ and RT, and the Ins/Cho ratio as explanatory factors and analyzed their relevance to PFS and OS. Abbreviations: BEV, bevacizumab; GTR, Gross total resection; Ins/Cho, ratio of myoinositol to total choline; OS, overall survival; PFS, progression free survival; RT, radiotherapy; STR, subtotal resection; TMZ, temozolomide. Table 4. Comparison of copy number aberrations by Ins/Cho ratio Ins/Cho ratio ≥0.7 Ins/Cho ratio <0.7 P value Ins/Cho ratio ≥0.7 Ins/Cho ratio <0.7 P value Ins/Cho ratio ≥0.7 (n = 9) Ins/Cho ratio <0.7 (n = 11) P value +7p11.2 7 (78%) 6 (55%) 0.37 −9p21.3 4 (44%) 3 (27%) 0.64 −10q23.3 4 (44%) 3 (27%) 0.64 Copy number aberrations identified by next generation sequencing are compared by the Ins/Cho ratio. We defined an effective change of gain or Copy number aberrations identified by next generation sequencing are compared by the Ins/Cho ratio. We defined an effective change of gain or Page 17/18 Page 17/18 Page 17/18 Figures Figure 1 Comparison of PFS (a) and OS (b) between the group with a high Ins/Cho ratio (n = 13) and that with a low ratio (n = 17). Analysis was by the Kaplan–Meier method, using the log-rank test. Abbreviations: Ins/Cho, ratio of myoinositol to total Choline; OS, overall survival; PFS, progression free survival. Figure 1 Comparison of PFS (a) and OS (b) between the gro low ratio (n = 17). Analysis was by the Kaplan–Me Figure 1 Comparison of PFS (a) and OS (b) between the group with a high Ins/Cho ratio (n = 13) and that with a low ratio (n = 17). Analysis was by the Kaplan–Meier method, using the log-rank test. Abbreviations: Ins/Cho, ratio of myoinositol to total Choline; OS, overall survival; PFS, progression free survival. Page 18/18 Page 18/18 Page 18/18
https://openalex.org/W3118879754	https://zenodo.org/records/2052797/files/article.pdf	English	null	The Ascent of Sap in Plants	Scientific American	1,908	public-domain	2,109	THE USE OF AFRICAN ELEPHANTS FOR TRANSPORT SE.RVICE. Variations in atmospheric pressure act in the same way. With a falling barometer the air in a expands and forces water into the vessel b above, and with a rising barometer the air in a contracts and "draws" water from the vessel b below, the valves opening and closing, as required, under the inftuence of the pr es flures in the various vessels. EXPERIME:I'TS have recently been made in the basin of the Congo, to train elephants for transport service, for the question of transport appears to be one of the most difficult with which the Congo administration has to deal. The African elephant, heretofore of value only for his ivory, may in future contribute in no small measure to a solution of the problem in regions difficult of access by other means. The experiments which have been carried out, up to the present, show that elephants can be used to advantage for porterage work in regions where the opening up of the country is most difficult, because of lack of transportation facil· ities. Contrary to the general belief that Central African elephants could not be tamed, and made to perform the same service as their Asiatic fellows in India, a bulIetin is stated by the Ainerican consul at Boma to have been issued by the Congo government, announcing the complete success of certain experi ments conducted at an "elephant farm" at Api, in the Uele district, in the northern seetion of the Congo State. Here a small herd of young elephants has been found in captivity for several years, and finally after much effort in training them satisfactory results have been obtained. The director of the elephant station, in an official report, says that these experiments dem on strate that the African elephants can live in captivity, and that by good treatment they can be induced to perform labor. Already the oldest members of the eIe· phant farm at Api execute the porterage and traction work of the station. They carry drivers on their backs, and pack saddles with loads. None of the animals is more than seven years old, and since the Indian ele phants are most efficient at the adult age, 15 years, it is believed that even better results may be looked for. BY DR. GUSTAV GLOCH.. NO.of No. persons Per persons. ill-Iodged. cent. Families of 3 persons ....... 527,000 72,000 or 13.6 Families of 4 or 5 persons .. 729,000 126,000 or 17.3 Families of 6 or 7 persons . . 282,000 93,000 or 32.9 Families of 8 to 15 persons . 126,000 50,000 01' 39.8 WHEN the ehern ist cannot determine the atomic structure of a new compound by analysis, he attempts to determine it by synthesis. Possibly the vessels, tubes, and valves of our ma chine may be represented by the intercellular spaces, tracheides, pore-canals and lenticels of the real plant. It is not to be supposed that sap is raised in one operation to the top of a California redwood tree, 450 feet in height, and the assumption that the sap is ele vated gradually is in accordance with the necessity of depositing nourishment and the known occurrence of respiration, as proved by the presence of pore canals, in all parts of the stern. But the strongest argument for the theory he re sketched is the fact that it explains the ascent of sap as a result of the generation of heat by the plant, and consequently makes the plant independent of passing weather changes. Whether true or false it at least deprives the ascent of sap of all mystery by showing how it can be produced by natural causes.ĪTranslated for the SCIENTIFIC AMERICAN SUPPLEMENT from Prome theus. The mechanism by which sap is moved in plants has not yet been revealed by anatomical study, but we may, perhaps, discover it by constructing an artificial plant capable of performing at least this single func tion with the aid of the natural forces which are at the disposal of the real plant. At least two such natural sources of energy exist: variations in the temperature, and variations in the pressure, of the atmosphere. This table shows that the proportion of the ill lodged increases rapidly with the size of the family, and that large families should be the first objects of benevolence. Dr. Bertillon observes that the lodging problem varies in different seetions of the city and that the rent paid by the ill-lodged in the heart of Paris would enable them to secure comfortable lodgings in the environs, which can now be quickly and cheaply reached by the Metropolitan underground railway. THE USE OF AFRICAN ELEPHANTS FOR TRANSPORT SE.RVICE. Mortality among the elephants newly captured has been great, and as yet it has not been possible to attempt to breed them in captivity, but experience is solving the problems of taming; the deaths are now few, and fresh recruits are constantly being added. With a beginning thus made the scope of the work at Api will doubt less be enlarged, and eventually it is expected elephant caravans will be established. Success in the under ta king means a great deal for the future of the coun try. Despite railways and steamship lines, the Congo will always be a country of forests and of savannas in tersected with swamps. European stock does not sur vive in the tropical heat, and native carriers can be employed only to a certain extent. The elenhant i8 This theory also explains the respiration of plants, which has hitherto been as puzzling as the ascent of sap. In animals, respiration is effected by the mus cular contraction and expansion of the ehest, and some equally powerful force is required for the circulation of air through the complex cellular structure of plants, in which simple "ventilation" is altogether inadequate. The apparatus described above works even better with gases than with liquids. Diagram of an artificial plant , in which sap i8 gradually elevated to any height by fluctuations in temperature. In our artificial plant we will, like Nature, make use of every force at our command. The device de scribed will not work in the roots, where change of temperature and access of air are almost prevented by the soil, but the osmotic pressure, which is known, in this connection, as root pressure, is quite able to raise the sap through the roots and to a considerable height above the ground. Our device is superftuous, also, at the top of the tree, where the partial vacuum produced by diffusion and evaporation suffices for the last stage of elevation of the sap. to obtain suitable lodgings in present conditions, and if the charity is restricted to large families it will cost less per capita, so that more good can be done with the same money. Dr. Bertillon's careful and minute researches prove that it is quite impossible to give relief to all persons who are now improperly lodged. BY DR. GUSTAV GLOCH.. But even in the outlying seetions there are more than 15,000 tenements, each of which is occupied by six or more persons. The first task, therefore, is to re lieve these 108,000 persons, and this can be done for about 100 million francs ($20,000,000). The sehe me of the artificial plant is shown in the illustration. It comprises an indefinite number of open vessels b, alternating in a vertical series, with closed vessels a. In the bottom of each closed vessel a is a sm all orifice m, which is covered by a valve opening upward and communicates with a tube c ex tending nearly to the bottom of the open vessel b be neath. The top of each closed vessel a is pierced by a tube d which extends downward nearly to the bottom of that vessel and terminates in a funnel-shaped ex pansion containing a valve n which opens upward. The tube d extends upward beyond the vessel b, above, and its curved end opens over the open mouth of that vessel. LODGING CONDITIONS IN PARIS AND OTHE.R FRENCH CITIES. In many other cities the conditions are similar to those of Paris, as is shown by the following table: DR. J. BERTILLON has been led by his study of 10dg ing conditions in cities to the conclusion that the cheap lodgings erected by the benevolent societies should be reserved for large families. There are three good reasons for this restriction. Large families are in greater distress than small ones, they are unable Percentage of Ill-Lodged Families (More than Two Percentage of Ill-Lodged Families (More than Two Persons Per Room.) Nnmber St. Dnll- Cher- in Farnily. Etienne. kerque. bourg. Reims. Lyoll. 3 24.5 19.4 25.1 8.6 23.6 4 20.3 15.2 17.4 5.8 11.3 6 69.7 53.1 49.9 42.0 21.8 6 69.1 53.3 48.8 41.3 5.7 7 73.9 67.2 57.8 59.8 35.6 Now let us fill the lowest of the open vessels b with water, and see what will happen. As the temperature falls, at night, the air in the closed vessel a next above, contracts, and "draws" water from the vessel b below, in which operation the valve m is open and the valve n is closed. As the temperature rises, in the morning, the air in a expands and forces water into the vessel b above, the valve m closing and the valve n opening automatically. This cycle of operations is repeated indefinitely. After all the vessels have been partly filled with water, they operate simultaneously, so that the water can be raised to any height. Hence a tree constructed on this plan would not be limited in height by any limit to the ascent of sap, but only by considerations of nutrition and resistance to the destroying force of the elements. c Diagram of an artificial plant , in which sap i8 gradually elevated to any height by fluctuations in temperature. From all this it appears evident that the cheap tene ments erected by philanthropie societles should be reserved, at least for the present, for families with three or more children. This rule has been adopted by the Society l'Abri, which owns two large model tenement houses in Paris.-Revue d'Hygiene. THE USE OF AFRICAN ELEPHANTS FOR TRANSPORT SE.RVICE. He makes a dis tinction between "insufficient" lodgings, wh ich have more than one occupant per room, and "overpopulated" 10dgings, with more than two occupants per room, and gives the following table for Paris, according to the census of 1901: The capacity of the apparatus, computed in accord· ance with the laws of expansion of gases, is too small for the proper nourishment of the plant, if it is oper ated solely by the daily alternation in temperature. But the flow of sap in plants shows several periods of ftuctuation in 24 hours, proving that there are other fore es at work. Plants breathe, oxidize part of their food and, consequently, generate heat. If we assurne that any vessel a in our apparatus contains, instead of water, root sap mixed with air and in process of oxidation, the heat genera ted in the vessel will force part of its contents into the vessel b above. After oxidation is completed the heated and expanded gases in the vessel a will cool and contract. Hence fresh sap will be drawn from the vessel b, beneath, and oxidation will recommence. The vessels a may there fore be regarded as simple heat engines, analogous to gasoline motors. When once started they con tinue to worlr independently of external changes in temperature, which serve only to start the machine after the winter rest or to keep it going slowly when fuel is lacking or oxidation is checked by cold. Overpopulated lodgings (more than 2 Overpopulated lodgings (more than 2 persons per room) ............... . Insufficient lodgings (from 1 to 2 per- sons per room) ................... . Sufficient lodgings (1 person per room) . Large lodgings (from 1 to 2 rooms per person) .......................... . Very large lodgings (more than 2 rooms per person) ..................... . 14.3 per cent 37.4 23.4 19.7 5.1 Hence one-seventh of the inhabitants of Paris live three or more in one room, five 01' more in two rooms, seven or more in three rooms, etc. To give "sufficient" lodgings to the 340,000 persons who are now living in this deplorable state of overcrowding and promiscuity would require the expenditure of 1,000 francs ($200) per capita, or 340 million francs ($68,000,000) in all. If the assistance is to be restricted to those who are most in need of it, the following table becomes of interest: © 1908 SCIENTIFIC AMRICAN, INC.
https://openalex.org/W2108550035	https://respiratory-research.biomedcentral.com/counter/pdf/10.1186/1465-9921-12-74	English	null	Expansion of CD4+CD25+helper T cells without regulatory function in smoking and COPD	Respiratory research	2,011	cc-by	6,144	RESEARCH Open Access © 2011 Roos-Engstrand 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. Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Abstract Background: Regulatory T cells have been implicated in the pathogenesis of COPD by the increased expression of CD25 on helper T cells along with enhanced intracellular expression of FoxP3 and low/absent CD127 expression on the cell surface. Method: Regulatory T cells were investigated in BALF from nine COPD subjects and compared to fourteen smokers with normal lung function and nine never-smokers. Results: In smokers with normal lung function, the expression of CD25+CD4+ was increased, whereas the proportions of FoxP3+ and CD127+ were unchanged compared to never-smokers. Among CD4+ cells expressing high levels of CD25, the proportion of FoxP3+ cells was decreased and the percentage of CD127+ was increased in smokers with normal lung function. CD4+CD25+ cells with low/absent CD127 expression were increased in smokers with normal lung function, but not in COPD, when compared to never smokers. Conclusion: The reduction of FoxP3 expression in BALF from smokers with normal lung function indicates that the increase in CD25 expression is not associated with the expansion of regulatory T cells. Instead, the high CD127 and low FoxP3 expressions implicate a predominantly non-regulatory CD25+ helper T-cell population in smokers and stable COPD. Therefore, we suggest a smoking-induced expansion of predominantly activated airway helper T cells that seem to persist after COPD development. Keywords: Bronchoalveolar lavage, BAL, CD25bright, CD127, FoxP3, lymphocyte subsets and CD8+ cells have been shown to be more activated in both smokers and in subjects with COPD [6]. CD25 is a constitutively expressed activation marker and CD4+ cells with “bright” or “high” expression of CD25+ have been suggested to be regulatory T cells, previously defined as suppressor T cells [7]. Their function is to suppress immune responses by the secretion of soluble inhibitory mediators, such as interleukin 10, or through direct cell-to-cell contact. The role of regulatory T cells in COPD is not well-known, but Smyth et al have reported that long-term cigarette smoking increases airway regulatory T cell numbers, in terms of CD4CD25bright cells [8]. In contrast, two other studies reported decreased levels of regulatory T-cells in subjects with emphysema and COPD compared to healthy con- trols [9,10]. Expansion of CD4+CD25+ helper T cells without regulatory function in smoking and COPD Ester Roos-Engstrand1, Jamshid Pourazar1, Annelie F Behndig1, Anders Bucht1,2 and Anders Blomberg1 Correspondence: ester.roos-engstrand@lung.umu.se 1Dept. of Public Health and Clinical Medicine, Division of Medicine, Umeå University, Sweden Full list of author information is available at the end of the article Methods Spirometry Dynamic spirometry (FVC and FEV1) was performed post-bronchodilatation using a Vitalograph spirometer (Vitalograph Ltd., Buckingham, UK), as outlined pre- viously [6]. Dynamic spirometry (FVC and FEV1) was performed post-bronchodilatation using a Vitalograph spirometer (Vitalograph Ltd., Buckingham, UK), as outlined pre- viously [6]. Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Page 2 of 8 Page 2 of 8 T cell marker. A mutation in the FoxP3 gene can cause immune dysfunction polyendocrinopathy enteropathy X-linked syndrome, IPEX, but also other autoimmune conditions such as diabetes, thyreoditis and inflammatory bowel diseases [12]. Recently, absent or low expression of the IL-7a receptor (CD127dim) has been reported as another unique marker for regulatory T cells [13]. As CD127 is an extracellular marker, it is more easily ana- lysed compared to FoxP3. Studies have shown that CD127 is down-regulated on all human T cells after acti- vation [14]. In a recent study, we have shown that airway T cells are highly activated in COPD as indicated by increased expression of CD69 and HLA-DR [6]. In addi- tion, CD4+ cells express high levels of CD25 in COPD and smokers, suggesting the presence of regulatory T-cells [6]. It is of importance to verify and evaluate reg- ulatory T cells in COPD in more detail, as these cells may play a role in the pathogenesis of COPD, as sug- gested by Barceló [10]. The aim of this study was there- fore to identify airway regulatory T cells in smokers and individuals with COPD, using flow cytometric analysis of CD127 and FoxP3 and their relation to CD25 expression. The COPD patients did not receiv any treatment with inhaled corticosteroids or oral anti-inflammatory drugs during at least four weeks prior to study start and neither regular long-acting b2-agonists nor long-acting anti-cholinergic drugs were allowed within two weeks prior to bronchoscopy. Short-acting b2-agonists and/or anti-cholinergic drugs were used on demand. All sub- jects were non-atopic and free from symptomatic airway infection within a six week-period prior to the study. None had a history of chronic bronchitis or frequent infectious exacerbations. All COPD patients had a post bronchodilator FEV1/FVC of less than 70% and were not reversible. Informed consent was obtained from all volunteers after verbal and written information and the study was approved by the local Ethics Review Board at Umeå University, Sweden, and performed according to the declaration of Helsinki. Introduction Chronic obstructive pulmonary disease (COPD) is char- acterized by progressive airway obstruction and airway inflammation. Tobacco smoking is the main risk factor for COPD. Smoking causes an inflammatory response in all smokers but only 50 percent develop COPD [1]. Increased numbers of neutrophils, macrophages and T lymphocytes have been found in the lungs of COPD patients [2,3]. A relationship has been shown between the number of cytotoxic CD8+ T-cells and a decline in lung function in patients with COPD [4,5] suggesting a role for these cells in the pathogenesis of COPD. The bal- ance between CD4+ helper T cells and CD8+ cytotoxic T-cells is altered in the lungs of COPD patients, which results in a decline in the CD4/CD8 ratio [4]. Both CD4+ However, CD25 bright is not a definite marker of regula- tory T cells [11]. Transcription factor fork head box P3, FoxP3, is considered a unique intra-nuclear regulatory * Correspondence: ester.roos-engstrand@lung.umu.se 1Dept. of Public Health and Clinical Medicine, Division of Medicine, Umeå University, Sweden Full list of author information is available at the end of the article Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Bronchoscopy B f b h Nine patients with COPD (four ex-smokers and five smokers), fourteen smokers with normal lung function (defined as smokers with normal dynamic spirometry, i.e. FEV1 and FVC values within 80-120% of predicted value) and nine healthy never-smokers were recruited, (table 1). All COPD subjects and smokers with normal lung function had a smoking history of at least ten pack-years. Current smokers were not allowed to smoke for at least 12 hours prior to bronchoscopy. The sub- jects were not allowed to have any other medical condi- tion apart from COPD. Before bronchoscopy atropine was given subcutaneously. Topical anaesthesia of the airways was obtained with lidocaine. All subjects were examined in the supine posi- tion using an Olympus BF IT160 video bronchoscope (Olympus, Tokyo, Japan). Bronchoalveolar lavage (BAL) was performed by infusing three aliquots of 60 ml of sterile sodium chloride (NaCl), pH 7.3 at 37°C that were gently sucked back after each infusion and pooled into a container placed in iced water. The recovered fluid was immediately transported to the laboratory for analysis. Table 1 Demographics and spirometry values Never-smokers n = 9 Smokers n = 14 COPD Ex-smokers n = 4 COPD Smokers n = 5 Male:Female 5:4 7:7 4:0 0:5 Age 65 ± 5.2 60 ± 6.6 67 ± 2.1 61 ± 2.4 Smoking (pack years) 0 (0-0) 30 (20-44) 50 (44-52) 46 (35-65) COPD stage (GOLD)+ NA NA 2 and 3 2 and 3 FEV1/FVC % Pre bronchodilatation 77 (74-83) 78 (75-82) 62 (60-67) 60 (56-67) FEV1/FVC % Post bronchodilatation NA 78 (77-81) 64 (62-67) 60 (59-66) FEV1 % of predicted Pre bronchodilatation 101 (89-110) 112 (104-120) 53 (40-64) 53 (47-66) FEV1 % of predicted Post bronchodilatation NA 114 (108-119) 63 (52-67) 60 (55-68) Data are shown as mean ± standard deviation for age, median and inter quartile range for all others. FEV1: Forced expiratory volume in one second; FVC: Forced vital capacity. NA: not applicable. + http://www.goldcopd.org. In the ex-smoking COPD patients, the smoking cessation was more than five years prior to study inclusion. Table 1 Demographics and spirometry values Table 1 Demographics and spirometry values Flow cytometry analysis were washed and centrifuged at 4°C for 10 minutes, 300 g. This washing procedure was performed twice. 10 μl of antibody solution was added to the cell suspension and allowed to bind for 30 minutes at 4°C in darkness. The cells were washed twice by adding permeabilisation buffer to the tubes and centrifuged at 4°C for 10 minutes, 300 g and 300 μl of PBS was added. To identify CD3+, CD4+ and CD25+ cells, the cells were stained with same antibodies as in the extracellular staining. To obtain FoxP3+ cells, phy- coerytrin (PE) conjugated anti-human FoxP3 was used in the same test tube. The percentage FoxP3 was determined out of gated CD3+ and CD4+ lymphocytes. BAL -lymphocyte subsets were determined using flow cytometry. BAL cells were centrifuged and diluted to a final concentration of 106 cells/ml. For each test, 10 μl of antibody solution was added to 200 μl of cell suspension and allowed to bind for 30 minutes at 4°C in darkness. Red blood cells were lysed with 2 ml FACS™Lysing solu- tion (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) for 10 minutes at room temperature. The remaining cells were then washed by adding PBS to the tubes and centrifuged at 4°C for 10 minutes, 300 g and repeated once. Cells were thereafter fixed with 500 μl Cell- FIX™(Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) before analysis using a FACSCalibur™ (Becton Dickinson) flow cytometer. The lymphocyte population was gated on their physical characteristics in a region according to their characteristic forward scatter (FCS) and side scatter (SSC) profiles, as previously reported [6]. 3,000 total events were collected in CD3+ gate per sample. Table 1 Demographics and spirometry values Data are shown as mean ± standard deviation for age, median and inter quartile range for all others. FEV1: Forced expiratory volume in one second; FVC: Forced vital capacity. NA: not applicable. + http://www.goldcopd.org. In the ex-smoking COPD patients, the smoking cessation was more than five years prior to study inclusion. Data are shown as mean ± standard deviation for age, median and inter quartile range for all others. FEV1: Forced expiratory volume in one second; FVC: Forced vital capacity. NA: not applicable. + http://www.goldcopd.org. In the ex-smoking COPD patients, the smoking cessation was more than five years prior to study inclusion. Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Page 3 of 8 Statistical analysis y Flow cytometry data were acquired and analysed using CellQuest Software (Becton Dickinson). Differences between three groups were tested using Kruskal-Wallis test and a p-value of less than 0.05 was considered signif- icant. If the Kruskal-Wallis test indicated significance, the Mann-Whitney U-test was used for post-hoc analysis for comparison between two groups, with corrections of p-values according to Bonferroni (a p-value less than 0.017 was considered significant). Whilst the number of COPD patients was small, the ex-smoking COPD group was compared to the smoking COPD group, using Mann-Whitney U-test. Here, a p-value of less than 0.05 was considered significant. To identify CD3+, CD4+, CD25+ and CD127+ cells, the cells were stained with Allophycocyanin (APC) conjugated anti-human CD3, fluorescein isothiocyanate (FITC) conju- gated anti-human CD4, phycoerytrin-Cy5 (PE Cy5) conju- gated anti-human CD25 and phycoerytrin (PE) conjugated anti-human CD127 in the same test tube (Becton Dickin- son, San Jose, CA, USA). The percentage of different cell types was counted out of gated CD3+ lymphocytes and furthermore out of gated CD4+. CD25bright cells were quantified as previously described [8,15]. Analyses of CD127-&dim are performed as shown in Figure 1. Results In smokers who maintain normal lung function, it has been implied that the upregulation of regulatory T cells would restrain cigarette smoke-induced inflammatory activation and, thus, the development of COPD [10]. In contrast, in smokers who develop COPD, the T regulatory response is supposed to be inappropriate, which enables an uncontrolled progress of the immunor- eaction, involving the activation of T cells into a cytotoxic phenotype. This further supports a potential involvement of the acquired immune response in the pathogenesis of COPD. To further evaluate the role of regulatory T cells in COPD and to clarify whether CD4+CD25bright cells really have regulatory properties, more specific biomar- kers are needed. The transcription factor FoxP3 is known to be highly expressed in regulatory T cells, whereas the cell surface marker CD127 is supposed to be low or absent on regulatory T cells [16,17]. Investiga- tions of these markers in COPD are rare and, to our knowledge, this is the first study addressing CD127 expression on BAL cells from smokers and subjects with COPD. Results The BAL recovery in subjects with COPD was (37%; 29- 52), (median; inter quartile range) in smokers with normal lung function (53%; 49-61) and in never-smokers (50%; 34-64). Intracellular staining of FoxP3 was conducted according to the recommended procedure obtained from eBioscience (San Diego, CA, USA). Cells were permeabilised with eBioscience FoxP3 Staining Buffer Set at 4°C for 30 min- utes. By adding permeabilisation buffer to the tubes, cells Smokers with normal lung function had increased total leukocyte numbers in BAL compared to never- smokers. Among leukocytes, the macrophage numbers Figure 1 Flow cytometry analysis of CD127 expression on BAL CD4+ T cells. Firstly, lymphocytes were gated in FSC and SSC. Secondly, CD4 + cells were gated in the histogram. CD25 and CD127 expression on the gated CD4 cells were analyzed in a dot plot. The grey area indicates CD25+CD127dim population. Figure 1 Flow cytometry analysis of CD127 expression on BAL CD4+ T cells. Firstly, lymphocytes were gated in FSC and SSC. Secondly, CD4 + cells were gated in the histogram. CD25 and CD127 expression on the gated CD4 cells were analyzed in a dot plot. The grey area indicates CD25+CD127dim population. Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Page 4 of 8 lung function compared to never-smokers, suggesting the presence of regulatory T cells [6,8]. In contrast, Barceló et al reported decreased percentages of CD4+CD25+ in patients with COPD compared to smokers with normal lung function [10] and Lee et al reported similar findings in patients with emphysema [9]. Recently, we found a decreased proportion of CD4+CD25bright cells in ex-smok- ing subjects with COPD compared to smoking COPD subjects [6]. However, despite more than five years after smoking cessation, the proportion of these cells was not normalized, suggesting a smoke-induced upregulation of CD4+CD25bright cells. Smoking habits in the other two stu- dies [8,10] were not clearly defined, which makes a full comparison between the studies difficult. CD4+CD25bright cells have been suggested to have regulatory features as key immunomodulators. In smokers who maintain normal lung function, it has been implied that the upregulation of regulatory T cells would restrain cigarette smoke-induced inflammatory activation and, thus, the development of COPD [10]. In contrast, in smokers who develop COPD, the T regulatory response is supposed to be inappropriate, which enables an uncontrolled progress of the immunor- eaction, involving the activation of T cells into a cytotoxic phenotype. Results This further supports a potential involvement of the acquired immune response in the pathogenesis of COPD. lung function compared to never-smokers, suggesting the presence of regulatory T cells [6,8]. In contrast, Barceló et al reported decreased percentages of CD4+CD25+ in patients with COPD compared to smokers with normal lung function [10] and Lee et al reported similar findings in patients with emphysema [9]. Recently, we found a decreased proportion of CD4+CD25bright cells in ex-smok- ing subjects with COPD compared to smoking COPD subjects [6]. However, despite more than five years after smoking cessation, the proportion of these cells was not normalized, suggesting a smoke-induced upregulation of CD4+CD25bright cells. Smoking habits in the other two stu- dies [8,10] were not clearly defined, which makes a full comparison between the studies difficult. CD4+CD25bright were increased. The number of macrophages was also increased in smokers with normal lung function, com- pared with COPD patients (table 2). To examine whether the difference in airway inflamma- tion between COPD patients and smokers with normal lung function was due to smoking habits, the group of COPD patients was divided into current smokers and ex- smokers. This subgroup analysis showed that smoking COPD patients had increased BAL macrophage numbers compared to ex-smokers (table 2). The median fluorescence intensity, MFI, were enhanced in smokers with normal lung function and in COPD, compared to never-smokers (Figure 2). The per- centages of CD4+CD25+ (data not shown) and CD4 +CD25bright (Figure 2) cells were enhanced in smokers with normal lung function, compared to never-smokers while the percentage of CD4+FoxP3+ and CD4+CD127+ cells was unchanged (Figure 3a and 3b). There were no significant difference in CD4+CD25+ cells between COPD patients and the other two groups. Among CD4+ T cells expressing CD25, smokers with normal lung func- tion had significantly decreased percentage of FoxP3 compared to never-smokers. CD127 expression on CD4+ T cells expressing CD25 was enhanced in subjects with COPD and smokers with normal lung function, com- pared to never-smokers (Figure 3). Ex-smoking COPD patients expressed decreased percentage of CD127+ cells in BALF compared to smoking COPD patients (Figure 3d). The expression of CD127-&dim on CD4+CD25+ T cells was increased in smokers with normal lung func- tion, compared to non-smokers (Figure 4). p cells have been suggested to have regulatory features as key immunomodulators. Discussion It has been suggested that regulatory T cells are important in the pathogenesis of COPD [8,10]. Recently published data have shown increased CD4+CD25bright cells in the air- ways of subjects with COPD and smokers with normal Table 2 Differential cell count of leukocytes in BAL fluid, given in number cells/ml104 Never smokers (NS) n = 9 Smokers (S) n = 14 COPD n = 9 p COPD ex-smokers n = 4 COPD smokers n = 5 p Total leukocytes 21 (13-26) 41 (34-52) 25 (18-37) P < 0.001 NS vs S 20 (10-26) 29 (22-52) NS Macrophages 19 (12-23) 37 (31-48) 22 (17-33) P < 0.001 NS vs S P < 0.014 S vs COPD 19 (9.6-22) 27 (20-42) P < 0.05 COPD ex-s vs COPD s Neutrophils 0.2 (0.045-0.31) 0.49 (0.23-1.1) 0.19 (0.06-0.93) NS 0.36 (0.04-1.07) 0.19 (0.07-5.2) NS Lymphocytes 1.7 (1.1-2.2) 2.3 (1.5-3.9) 1.5 (0.90-2.8) NS 1.5(0.8-2.2) 1.5 (0.7-4.1) NS Eosinophils 0.08 (0-0.54) 0.05 (0-0.3) 0.06 (0.01-0.66) NS 0.015 (0.00- 0.52) 0.47 (0.05- 0.96) NS Mast cells 0.06 (0.005-0.12) 0.06 (0.03- 0.10) 0.03 (0.005- 0.065) NS 0.005 (0.00- 0.04) 0.04 (0.02- 0.10) NS Data are given as median and IQR. Table 2 Differential cell count of leukocytes in BAL fluid, given in number cells/ml104 Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Page 5 of 8 Figure 2 Flow cytometry analyses of BAL T cells of never-smokers (NS), smokers with normal lung function (S) and COPD. CD4 +CD25bright are given as percent of gated CD3 (a). CD25+ cells out of CD4+ cells are given as median fluorescence intensity, MFI (b). Significance levels are noted as p < 0.01, * p < 0.001. Data are given as median and IQR. Figure 2 Flow cytometry analyses of BAL T cells of never-smokers (NS), smokers with normal lung function (S) and COPD. CD4 +CD25bright are given as percent of gated CD3 (a). CD25+ cells out of CD4+ cells are given as median fluorescence intensity, MFI (b). Significance levels are noted as p < 0.01, * p < 0.001. Data are given as median and IQR. Figure 3 Flow cytometry analyses of BAL T cells from never-smokers (NS), smokers with normal lung function (S) and COPD subjects. The proportion of CD4+ T cells expressing FoxP3 (a) or CD127 (b) are given as percent of total T cells (CD3+). Discussion Percentage of FoxP3+ (c) or CD127+ (d) among CD4+ T cells expressing CD25. The CD127+ population includes the CD127dim cells. Within the COPD group, ●indicates ex- smokers and Δ smokers. Significance levels are noted as p < 0.01 and * p < 0.001. COPD smokers have increased proportions CD127/CD25 among CD4+ cells compared to COPD ex-smokers (p = 0.027) and to never-smokers (p = 0.003). Data are given as median and IQR. Figure 3 Flow cytometry analyses of BAL T cells from never-smokers (NS), smokers with normal lung function (S) and COPD subjects. The proportion of CD4+ T cells expressing FoxP3 (a) or CD127 (b) are given as percent of total T cells (CD3+). Percentage of FoxP3+ (c) or CD127+ (d) among CD4+ T cells expressing CD25. The CD127+ population includes the CD127dim cells. Within the COPD group, ●indicates ex- smokers and Δ smokers. Significance levels are noted as p < 0.01 and * p < 0.001. COPD smokers have increased proportions CD127/CD25 among CD4+ cells compared to COPD ex-smokers (p = 0.027) and to never-smokers (p = 0.003). Data are given as median and IQR. Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Page 6 of 8 Figure 4 Flow cytometry analysis of CD127 expression on BAL T cells from never-smokers (NS), smokers with normal lung function (S) and COPD. The combined CD127- and CD127dim populations are given as percent of gated CD25+CD4+ cells. Among the COPD group, ●indicates ex-smokers and Δ smokers. Significance levels are noted as p < 0.01 Data are given as median and IQR. ex-smokers, we observed that smokers had increased proportions of CD127 on CD4+CD25+ cells compared to ex-smokers (Figure 3d). The group of ex-smoking COPD subjects is small, yet the present data imply that tobacco smoking may induce an activation of airway CD4+ cells, in terms of increased CD127 expression and that the CD127 expression appears to decline after smoking cessation. Despite more than five years since smoking cessation, the expression of CD127 among the CD25 helper T cells tended to be higher in COPD patients compared to never-smokers, indicating a pro- longed immune activation. No differences were found between the groups in helper T cells expressing FoxP3+ (Figure 3a). Among CD25 expressing helper T cells, the percentage of FoxP3+ was decreased in smokers compared to never-smokers. Discussion The data suggest that a large proportion of CD4+CD25+ cells in smokers do not express FoxP3 and, thus, have not a regulatory T cell function. Compared to smokers and non-smokers, a decrease in the expression of FoxP3 has been found in the smaller airways in COPD, whereas FoxP3 expression was increased in large airways in both smokers and subjects with COPD [20]. Another study reported increased regulatory T cell numbers in lymphoid follicles and bronchial tissue in subjects with moderate COPD [21]. Within the lung tissue, regulatory T cells expressing FoxP3 seem to be more abundant in larger air- ways compared to smaller airways. However, the role for FoxP3 in regulating the immune defence in different regions of the lungs in smoking and COPD needs to be further elucidated. Figure 4 Flow cytometry analysis of CD127 expression on BAL T cells from never-smokers (NS), smokers with normal lung function (S) and COPD. The combined CD127- and CD127dim populations are given as percent of gated CD25+CD4+ cells. Among the COPD group, ●indicates ex-smokers and Δ smokers. Significance levels are noted as p < 0.01 Data are given as median and IQR. CD127 expressing cells have been studied in allergic asthma, gastric cancer and glioma [13,16,18]. Expression of CD25 and CD127 on CD4+ cells has been suggested to discriminate between regulatory and activated T cells [17]. FoxP3 is strongly expressed in CD25bright cells, whilst CD127 is down-regulated on these cells. CD127 expression is shown to be inversely associated with FoxP3 and suppressive function of human CD4+ regula- tory T cells in peripheral blood [14]. In the present study of BAL T cells, a similar pattern was found supporting an inverse association between FoxP3 and CD127 expres- sion also on BAL T cells (Figure 3c, d). CD25+CD127dim cells are suggested to have immunore- gulatory properties, whilst CD25+CD127bright have not [17]. Here, the proportion of CD4+CD25+ with low or absent expression of CD127 was increased in smokers with normal lung function compared to non-smokers. However, from our data (Figure 4), it appears that the smokers might be divided into two subpopulations, one with increased CD127-&dim on CD4+CD25+ cells and one subpopulation with unchanged CD127-&dim expression, suggesting an increased presence of regulatory T cells in some “healthy” smokers. References 1. Lundback B, Lindberg A, Lindstrom M, Ronmark E, Jonsson AC, Jonsson E, Larsson LG, Andersson S, Sandstrom T, Larsson K: Not 15 but 50% of smokers develop COPD?–Report from the Obstructive Lung Disease in Northern Sweden Studies. Respiratory medicine 2003, 97(2):115-122. 1. Lundback B, Lindberg A, Lindstrom M, Ronmark E, Jonsson AC, Jonsson E, Larsson LG, Andersson S, Sandstrom T, Larsson K: Not 15 but 50% of smokers develop COPD?–Report from the Obstructive Lung Disease in Northern Sweden Studies. Respiratory medicine 2003, 97(2):115-122. The differential cell count confirms previously pub- lished data [6]. Macrophages were increased in smokers with normal lung function and in smoking patients with COPD. This is not surprising as macrophages play a key role in the inflammatory response to noxious particles and gases, such as tobacco smoke exposure. The lack of increase in neutrophils in the COPD subjects further implies that these subjects were without any history of bronchitis or frequent infectious exacerbations. There was no difference in lymphocyte numbers between the three groups; the difference was within the lymphocyte population, mainly related to the T lymphocyte subtypes. p y 2. Di Stefano A, Caramori G, Ricciardolo FL, Capelli A, Adcock IM, Donner CF: Cellular and molecular mechanisms in chronic obstructive pulmonary disease: an overview. Clin Exp Allergy 2004, 34(8):1156-1167. 3. Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, Cherniack RM, Rogers RM, Sciurba FC, Coxson HO, et al: The nature of small-airway obstruction in chronic obstructive pulmonary disease. The New England journal of medicine 2004, 350(26):2645-2653. 4. O’Shaughnessy TC, Ansari TW, Barnes NC, Jeffery PK: Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1. American journal of respiratory and critical care medicine 1997, 155(3):852-857. p y 5. Saetta M, Di Stefano A, Turato G, Facchini FM, Corbino L, Mapp CE, Maestrelli P, Ciaccia A, Fabbri LM: CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. American journal of respiratory and critical care medicine 1998, 157(3 Pt 1):822-826. In conclusion, we demonstrate that smoking subjects with COPD have increased proportions of CD127+ helper T cells in the airways. Smoking cessation may reduce the proportion of these cells but this has to be confirmed in longitudinal studies. Discussion Based on the present data, we hypothesise that, within the group of smokers with normal lung function, there may be subjects with insufficient expansion of regulatory T cells, who will be at risk for developing COPD [1]. If this was the case, it would be pos- sible to distinguish between smoking subjects with differ- ent susceptibility to develop COPD. This issue needs to be addressed in future prospective studies. It cannot be excluded that T-lymphocytes isolated from peripheral blood or other lung compartments, such as bronchial mucosa or peripheral lung tissue, may show different phe- notypic characteristics compared with BAL-cells. It has CD25 is of importance in mediating immune tolerance and protection from autoimmune disease [19]. As indi- cated above, CD25bright expression on CD4+ cells is usually implied as regulatory T cells. The present study shows that an increased percentage of of CD4+ CD25bright cells is associated to current smoking (Figure 2a) and that increased cell surface expression of CD25, expressed as median fluorescence intensity, is associated to both cur- rent smoking and COPD (Figure 2b). Even though the number of patients in the present study is rather small, the data are consistent with previously published results [6]. When it comes to the proportion of CD127+ helper T cells among CD3+ cells in BAL fluid, there was no dif- ference between the three groups (Figure 3b). However, in subjects with COPD and smokers with normal lung function, the expression of CD127+/CD4+CD25+ cells was increased compared to never-smokers (Figure 3d). When COPD subjects were divided into smokers and Page 7 of 8 Page 7 of 8 Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 been suggested that lung lymphoid tissue contains more T regulatory cells in COPD compared to smokers and healthy subjects [21]. bronchoscopies and manuscript preparation. ABu contributed with scientific expertise and manuscript preparation. ABl was responsible for study design, subject recruitment, bronchoscopies and manuscript preparation. All authors read and approved the final manuscript. The COPD subjects included in this study were clini- cally stable, i.e. with no history of recurrent infectious exacerbations and in no need of regular medications, apart from short acting bronchodilators on demand. Author details 1 15. Crispin JC, Martinez A, Alcocer-Varela J: Quantification of regulatory T cells in patients with systemic lupus erythematosus. Journal of autoimmunity 2003, 21(3):273-276. 1Dept. of Public Health and Clinical Medicine, Division of Medicine, Umeå University, Sweden. 2Swedish Defence Research Agency, Division of CBRN Defence and Security, Umeå, Sweden. 16. Ardon H, Verbinnen B, Maes W, Beez T, Van Gool S, De Vleeschouwer S: Technical advancement in regulatory T cell isolation and characterization using CD127 expression in patients with malignant glioma treated with autologous dendritic cell vaccination. Journal of immunological methods 2009. Discussion Also, the ex-smoking COPD subjects stopped smoking more than five years prior to study inclusion, whereas all smokers with normal lung function were current smo- kers, with at least a smoking history of ten pack years. Competing interests Th h d l h The authors declare that they have no competing interests. Received: 22 December 2010 Accepted: 8 June 2011 Published: 8 June 2011 Received: 22 December 2010 Accepted: 8 June 2011 Published: 8 June 2011 Acknowledgements Thi t d This study was supported by Swedish Heart-Lung Foundation, the Swedish Heart and Lung Association, King Gustaf V’s and Queen Victoria’s foundation and Umeå University. 13. Shen LS, Wang J, Shen DF, Yuan XL, Dong P, Li MX, Xue J, Zhang FM, Ge HL, Xu D: CD4(+)CD25(+)CD127(low/-) regulatory T cells express Foxp3 and suppress effector T cell proliferation and contribute to gastric cancers progression. Clinical immunology (Orlando, Fla 2009, 131(1):109-118. Anders Blomberg is the holder of the Lars Werkö Distinguished Research Fellowship from the Swedish Heart-Lung Foundation. The authors would like to thank Ann-Britt Lundström, Elisabeth Åslund, Annika Johansson, Helena Tjällgren-Bogseth and Frida Holmström for their contribution to the project. 14. Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, Gottlieb PA, Kapranov P, Gingeras TR, Fazekas de St Groth B, et al: CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4 + T reg cells. The Journal of experimental medicine 2006, 203(7):1701-1711. References These data therefore indicate the expansion of a T cell population without a regulatory function, which may contribute to the persistent cyto- toxic T cells responses previously reported in COPD. However, a fraction of smokers without clinical signs of COPD had an increased population of helper T cells with low or absent CD127 expression, suggesting the presence of regulatory T cells that potentially can modulate the smoke-induced immune responses. Whether such a T cell population would play a role in the protection of COPD development in smokers remains to be elucidated. 6. Roos-Engstrand E, Ekstrand-Hammarstrom B, Pourazar J, Behndig AF, Bucht A, Blomberg A: Influence of smoking cessation on airway T 6. Roos-Engstrand E, Ekstrand-Hammarstrom B, Pourazar J, Behndig AF, Bucht A, Blomberg A: Influence of smoking cessation on airway T lymphocyte subsets in COPD. Copd 2009, 6(2):112-120. oos-Engstrand E, Ekstrand-Hammarstrom B, Pourazar J, Behndig AF, 7. Mills KH: Regulatory T cells: friend or foe in immunity to infection? Nat Rev Immunol 2004, 4(11):841-855. 8. Smyth LJ, Starkey C, Vestbo J, Singh D: CD4-regulatory cells in COPD patients. Chest 2007, 132(1):156-163. 9. Lee SH, Goswami S, Grudo A, Song LZ, Bandi V, Goodnight-White S, Green L, Hacken-Bitar J, Huh J, Bakaeen F, et al: Antielastin autoimmunity in tobacco smoking-induced emphysema. Nature medicine 2007, 13(5):567-569. 10. Barcelo B, Pons J, Ferrer JM, Sauleda J, Fuster A, Agusti AG: Phenotypic characterisation of T-lymphocytes in COPD: abnormal CD4+CD25+ regulatory T-lymphocyte response to tobacco smoking. Eur Respir J 2008, 31(3):555-562. 11. Wan YY: Regulatory T cells: immune suppression and beyond. Cellular & molecular immunology 7(3):204-210. 12. Gambineri E, Torgerson TR, Ochs HD: Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX), a syndrome of systemic autoimmunity caused by mutations of FOXP3, a critical regulator of T-cell homeostasis. Current opinion in rheumatology 2003, 15(4):430-435. Anders Blomberg is the holder of the Lars Werkö Distinguished Research Fellowship from the Swedish Heart-Lung Foundation. This study was supported by Swedish Heart-Lung Foundation, the Swedish Heart and Lung Association, King Gustaf V’s and Queen Victoria’s foundation and Umeå University. g This study was supported by Swedish Heart-Lung Foundation, the Swedish Heart and Lung Association, King Gustaf V’s and Queen Victoria’s foundation and Umeå University. Anders Blomberg is the holder of the Lars Werkö Distinguished Research Fellowship from the Swedish Heart-Lung Foundation. The authors would like to thank Ann-Britt Lundström, Elisabeth Åslund, Annika Johansson, Helena Tjällgren-Bogseth and Frida Holmström for their contribution to the project. Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 17. Seddiki N, Santner-Nanan B, Martinson J, Zaunders J, Sasson S, Landay A, Solomon M, Selby W, Alexander SI, Nanan R, et al: Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. The Journal of experimental medicine 2006, 203(7):1693-1700. 18. Nguyen KD, Fohner A, Booker JD, Dong C, Krensky AM, Nadeau KC: XCL1 enhances regulatory activities of CD4+ CD25(high) CD127(low/-) T cells in human allergic asthma. J Immunol 2008, 181(8):5386-5395. 19. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M: Immunologic self- tolerance maintained by activated T cells expressing IL-2 receptor alpha- chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995, 155(3):1151-1164. 20. Isajevs S, Taivans I, Strazda G, Kopeika U, Bukovskis M, Gordjusina V, Kratovska A: Decreased FOXP3 expression in small airways of smokers with COPD. Eur Respir J 2009, 33(1):61-67. 21. Plumb J, Smyth LJ, Adams HR, Vestbo J, Bentley A, Singh SD: Increased T- regulatory cells within lymphocyte follicles in moderate COPD. Eur Respir J 2009, 34(1):89-94. doi:10.1186/1465-9921-12-74 Cite this article as: Roos-Engstrand et al.: Expansion of CD4+CD25+ helper T cells without regulatory function in smoking and COPD. Respiratory Research 2011 12:74. 17. Seddiki N, Santner-Nanan B, Martinson J, Zaunders J, Sasson S, Landay A, Solomon M, Selby W, Alexander SI, Nanan R, et al: Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. The Journal of experimental medicine 2006, 203(7):1693-1700. 18. Nguyen KD, Fohner A, Booker JD, Dong C, Krensky AM, Nadeau KC: XCL1 enhances regulatory activities of CD4+ CD25(high) CD127(low/-) T cells in human allergic asthma. J Immunol 2008, 181(8):5386-5395. 18. Nguyen KD, Fohner A, Booker JD, Dong C, Krensky AM, Nadeau KC: XCL1 enhances regulatory activities of CD4+ CD25(high) CD127(low/-) T cells in human allergic asthma. J Immunol 2008, 181(8):5386-5395. 19. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M: Immunologic self- tolerance maintained by activated T cells expressing IL-2 receptor alpha- chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995, 155(3):1151-1164. 20. Isajevs S, Taivans I, Strazda G, Kopeika U, Bukovskis M, Gordjusina V, Kratovska A: Decreased FOXP3 expression in small airways of smokers with COPD. Eur Respir J 2009, 33(1):61-67. 21. Plumb J, Smyth LJ, Adams HR, Vestbo J, Bentley A, Singh SD: Increased T- regulatory cells within lymphocyte follicles in moderate COPD. Authors’ contributions ERE was responsible for preparation and analysis of BAL-samples, statistical analyses, evaluation of data and manuscript preparation. JP contributed with scientific know-how of FACS analyses. AFB took part in subject recruitment, Page 8 of 8 Page 8 of 8 Roos-Engstrand et al. Respiratory Research 2011, 12:74 http://respiratory-research.com/content/12/1/74 Eur Respir J 2009, 34(1):89-94. doi:10.1186/1465-9921-12-74 Cite this article as: Roos-Engstrand et al.: Expansion of CD4+CD25+ helper T cells without regulatory function in smoking and COPD. Respiratory Research 2011 12:74. 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
https://openalex.org/W3215514743	https://www.mdpi.com/2218-273X/11/12/1765/pdf?version=1637835252	English	null	Natural Bioactive Cinnamoyltyramine Alkylamides and Co-Metabolites	Biomolecules	2,021	cc-by	26,078	Review Natural Bioactive Cinnamoyltyramine Alkylamides and Co-Metabolites Natural Bioactive Cinnamoyltyramine Alkylamides Co-Metabolites Antonio Evidente * and Marco Masi Antonio Evidente * and Marco Masi De a t e t of C Antonio Evidente * and Marco Masi Department of C Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte Sant’Angelo, Via Cintia 4, 80126 Naples, Italy; marco.masi@unina.it * Correspondence: evidente@unina.it; Tel.: +39-081-2539178 Sant Angelo, Via Cintia 4, 80126 Naples, Italy; marco.masi@unina.it * Correspondence: evidente@unina.it; Tel.: +39-081-2539178 Abstract: Natural products are a vital source for agriculture, medicine, cosmetic and other fields. Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte Sant’Angelo, Via Cintia 4, 80126 Naples, Italy; marco.masi@unina.it * Correspondence: evidente@unina.it; Tel.: +39-081-2539178 Sant Angelo, Via Cintia 4, 80126 Naples, Italy; marco.masi@unina.it * Correspondence: evidente@unina.it; Tel.: +39-081-2539178 Abstract: Natural products are a vital source for agriculture, medicine, cosmetic and other fields. Abstract: Natural products are a vital source for agriculture, medicine, cosmetic and other ﬁelds. Among them alkylamides are a broad and expanding group found in at least 33 plant families. Frequently, they possess a simple carbon skeleton architecture but show broad structural variability and important properties such as immunomodulatory, antimicrobial, antiviral, larvicidal, insecticidal and antioxidant properties, amongst others. Despite to these several and promising biological activities, up to today, only two reviews have been published on natural alkylamides. One focuses on their potential pharmacology application and their distribution in the plant kingdom and the other one on the bioactive alkylamides speciﬁcally found in Annona spp. The present review is focused on the plant bioactive cinnamoyltyramine alkylamides, which are subject of several works reported in the literature. Furthermore, the co-metabolites isolated from the same natural sources and their biological activities are also reported. Among them alkylamides are a broad and expanding group found in at least 33 plant families. Fre- quently, they possess a simple carbon skeleton architecture but show broad structural variability and important properties such as immunomodulatory, antimicrobial, antiviral, larvicidal, insecti- cidal and antioxidant properties, amongst others. Despite to these several and promising biological activities, up to today, only two reviews have been published on natural alkylamides. One focuses on their potential pharmacology application and their distribution in the plant kingdom and the other one on the bioactive alkylamides specifically found in Annona spp. The present review is fo- cused on the plant bioactive cinnamoyltyramine alkylamides, which are subject of several works reported in the literature. biomolecules biomolecules 1. Introduction 1. Introduction Alkyla ide Alkylamides are a broad and expanding group of bioactive natural compounds grouped at least in 33 plant families as Aristolochiaceae, Asteraceae, Brassicaceae, Convolvulaceae, Euphorbiaceae, Menispermaceae, Piperaceae, Poaceae, Rutaceae and Solanaceae [1]. Many of these species were used in folk medicine for the broad spectra of biological activities as immunomodulatory, antimicrobial, antiviral, larvicidal, insecticidal, diuretic, analgesic, cannabimimetic and antioxidant activities. They are also involved in the antibiotic’s potentiation, the prostaglandin biosynthesis inhibition, RNA synthesis and the arachidonic acid metabolism. Alkylamides possess a broad range of pharmacological effects [2] and thus their potential application in the pharmaceutical, cosmetic and food industries could be planned. Alkylamides are found in different organs of the plants such as roots, leaves, stems, fruits, ﬂowers, seeds and tubers. Alkylamides were also formulated as plant growth regulators, which affect the growth, roots development and inducing of plant biomass production [3]. Alkylamides are a broad and expanding group of bioactive natural compounds grouped at least in 33 plant families as Aristolochiaceae, Asteraceae, Brassicaceae, Con- volvulaceae, Euphorbiaceae, Menispermaceae, Piperaceae, Poaceae, Rutaceae and Sola- naceae [1]. Many of these species were used in folk medicine for the broad spectra of bio- logical activities as immunomodulatory, antimicrobial, antiviral, larvicidal, insecticidal, diuretic, analgesic, cannabimimetic and antioxidant activities. They are also involved in the antibiotic’s potentiation, the prostaglandin biosynthesis inhibition, RNA synthesis and the arachidonic acid metabolism. Alkylamides possess a broad range of pharmaco- logical effects [2] and thus their potential application in the pharmaceutical, cosmetic and food industries could be planned. Alkylamides are found in different organs of the plants such as roots, leaves, stems, fruits, flowers, seeds and tubers. Alkylamides were also for- mulated as plant growth regulators, which affect the growth, roots development and in- ducing of plant biomass production [3]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Natural alkylamides are constituted by an aliphatic, cyclic or aromatic amine residue (R1), and a C8 to C18 saturated or unsaturated chain acid, which can also be aromatic (R2). The structural formula representing all the alkylamides is reported in Figure 1. Natural alkylamides are constituted by an aliphatic, cyclic or aromatic amine residue (R1), and a C8 to C18 saturated or unsaturated chain acid, which can also be aromatic (R2). ion: Evidente Citation: Evidente, A.; Masi, M. Natural Bioactive Cinnamoyltyramine Alkylamides and Co-Metabolites. Biomolecules 2021, 11, 1765. https://doi.org/10.3390/ biom11121765 Natural Bioactive Cinnamoyltyramine Alkylamides and Co-Metabolites. Biomolecules 2021, 11, x. https://doi.org/ 10.3390/xxxxx Review Natural Bioactive Cinnamoyltyramine Alkylamides and Co-Metabolites Natural Bioactive Cinnamoyltyramine Alkylamides Co-Metabolites Antonio Evidente * and Marco Masi Furthermore, the co-metabolites isolated from the same natural sources and their biological activities are also reported. Keywords: alkylamide; cinnamoyltyramine; plant sources; different carbon skeleton; biological ac- Keywords: alkylamide; cinnamoyltyramine; plant sources; different carbon skeleton; biological activity tivity Citation: Evidente, A.; Masi, M. Natural Bioactive Cinnamoyltyramine Alkylamides and Co-Metabolites. Biomolecules 2021, 11, 1765. https://doi.org/10.3390/ biom11121765 Academic Editor: Anna Sparatore Received: 8 November 2021 Accepted: 21 November 2021 Published: 25 November 2021 Citation: Evidente, A.; Masi, M. Natural Bioactive Cinnamoyltyramine Alkylamides and Co-Metabolites. Biomolecules 2021, 11, x. https://doi.org/ 10.3390/xxxxx Academic Editor: Anna Sparatore Received: 8 November 2021 Accepted: 21 November 2021 Published: 25 November 2021 Publi he ’ Note MDPI tay eu Citation: Evidente, A.; Masi, M. Natural Bioactive Cinnamoyltyramine Alkylamides and Co-Metabolites. Biomolecules 2021, 11, 1765. https://doi.org/10.3390/ biom11121765 Academic Editor: Anna Sparatore Received: 8 November 2021 Accepted: 21 November 2021 Published: 25 November 2021 Citation: Evidente, A.; Masi, M. Natural Bioactive Cinnamoyltyramine Alkylamides and Co-Metabolites. Biomolecules 2021, 11, x. https://doi.org/ 10.3390/xxxxx Academic Editor: Anna Sparatore Received: 8 November 2021 Accepted: 21 November 2021 Published: 25 November 2021 Publisher’s Note: MDPI stays neu 1. Introduction 1. Introduction Alkyla ide The structural formula representing all the alkylamides is reported in Figure 1. Copyright: © 2021 Submitted for pos Copyright: © 20 Submitted for po R2 N H O R1 Figure 1. General structure of an alkylamide. Figure 1. General structure of an alkylamide. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). p p publication under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https://cre- ativecommons.org/licenses/by/4.0/). Figure 1. General structure of an alkylamide. Figure 1. General structure of an alkylamide. The nature of the acid and the amine residues are characteristic of each plant family and species. They are also classiﬁed as protoalkaloid or pseudoalkaloid compounds and creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 1765. https://doi.org/10.3390/biom11121765 Biomolecules 2021, 11, 1765 2 of 43 2 of 43 represent a group of lipidic compounds structurally related to animal endocannabinoids and are strongly active metabolites in the central nervous system. Some previous reviews reported the chemistry and the biological activity of alkylamides, and although they cover a broad range of literature, they are organized differently. One was organized accordingly to the family of the plant source [4], and another one reported the chemistry and the detailed description of their biological activities [5]. p y p p p represent a group of lipidic compounds structurally related to animal endocannabinoids and are strongly active metabolites in the central nervous system. Some previous reviews reported the chemistry and the biological activity of alkylamides, and although they cover a broad range of literature, they are organized differently. One was organized accordingly to the family of the plant source [4], and another one reported the chemistry and the de- tailed description of their biological activities [5]. The present review is focused on the cinnamoyltyramine subgroup of the alkylamides, reporting their biosynthesis, chemical structures, biological activities, hemisynthetic deriva- tives and structure activity studies. Furthermore, the co-metabolites isolated from the same natural sources and their biological activity are also described. p g The present review is focused on the cinnamoyltyramine subgroup of the alkyla- mides, reporting their biosynthesis, chemical structures, biological activities, hemisyn- thetic derivatives and structure activity studies. Furthermore, the co-metabolites isolated from the same natural sources and their biological activity are also described. 2. Biosynthesis of N-trans-Cinnamoyltyramine 2. Biosynthesis of N-trans-Cinnamoyltyramine The biosynthesis of N-trans-cinnamoyltyramine (1) in plants could occur in several steps. The biosynthetic pathway starts from trans-cinnamic acid and tyramine, which were, respectively, generated from phenylalanine (L-Phe), as were the other cinnamic acids (i.e., p-coumaric, caffeic, ferulic, 5-hydroxyferulic and sinapic acids) and tyrosine (Tyr), as reported in Scheme 1. y y y The biosynthesis of N-trans-cinnamoyltyramine (1) in plants could occur in several steps. The biosynthetic pathway starts from trans-cinnamic acid and tyramine, which were, respectively, generated from phenylalanine (L-Phe), as were the other cinnamic ac- ids (i.e., p-coumaric, caffeic, ferulic, 5-hydroxyferulic and sinapic acids) and tyrosine (Tyr), as reported in Scheme 1. Scheme 1. Biosynthesis of cynnamic acids and tyramine from phenylalanine and tyrosine, respec- Scheme 1 Biosynthesis of cynnamic acids and tyramine from phenylalanine and tyrosine respectively Scheme 1. Biosynthesis of cynnamic acids and tyramine from phenylalanine and tyrosine, respec- ti ely Scheme 1. Biosynthesis of cynnamic acids and tyramine from phenylalanine and tyrosine, respectively. y Both aromatic amino acids (Phe and Tyr) were synthesized from prefenic acid, which was, in turn, generate from shikimic acid according to the shikimate pathway [6,7] re- Both aromatic amino acids (Phe and Tyr) were synthesized from prefenic acid, which was, in turn, generate from shikimic acid according to the shikimate pathway [6,7] reported in Scheme 2. ported in Scheme 2. In particular, Phe was converted by phenylalanine ammonia-lyase into cinnamic acid according to [7,8], and tyramine was synthesized by decarboxylation of tyrosine as re- ported in Scheme 1 [7,9]. As reported in Scheme 3, cinnamic acid was converted by COA ligase into the corresponding activate form [10]. The final step provides the conjugation of cinnamoylCoA and tyramine catalyzed by the tyramine n-hydroxycinnamoyl transfer- ase (THT): this enzyme is not specific to cinnamoylCoA and tyramine, but also catalyzes In particular, Phe was converted by phenylalanine ammonia-lyase into cinnamic acid according to [7,8], and tyramine was synthesized by decarboxylation of tyrosine as reported in Scheme 1 [7,9]. As reported in Scheme 3, cinnamic acid was converted by COA ligase into the corresponding activate form [10]. The ﬁnal step provides the conjugation of cinnamoylCoA and tyramine catalyzed by the tyramine n-hydroxycinnamoyl transferase (THT): this enzyme is not speciﬁc to cinnamoylCoA and tyramine, but also catalyzes the conjugation of tyramine with the other CoA-activated cinnamic acids cited above [11,12]. Biomolecules 2021, 11, 1765 3 of 43 above Scheme 2. 2. Biosynthesis of N-trans-Cinnamoyltyramine 2. Biosynthesis of N-trans-Cinnamoyltyramine Biosynthesis of phenylalanine and tyrosine (Phe and Tyr) according to shikimic acid pathway Scheme 2. Biosynthesis of phenylalanine and tyrosine (Phe and Tyr) according to shikimic acid pathway. Scheme 2. Biosynthesis of phenylalanine and tyrosine (Phe and Tyr) according to shikimic acid pathway. S he e 2 Bio y the i of he yla Scheme 2. Biosynthesis of phenylalanine and tyrosine (Phe and Tyr) according to shikimic acid pathway Scheme 2. Biosynthesis of phenylalanine and tyrosine (Phe and Tyr) according to shikimic acid pathway Scheme 2. Biosynthesis of phenylalanine and tyrosine (Phe and Tyr) according to shikimic acid pathway. CO2H CoA Ligase SHCoA OH H2N THT HSCoA Cinnamoyl COAs Tyramine N H SCoA O O OH trans-Cinnamoyltyramine (1) Scheme 3. Biosinthesis of N-trans-cinnamoyltyramine (1) by conjugation of cinnamoylCoA and ty- CO2H CoA Ligase SHCoA OH H2N THT HSCoA Cinnamoyl COAs Tyramine N H SCoA O O OH trans-Cinnamoyltyramine (1) Scheme 3. Biosinthesis of N-trans-cinnamoyltyramine (1) by conjugation of cinnamoylCoA and ty- i Scheme 3. Biosinthesis of N-trans-cinnamoyltyramine (1) by conjugation of cinnamoylCoA and tyramine. CO2H CoA Ligase SHCoA Cinnamoyl COAs SCoA O CO2H CoA Ligase SHCoA Cinnamoyl COAs SCoA O CO2H CoA Ligase SHCoA OH H2N THT HSCoA Cinnamoyl COAs Tyramine N H SCoA O O OH trans-Cinnamoyltyramine (1) S h 3 Bi i th i f N t i lt i (1 CO2H CoA Ligase SHCoA OH H2N THT HSCoA Cinnamoyl COAs Tyramine N H SCoA O O OH trans-Cinnamoyltyramine (1) Scheme 3 Biosinthesis of N trans cinnamoyltyramine (1 h f l ( ) b CoA Ligase CoA Ligase OH H2N Tyramine OH H2N Tyramine SC A SCoA H N H2N Tyramine Tyramine trans-Cinnamoyltyramine (1) trans-Cinnamoyltyramine (1) Scheme 3. Biosinthesis of N-trans-cinnamoyltyramine (1) by conjugation of cinnamoylCoA and ty- i Scheme 3. Biosinthesis of N-trans-cinnamoyltyramine (1) by conjugation of cinnamoylCoA and ty- a i e Scheme 3. Biosinthesis of N-trans-cinnamoyltyramine (1) by conjugation of cinnamoylCoA and tyramine. Scheme 3. Biosinthesis of N-trans-cinnamoyltyramine (1) by conjugation of cinnamoylCoA and ty- i Scheme 3. Biosinthesis of N-trans-cinnamoyltyramine (1) by conjugation of cinnamoylCoA and ty i Scheme 3. Biosinthesis of N-trans-cinnamoyltyramine (1) by conjugation of cinnamoylCoA and tyramine Scheme 3. Biosinthesis of N-trans-cinnamoyltyramine (1) by conjugation of cinnamoylCoA and ty- i Scheme 3. Biosinthesis of N-trans-cinnamoyltyramine (1) by conjugation of cinnamoylCoA and ty- ramine Scheme 3. Biosinthesis of N-trans-cinnamoyltyramine (1) by conjugation of cinnamoylCoA and tyramine. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources g y y y y Co-Metabolites Isolated from the Same Natural Sources This section describes the structure and stereostructure determination of both E- and Z-diastereomers of p-coumaroyl-, caffeoyl-, feruloyl-, 5-hydroxyferuloyl- and sinapoyl- tyramine alkylamides, including a few uncommon analogues and their biological g y y y y Co-Metabolites Isolated from the Same Natural Sources This section describes the structure and stereostructure determination of both E- and Z-diastereomers of p-coumaroyl-, caffeoyl-, feruloyl-, 5-hydroxyferuloyl- and sinapoyl- tyramine alkylamides, including a few uncommon analogues and their biological This section describes the structure and stereostructure determination of both E- and Z-diastereomers of p-coumaroyl-, caffeoyl-, feruloyl-, 5-hydroxyferuloyl- and sinapoyl- tyramine alkylamides, including a few uncommon analogues and their biological activities. Their promising practical applications are also described. Furthermore, chemical and biological aspects of the co-metabolites isolated from the same sources are described. g p N-cis-feruloyltyramine (NCFT) and grossamide (2 and 3, Figure 2), two previously undescribed phenolic amides, were isolated from the roots of bell pepper (Capsicum annuum var. grossum, Solanaceae) together with p-aminobenzaldehyde and other alkylamides as Biomolecules 2021, 11, 1765 4 of 43 viously cum an- N-trans-p-coumaroyltyramine (NTCT, also called prapazine), N-trans-feruloyltyramine (NTFT), N-trans-p-coumaroyloctopamine (NTCO) and N-trans-feruloyloctopamine (NTFO) (4–7, Figure 2) [13,14]. These latter compounds were previously isolated from the roots of eggplant (Solanum melongena L., Solanacee) [15]. The structure of grossamide was conﬁrmed by its synthesis starting from N-trans-feruloyltyramine by an oxidative radical coupling. It is classiﬁed into a group of lignin accordingly McCredie et al. (1969) [16], who suggested to include in the lignin group all low molecular weight natural compounds that were generated from the oxidative coupling of p-hydroxyphenylpropene [13]. mides as N trans p coumaroyltyramine (NTCT, also called prapazine), N trans feruloylty ramine (NTFT), N-trans-p-coumaroyloctopamine (NTCO) and N-trans-feruloyloctopa- mine (NTFO) (4–7, Figure 2) [13,14]. These latter compounds were previously isolated from the roots of eggplant (Solanum melongena L., Solanacee) [15]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources The structure of grossa- mide was confirmed by its synthesis starting from N-trans-feruloyltyramine by an oxida- tive radical coupling. It is classified into a group of lignin accordingly McCredie et al. (1969) [16], who suggested to include in the lignin group all low molecular weight natural compounds that were generated from the oxidative coupling of p-hydroxyphenylpropene [13]. Figure 2. The structures of compounds 2–19. Figure 2. The structures of compounds 2–19. Figure 2. The structures of compounds 2–19. Figure 2. The structures of compounds 2–19. Very few studies have been reported on oxidative coupling products possessing am- ide groups. Among them there are hordatines A, B and M (8–10, Figure 2) found as anti- fungal factors in barley (Hordeum vulgare, Graminacee) [17]. Hordatin M is a mixture of glucosides of hordatins A and B. Hordatins belong to polyammide, whose biosynthesis Very few studies have been reported on oxidative coupling products possessing amide groups. Among them there are hordatines A, B and M (8–10, Figure 2) found as antifungal factors in barley (Hordeum vulgare, Graminacee) [17]. Hordatin M is a mixture of glucosides of hordatins A and B. Hordatins belong to polyammide, whose biosynthesis started from p-hydrocynnamic acid CoA and agamatine obtained from decarboxylation of arginine. Then agmatinecoumaroyl transerase (ACT) catalyzes agamatine conjugates from coumaroyl- or feruloyl-CoA to give the corresponding p-hydrocinnamoylagamantine amide. The latter generate the dimeric hordatines by peroxidase oxidation [18]. Hordatines showed signif- icant antifungal activities [19,20] and are biosynthesized as pro-defense compounds in barley seedlings or are accumulated in plants after a pathogen attack [21,22]. Biomolecules 2021, 11, 1765 5 of 43 NTCT and N-cis-p-cumaroyltyramine (NCCT, 11, Figure 2), lunularic acid (12, Figure 2) and p-coumaric acid were isolated from bulbs of Allium chinense (Amaryllidaceae), which is used in Chinese folk medicine [23]. They are well known as inhibitors of prostaglandin (PG) and thromboxane synthetases. Rhapontigenin, piceatannol, rhaponticin and piceatannol glucoside (13–16, Figure 2) are stilbene derivatives structurally related to lunularic acid and obatined from rhubarb (Rheum rhabarbarum, Polygonaceae) [24]. They were tested among other analogues to evaluate their effect on prostaglandin synthetase, using platelet-rich plasma (PRP) obtained from blood collected from the main leg artery of a male albino rabbit. Rhapontigenin showed the most potent inhibition on PG-ase and strongly inhib- ited platelet aggregation induced by arachidonic acid and collagen. Platelet aggregation was demonstrated in in vivo studies. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources The balance between thromboxane (TX) A2 and prostaglandin (PG) I2 (prostacyclin) plays a very important role in the regulation of blood ﬂow. In fact, an excessive platelet aggregation is responsible to co-cause thrombosis and arteriosclerosis. Consequently, the inhibitory effect against PG or TX biosynthesis showed by the stilbene metabolites isolated from A. chinese could have an important therapeutic potential [23]. p NTFT was successively isolated together with new alkaloids, papracinine and pa- prazine, and six already known ones, fumaritine N-oxide, parfumine, lastourvilline, fumariﬂorine and N-methyl corydaldine from the aerial parts of Fumaria indica (Papaver- aceae), which is diffused in Europe, Central Asia and Africa. However, no activity was reported [25]. In the same year, but from the bark ethanolic extract of Asimina triloba L. (Annonaceae), NTCT and NTFT were isolated by a bio-guided fractionation testing brine shrimp lethality, together with a previously undescribed cytotoxic compound named acetogenin, and some known compounds such as asimicin, bullatacin, bullatacinone and (+)-syringaresinol. A. triloba L., an Annonaceae, commonly known as the pawpaw tree, which is native to the United States and spread in Europe, has been prized for its delicious, custard-like fruit. Trilobacin is a diastereomer of asimicin and both compounds showed potent and selective cytotoxicities in the NCI human tumor cell line screen [26]. p y NTCT was isolated from the stem bark extracts of Isolona maitlandii (Annonaceae), together with hexalobine-type, aporphinoids, amides and sterols. The leaf extract contained only hexalobines including ent-hexalobine C and ﬁve previously undescribed hexalobines. Any biological activity was reported [27]. NTCT and NCCT were isolated also from Aristolochia mollissima belong to Aristolochi- aceae. Aristolochia is a genus constituted by ca. 400 species that are widely distributed from the tropics to temperate regions. The roots and fruits of A. mollissima are used in Chinese folk medicine as analgesic, anticancer, antimalarial and anti-inﬂammatory agents, and also for the treatment of stomachache, abdominal pain and rheumatism. New sesquiterpenes, named mandolins S, R, U (17–19, Figure 2), W and X (20 and 21, Figure 3), together with 38 already known compounds belonging to different groups of natural compounds, were isolated from this plant [28]. p NCFT, NTCT and NTFT were again isolated together with NCCT (11) and the already known lariciresinol, 13-hydroxycapsidiol, lubiminol and drummondol from red pepper (Capsicum annuum) (Solanaceae). However, the main metabolite isolated from C. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources annnum was capsaicin, a compound known to be responsible of pungent activity, and the plant was studied for its components, dietary effects and analgesic antioxidant activity [29,30]. Furthermore, 10 previously undescribed co-metabolites (eight bicyclic and two spiranic sesquiterpenes) were isolated from the same plant and named canusesnol A–J (22–31, Figure 3). The sesquiterpenes and the known compounds showed scant cytotoxic and anti-HIV activity [31]. NTFT and NTCT were isolated together with an azanthracene alkaloid, characterized as 1-aza-9,10-dimethoxy-4-methyl-2-oxo-1,2-dihydroanthracene and named kalasinamide, from the stems of Polyalthia suberosa (Annonaceae), which is a shrubby tree spread be- tween southeast Asia and south China [32]. From the organic extract of its stems and leaves collected in China, a triterpene was previously isolated, named suberosol, with anti- Biomolecules 2021, 11, 1765 6 of 43 HIV activity [33]. Successively, from the same plant together with NTFT and NTCT, two undescribed 2-substituted furans, 1-(2-furyl)pentacosa-16,18-diyne and 23-(2-furyl)tricosa- 5,7-diynoic acid [34], were also isolated. As NTCT was isolated in limited amount not sufﬁcient to investigate its biological activity, its synthesis was realized in one step starting from coumaric acid and tyramine with a ﬁnal yield about of 55%. It showed suppression of growth of human tumor cells, such as U937 and Jurkat cells, which appeared asso- ciated with an increased percentage of cells in the S phase of the cell cycle progression. Furthermore, NTCT was able to inhibit the protein tyrosine kinases including epidermal growth factor receptor (EGFR) [35]. REVIEW 6 of 42 (22–31, Figure 3). The sesquiterpenes and the known compounds showed scant cytotoxic and anti-HIV activity [31]. Figure 3. The structure of compounds 20–37. Figure 3. The structure of compounds 20–37. Figure 3. The structure of compounds 20–37. Figure 3. The structure of compounds 20–37. Figure 3. The structure of compounds 20–37. Figure 3. The structure of compounds 20–37. NTFT and NTCT were isolated together with an azanthracene alkaloid, characterized as 1-aza-9,10-dimethoxy-4-methyl-2-oxo-1,2-dihydroanthracene and named kalasina- mide, from the stems of Polyalthia suberosa (Annonaceae), which is a shrubby tree spread between southeast Asia and south China [32]. From the organic extract of its stems and NTCT was isolated from twigs of Celtis chinensis, which was used in folk medicine in Korea, Japan and China to treat lumbago, irregular menstruation and gastric diseases [36]. Furthermore, NTCT inhibited acetylcholinesterase (ACHE), a well-known enzyme that plays an important role in Alzheimers disease [37]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources g leaves collected in China, a triterpene was previously isolated, named suberosol, with anti-HIV activity [33]. Successively, from the same plant together with NTFT and NTCT, two undescribed 2-substituted furans, 1-(2-furyl)pentacosa-16,18-diyne and 23-(2-fu- ryl)tricosa-5,7-diynoic acid [34], were also isolated. As NTCT was isolated in limited amount not sufficient to investigate its biological activity, its synthesis was realized in one step starting from coumaric acid and tyramine with a final yield about of 55%. It showed y The same four alkymides, NCFT, NTCT, NTFT and NCCT, were again isolated to- gether with other already known compounds, belonging to different classes of naturally occurring compounds, from the root and stem of Aristolochia elegans [38]. A. elegans belong to the genus Aristolochia (Aristolochiaceae), and the alcoholic extracts of some species were investigated for their uterus contraction stimulating [39], antimitotic and antiviral properties [40]. A. elegans also produced previously undescribed compounds characterized Biomolecules 2021, 11, 1765 7 of 43 as two aristolactams, aristolactam E and aristolactam-AIIIa-6-O-β-D-glucoside (32 and 33, Figure 3), three benzoyl benzyltetrahydroisoquinoline ether N-oxide alkaloids, aristo- quinolines A–C (34–36, Figure 3), as well as a biphenyl ether, aristogin F (37, Figure 3). All the metabolites were tested to evaluate their potential antioxidative and antityrosinase properties, but neither the four alkylamines or the new metabolites showed activity [38]. NTCT and NTFT were isolated from the organic extract of leaves and stems of Piper sanctum (Piperaceae) collected in Mexico together with nine monosubstituted 8-benzo[d][1, 3]dioxole (38–46, Figure 4), three monosubstituted alkylbenzene, a 2,6-disubstituted tetrahy- dropyranone and a 2,5-disubstituted tetrahydrofuranone. From the same extract were also isolated p-eugenol, methyleugenol, Z-piperolide, demethoxyyangonin 5,6-dehydro-7,8- dihydromethysticin, cepharanone B, piperolactam A, cepharadione B and safrol, which was the major component of the oily extract. Compounds 38, 39, 43, demethoxyyangonin, 5,6-dehydro-7,8-dihydromethysticin, cepharanone B, piperolactam A and NTCT inhibited the growth of Mycobacterium tuberculosis when tested by the Microplate Alamar Blue Assay (MABA), with MIC values ranging from 4 to 64 µg/mL [41]. REVIEW 8 of 42 aminobenzaldehyde; the three flavonoids, such as (2S)-3′,7-dihydroxy-4′-methoxyflavan, 7-hydroxyflavanone and 4′,7-dihydroxyflavone; and the five phenolic compounds, such as trans-caffeic acid, 4-coumaric acid, 4-hydroxybenzonic acid, ethyl 4-hydroxybenzoate and 2-(3,4-dihydroxyphenyl)-1,3-benzodioxole-5-carboxaldehyde. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources When the alkaloids 60 and 61 and the other alkaloids were assayed for their cytotoxic activities against the hu- man tumor cell lines A549, LOVO, HL-60 and 6T-CEM, only crinumaquine, lycorine, un- geremine, 11-O-methylcrinamine, 3-O-acetylhamayne and crinamine showed inhibition against one or more of the tested cell lines [52]. as two aristolactams, aristolactam E and aristolactam-AIIIa-6-O-β-D-glucoside (32 and 33, Figure 3), three benzoyl benzyltetrahydroisoquinoline ether N-oxide alkaloids, aristo- quinolines A–C (34–36, Figure 3), as well as a biphenyl ether, aristogin F (37, Figure 3). All the metabolites were tested to evaluate their potential antioxidative and antityrosinase properties, but neither the four alkylamines or the new metabolites showed activity [38]. REVIEW 8 of 42 as two aristolactams, aristolactam E and aristolactam-AIIIa-6-O-β-D-glucoside (32 and 33, Figure 3), three benzoyl benzyltetrahydroisoquinoline ether N-oxide alkaloids, aristo- quinolines A–C (34–36, Figure 3), as well as a biphenyl ether, aristogin F (37, Figure 3). All the metabolites were tested to evaluate their potential antioxidative and antityrosinase properties, but neither the four alkylamines or the new metabolites showed activity [38]. REVIEW 8 of 42 y y NTCT and NTFT were isolated from the organic extract of leaves and stems of Piper sanctum (Piperaceae) collected in Mexico together with nine monosubstituted 8-benzo[d][1, 3]dioxole (38–46, Figure 4), three monosubstituted alkylbenzene, a 2,6-disubstituted tetrahy- dropyranone and a 2,5-disubstituted tetrahydrofuranone. From the same extract were also isolated p-eugenol, methyleugenol, Z-piperolide, demethoxyyangonin 5,6-dehydro-7,8- dihydromethysticin, cepharanone B, piperolactam A, cepharadione B and safrol, which was the major component of the oily extract. Compounds 38, 39, 43, demethoxyyangonin, 5,6-dehydro-7,8-dihydromethysticin, cepharanone B, piperolactam A and NTCT inhibited the growth of Mycobacterium tuberculosis when tested by the Microplate Alamar Blue Assay (MABA), with MIC values ranging from 4 to 64 µg/mL [41]. aminobenzaldehyde; the three flavonoids, such as (2S)-3′,7-dihydroxy-4′-methoxyflavan, 7-hydroxyflavanone and 4′,7-dihydroxyflavone; and the five phenolic compounds, such as trans-caffeic acid, 4-coumaric acid, 4-hydroxybenzonic acid, ethyl 4-hydroxybenzoate and 2-(3,4-dihydroxyphenyl)-1,3-benzodioxole-5-carboxaldehyde. When the alkaloids 60 and 61 and the other alkaloids were assayed for their cytotoxic activities against the hu- man tumor cell lines A549, LOVO, HL-60 and 6T-CEM, only crinumaquine, lycorine, un- geremine, 11-O-methylcrinamine, 3-O-acetylhamayne and crinamine showed inhibition against one or more of the tested cell lines [52]. Figure 4. The structures of compounds 38–52. Figure 4. The structures of compounds 38–52. Figure 4. The structures of compounds 38–52. Figure 4. The structures of compounds 38–52. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources Biomolecules 2021, 11, 1765 8 of 43 N-trans-sinapoyltyramine (NTST, 52, Figure 4), NCFT, NTFT and NTCT were iso- lated together with 23 known compounds from the bark stems of Polyalthia longifolia var. pendula [40], while NTFT and NTCT were also isolated from Sparattanthelium tupiniquinorum (Hernandiaceae) collected in Brazil [42,43]. NTCT, NCCT and NTFT were isolated together with six previously undescribed lignans (53–58, Figure 5), and 11 other types of known compounds from Peperomia duclouxii (Piperaceae), which is a plant used in folk medicine as an anticancer agent in mainland China. When these compounds were tested in cytotoxic and MDR (multidrug resistance) reversal cell activity assays, only compound 55 inhibited the growth of VA-13 and HepG2 cancer cells, with IC50 values of 5.3 and 13.2 µg/mL, respetively. Compound 55 also showed potent effects on calcein accumulation in MDR 2780AD cells than verapamil, which was used as a positive control. Compound 58 exhibited anti-inﬂammatory activity using an ICAM-1 assay (induction of the intercellular adhesion molecule-1) and stimulated IL-1α (Interleukin 1 alpha) and TNF-α (tumor necrosis factor alpha) with IC50 values of 107 and 13.4 µM, respectively, and without cytotoxicity against A549 cells [44]. REVIEW 9 of 42 palmatrubin and jatrorrhizine. All the compounds were assayed for antileishmanial ac- tivity against Leishmania donovani testing the effects of promastigotes and intracellular amastigotes, and only compound 63 exhibited the highest in vitro antileishmanial activity, whereas compounds 62, palmatine and palmatrubin showed moderate activity. The other compounds were found to be inactive [54] Figure 5. The structures of compounds 53–68. Piper sarmentosum and Piper nigrum (Piperaceae) are well known for thei Figure 5. The structures of compounds 53–68. Figure 5. The structures of compounds 53–68. Figure 5. The structures of compounds 53–68. Biomolecules 2021, 11, 1765 9 of 43 NTCT was isolated together with cannabisin G and (±)-lyoniresinol from the or- ganic extract of the root bark of Berberis vulgaris L. (Berberidaceae). Different parts of this species were used for the treatment of diarrhea, gallbladder and liver dysfunctions, leishmaniasis, malaria, stomach problems and urinary tract diseases [45]. Cannabisin G and (±)-lyoniresinol, using a hydroxyl radical scavenging assay, exhibited antioxidant activity, while cannabisin G showed cytoprotective activity in cultured MCF-7 cells [46]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources NTCT and N-trans-caffeoyltyramine (NTCAT, 59, Figure 5) were isolated together with two new alkaloids, named asiaticumines A and B (60 and 61, Figure 5), and 18 other known compounds from the CHCl3 and EtOAc extracts of Crinum asiaticum var. sinicum Baker bulbs. This plant belongs to a well-known subgroup of Amaryllid- ceae, which synthesize alkaloids with several biological activities [47–51]. This species was used in traditional Chinese medicine for the treatment of abscesses, aching joints and sores. The already known metabolites were identiﬁed as the alkaloids crinumaquine, lycorine, hippacine, ungeremine, 11-O-methylcrinamine, 3-O-acetylhamayne, crinamine, criwelline and 4-hydroxystyryolamine. The other metabolites were identiﬁed as follows: 4-aminobenzaldehyde; the three ﬂavonoids, such as (2S)-3′,7-dihydroxy-4′-methoxyﬂavan, 7-hydroxyﬂavanone and 4′,7-dihydroxyﬂavone; and the ﬁve phenolic compounds, such as trans-caffeic acid, 4-coumaric acid, 4-hydroxybenzonic acid, ethyl 4-hydroxybenzoate and 2-(3,4-dihydroxyphenyl)-1,3-benzodioxole-5-carboxaldehyde. When the alkaloids 60 and 61 and the other alkaloids were assayed for their cytotoxic activities against the human tumor cell lines A549, LOVO, HL-60 and 6T-CEM, only crinumaquine, lycorine, ungeremine, 11-O-methylcrinamine, 3-O-acetylhamayne and crinamine showed inhibition against one or more of the tested cell lines [52]. g NTCT was isolated together with two previously undescribed compounds, namely 4-methyl-heptadec-6-enoic acid ethyl ester and 3-hydroxy-2,9,11-trimethoxy-5,6-dihydro isoquino[3,2-a]isoquinolinylium (62 and 63, Figure 5), and other ﬁve already known metabolites from an ethanolic extract of the stems of Tinospora sinensis (syn: Tinospora malabarica) (Menispermaceaeis). This plant is well known for its therapeutic value in treat- ing debility, dyspepsia, fever, inﬂammation, syphilis, ulcers, bronchitis and immunomod- ulatory properties, as well as urinary, skin and liver diseases [53]. The ﬁve known com- pounds were identiﬁed as lirioresino-β-dimethyl ether, β-sitosterol, palmatine, palmatrubin and jatrorrhizine. All the compounds were assayed for antileishmanial activity against Leishmania donovani testing the effects of promastigotes and intracellular amastigotes, and only compound 63 exhibited the highest in vitro antileishmanial activity, whereas com- pounds 62, palmatine and palmatrubin showed moderate activity. The other compounds were found to be inactive [54]. Piper sarmentosum and Piper nigrum (Piperaceae) are well known for their therapeutic effects and content of alkaloid and amides [55]. P. nigrum has showed CNS (central nervous system) stimulant, analgesic, antipyretic and antifeedant activities [56], while the P. sarmentosum leaves were used to treat malaria, coughs and colds, as well as toothache, and showed antituberculosis and antiplasmodial activities [57]. NTCT was isolated together with ﬁve known amides, namely pellitorine (E)-1-[30,40-(methylenedioxy)cinnamoyl]pipe- ridine 2,4-tetradecadienoic acid isobutyl amide, piperine, sylvamide, cepharadione A and piperolactam D from P. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources nigrum, while a previously undescribed aromatic compound characterized as 1-nitrosoimino-2,4,5-trimethoxybenzene (64, Figure 5) was obtained from P. sarmentosum. The organic extracts of both plants showed cytotoxic activity against HeLa and MCF-7v cancer cell lines, with a signiﬁcant contribution of compound 64 for the activity of the extract of P. sarmentosum [58]. y NTCT and NTFT were isolated together with four previously undescribed alkaloids, namely 3-(2-(7,7-dimethyl-3,7-dihydropyrano[3,2-e]indol-1-yl)ethyl)quinazoline-2,4(1H,3H)- dione, 3-(2-(7,7-dimethyl-3,7-dihydropyrano-[3,2-e]indol-1-yl)ethyl)-1-hydroxyquinazoline- 2,4(1H,3H)-dione, 3-(2-(7,7-dimethyl-3,7-dihydropyrano [3,2-e]indol-1-yl)ethyl-1-methylqu- inazoline-2,4(1H,3H)-dione and (E)-3-(6,7-dihydroxy-3,7-dimethyloct-2-enyl)-4-methoxy- 1-methylquinolin-2(1H)-one (65–68, Figure 5), from the methanol extract of Conchocarpus Biomolecules 2021, 11, 1765 10 of 43 10 of 43 gaudichaudianus stems (Rutaceae). This tree is used by the native people in northern Brazil [59]. NTCT and NTFT were isolated together with 11 new diglycosides, named erycibo- sides A–L (69–80, Figure 6), 4 new chlorogenic acid derivatives (81–84, Figure 6), a new biscoumarin (85, Figure 6), and 21 other known compounds, from the roots and stems ethanol extract of Erycibe hainanesis (Convolvulaceae) [60]. This genus consists of about 66 species, with 11 species found in China. Compounds belonging to ﬂavonoids, coumarins, chlorogenic acids, alkaloids and several other components were previously extracted from Erycibe species [61]. Some of them have been shown to exhibit anti-inﬂammatory, muscarinic agonistic and cytotoxic activities and have been used in Chinese folk medi- cine [62,63]. Erycibosides B, F and L (70 and 74, Figure 6) and the new biscoumarin (85, Figure 6) showed strong hepatoprotective activities at concentrations of 1 × 10−5 to 1 × 10−4 M [58]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources 69, R1=syringoyl, R2=R3=H, 70, R1=R3=H, R2=syringoyl, 73, R1=vanilloyl, R2=R3=H O OH OR2 O R1O O OH O OH OH O MeO O O R3 O OH OR2 O R1O O OH O OH OH O 75, R1=syringoyl, R2=H O OH OR2 O R1O O OH O OH OH O 76, R1=syringoyl, R2=H OH 77, R1=syringoyl, R2=R3=H 78, R1=syringoyl, R2=H, R3=OH O OH OR2 O R1O O OH OH OH O OH HO OH R3 O OH OR2 O R1O O OH O OH OH O OMe OMe OMe O OH HO OH OH OH O R O O MeO O O 72, R=syringoyl 74, R=vanilloyl 72, R=syringoyl 74, R=vanilloyl OH OH OH 75, R1=syringoyl, R2=H 76, R1=syringoyl, R2=H 77, R1=syringoyl, R2=R3=H 78, R1=syringoyl, R2=H, R3=OH O OH OR2 O R1O O OH OH OH O OH HO OH R3 O OH OR2 O R1O O OH O OH OH O OMe OMe OMe 75, R1=syringoyl, R2=H 75, R1=syringoyl, R2=H 79, R1=syringoyl, R2=H O OH OR2 O R1O O OH OH OH O OH HO OH OR1 OH OR3 R2O OR 79, R1= O OH OR2 O R1O O OH OH OH O OH HO O 79, R1=syringoyl, R2=H OR1 OH OR3 R2O O OR4 80, R1=syringoyl, R2=H 81, R1=R4=H, R2=syringoyl, R3=caffeoyl 82, R1=syringoyl, R2=R4=H, R3=caffeoyl 83, R1=syringoyl, R2=caffeoyl, R3=R4=H 84, R1=H, R2=vanilloyl, R3=caffeoyl, R4=Me O HO MeO O O O MeO O 85 85 Figure 6. The structures of compounds 69–85. Figure 6. The structures of compounds 69–85. NTCT and NCCT, 1,7-bis(4-hydroxyphenyl)heptane-3,5-diol and 6-hydroxy-2,4,7- trimethoxyphenanthrene were isolated from the fresh tuberous rhizomes of Chinese yam (Dioscorea opposita Thunb.) (Dioscoreaceae) [64]. This plant has a noteworthy interest in agriculture, food and pharmaceutical fields [65,66]. NTCT, NTCT and the hepatanediol de i ati e e e i olated fo the fi t ti e f o D o osita The i hibito y a ti itie of NTCT and NCCT, 1,7-bis(4-hydroxyphenyl)heptane-3,5-diol and 6-hydroxy-2,4,7- trimethoxyphenanthrene were isolated from the fresh tuberous rhizomes of Chinese yam (Dioscorea opposita Thunb.) (Dioscoreaceae) [64]. This plant has a noteworthy interest in agriculture, food and pharmaceutical ﬁelds [65,66]. NTCT, NTCT and the hepatanediol NTCT and NCCT, 1,7-bis(4-hydroxyphenyl)heptane-3,5-diol and 6-hydroxy-2,4,7- trimethoxyphenanthrene were isolated from the fresh tuberous rhizomes of Chinese yam (Dioscorea opposita Thunb.) (Dioscoreaceae) [64]. This plant has a noteworthy interest in agriculture, food and pharmaceutical fields [65,66]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources REVIEW 11 of 42 69, R1=syringoyl, R2=R3=H, 70, R1=R3=H, R2=syringoyl, 73, R1=vanilloyl, R2=R3=H O OH OR2 O R1O O OH O OH OH O MeO O O R3 O OH OR2 O R1O O OH O OH OH O 75, R1=syringoyl, R2=H O OH OR2 O R1O O OH O OH OH O 76, R1=syringoyl, R2=H OH 79, R1=syringoyl, R2=H 80, R1=syringoyl, R2=H 77, R1=syringoyl, R2=R3=H 78, R1=syringoyl, R2=H, R3=OH O OH OR2 O R1O O OH OH OH O OH HO OH R3 O OH OR2 O R1O O OH OH OH O OH HO OH O OH OR2 O R1O O OH O OH OH O OMe OMe OMe O OH HO OH OH OH O R O O MeO O O 72, R=syringoyl 74, R=vanilloyl OR1 OH OR3 R2O O OR4 81, R1=R4=H, R2=syringoyl, R3=caffeoyl 82, R1=syringoyl, R2=R4=H, R3=caffeoyl 83, R1=syringoyl, R2=caffeoyl, R3=R4=H 84, R1=H, R2=vanilloyl, R3=caffeoyl, R4=Me O HO MeO O O O MeO O 85 Figure 6. The structures of compounds 69–85. Figure 6. The structures of compounds 69–85. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources NTCT, NCCT and NTFT were isolated together with ferul aldehyde, 6,7-dimethoxyco- umarin and ﬁcusal from the organic extract of Solanum melongena L. (Solanaceae) root [76]. The roots of this plant, called “Qie gen” in China, were used in folk Chinese medicine for the treatment of toothache, chilblains and beriberi. Other studies showed that the extracts of S. melongena had anti-inﬂammatory, analgesic and antiatherosclerosis activities [77,78]. Only the three alkylamides NTCT, NCCT and NTFT inhibited α-glucosidase with IC50 values of 500.6, 5.3 and 46.3 µM, respectively, and they were not competitive inhibitors. Thus, the plant could be proposed for pharmacological application [76]. NTCT, NTFT and NTCAT were isolated as the main component from the organic extract of Polygonum hyrcanicum (Polygonaceae) aerial parts, which showed high activity against Trypanosoma brucei rhodesiense (IC50 = 3.7 µg/mL). This protozoan parasite induces sleeping sickness, also known as human African trypanosomiasis (HAT). HAT infects more than 50,000 people each year and about 60 million people are at risk of trypanosomiasis [79]. The three alkylamides, NTCT, NTFT and NTCAT, showed activity with C50s ranging from 2.2 to 13.3 µM [80]. P. hyrcanicum is an endemic plant growing in northern areas of Iran and is known as Gheq-buqun in the Turkmen Sahra region, where its decoction has been used for the treatment of liver problems, anemia, hemorrhoids and kidney stones [81]. From the same organic extract, some other known and lesser active compounds were also isolated as cannabisin B, tyrosol, p-coumaric and ferulic acids, and NCFT and N-trans-3,4-dimethoxycinnamoyldopamine (90, Figure 7). This data again showed that E stereoisomer is more active than the Z one (NCFT). However, it is important to remember that cinnamoylphenethyl amides rapidly isomerize when exposed to UV light and therefore NCFT could be an artifact formed during the isolation procedure [82]. NTCT, NTFT, NTCAT and N-cis-feruloyloctopamine (NCFO (91, Figure 7)) were isolated together with 7 new neolignanamides (92–98, Figure 7), a new lignanamide (99, Figure 8) and 17 known phenolic compounds from the organic extract of Lycium chinense [83]. This plant belongs to genus Lycium (Solanaceae family) mainly distributed in South America, South Africa and temperate Europe and Asia. It was used in traditional Chinese medicine as an antipyretic and for the treatment of pneumonia, night-sweats, cough, hematemesis, inﬂammation and diabetes mellitus [84]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources NTCT, NTCT and the hepatanediol d i ti i l t d f th fi t ti f D it Th i hibit ti iti f NTCT and NCCT, 1,7-bis(4-hydroxyphenyl)heptane-3,5-diol and 6-hydroxy-2,4,7- trimethoxyphenanthrene were isolated from the fresh tuberous rhizomes of Chinese yam (Dioscorea opposita Thunb.) (Dioscoreaceae) [64]. This plant has a noteworthy interest in agriculture, food and pharmaceutical ﬁelds [65,66]. NTCT, NTCT and the hepatanediol Biomolecules 2021, 11, 1765 11 of 43 11 of 43 derivative were isolated for the ﬁrst time from D. opposita. The inhibitory activities of crude extracts as well as those of puriﬁed constituents were evaluated against yeast α-glucosidase to search for the active principles for treatment of diabetes. NTCT, the heptanediol and the phenanthrene derivative showed a signiﬁcant activity with IC50 = 0.40, 0.38 and 0.77 µM, respectively, while NCCT was inactive suggesting that the stereochemistry of the double bond of this alkylamide is a structural feature important for the activity [64]. NTFT, NTCT and 3′methoxy-NTFT and kaempferol (86 and 87, Figure 7) were isolated from Welsh onion (Allium ﬁstulosum L.) (Amaryllidaceae) organic extracts [67]. A. ﬁstulosum L. is a perennial herb that is classiﬁed as an Allium species, which is a popular ﬂavoring vegetable in China, Japan and Korea [68]. This plant is widely cultivated in south- ern areas of Korea and is traditionally used for salads and cooking. In the same country, its roots and trunks were used in traditional folk medicine for the treatment of febrile disease, headache, abdominal pain, diarrhea and habitual abortion [69]. Successive studies reported that Welsh onion showed anti-aggregation [70,71] and anti-hypertensive [70–74] activities. The three alkylamides NTFT, NTCT and N-cis-feruloyl-3’-methoxytyramine were isolated for the ﬁrst time from the Welsh onion. NTFT and its 3′-methoxy analogue exhibited signiﬁcantly (p < 0.05) higher DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity than the compound NTCT [67]. REVIEW 13 of 42 Figure 7. The structures of compounds 86–98. Figure 7. The structures of compounds 86–98. Figure 7. The structures of compounds 86–98. Figure 7. The structures of compounds 86–98. Biomolecules 2021, 11, 1765 12 of 43 12 of 43 NTCT and NTCAT were isolated together their 4′-O-methyl derivatives (88 and 89, Figure 7), β-sitostenone, ferulic, hydroferulic, 5-hydroxy-3,4-dimethoxycinnamic veratic, vanillic, isovanillic and syringic acids, as well as (+)-syringaresinol and pheophorbide D from the stems of Capsicum annuum (Solanaceae) [75]. Compound 88 was isolated for the ﬁrst time as a naturally occurring compound [75]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources nucifera leaves has potential as an anti-obesity agent [88]. NTCAT, NTFT, NTCT and N-trans-feruloyldopamine were isolated together with the well-known 13-hydroxysolavetivone, betulinic acid, 3′-O-methoxydopamine, alangiligno- side C, isolariciresinol, polistachiol, (+)-(8R,7′S,8′S)-3α-O-(β-D-glucopiranosyl)-lioniresinol, (−)-(8S,7′R,8′R)-3α-O-(β-D-glucopiranosyl)-lioniresinol and solamargine from the organic extract of Solanum buddleifolium Sendtn (Solanaceae) stems [90]. S. buddleifolium is widely distributed in the northeast of Brazil, where it is used in folk medicine [91]. NTFT was isolated together with two bis-alkaloids, ﬂaviﬂoramides A and B (104 and 105, Figure 8), and paprazine from the aerial part of Piper ﬂaviﬂorum [92]. This plant belongs to the Piper genus, which is well known as a rich source of a variety of alkaloids, having interesting pharmacological activities, such as anti-inﬂammatory, antino-ciceptive, anticancer and antidepressant properties [92–94]. N-trans-Cinnamoyltyramine (1, Scheme 3) and NTCT were isolated together with two sesquiterpenes, named aristoyunnolins I and J (106 and 107, Figure 8), and six other known compounds from the roots of Aristolochia yunnanensis (syn. Aristolochia grifﬁthii) (Aristolochiaceae) [95]. This plant is endemic to Yunnan Province of China, known as “Nan Mu Xiang”, and is used in Chinese medicine for the treatment of trichomoniasis, gastrointestinal diseases and rheumatic pain [94]. All the compounds were evaluated against P-388 and A-549 cell lines, and among them costunolide (108, Figure 8) exhibited moderate activity [95]. NTCT, NTFT, NTCAT, dihydro-NTCAT and three neolignanamides and two lig- nanamides were isolated from the root bark of Lycium chinense Miller, Lycii Radicis Cortex (Solanacee). This plant was used in traditional Chinese medicine to treat different inﬂam- mation symptoms and diabetes mellitus [96]. The results of biological assays showed that akylamides, as main components of L. chinese, were responsible for NF-κB inhibition. The SAR study also suggests that the NF-κB inhibitory activity of NTCAT could be due to its Michael acceptor-type structure (α,β-unsaturated carbonyl group) [97]. NCCT, NTCT, 8 carbazole alkaloids, claulamines C, D and E (109–111, Figure 8) and clausenalines B−F (112–114, Figure 8, 115–116, Figure 9), as well as 4 coumarins, clausemarins A−D (117–120, Figure 9), and 41 already known compounds were isolated from the roots of Clausena lansium (Rutaceae) [98]. This plant, also known as “wampee”, is a native species of southern mainland China and it was used in folk medicine in China, Taiwan and the Philippines. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources The known compounds were identified as thoreliamide B, gentisic, vanillic, p-coumaric caffeic, ferulic, sinapic and dihydrocaffeic acids, as well as isoscopoletin, fraxidin, aquillochin, scopolin, kaempferide, apigenin and luteolin. The cinnamic acid amides, neolignanamides and lignanamides showed moderate radical scavenging activity towards the DPPH and superoxide radicals [83]. NTCT and NTFT were isolated from the organic extract of P. oleracea (Portulacaceae) together with a pyrrole alkaloid named portulacaldehyde (100, Figure 8), N-(E)-feruloyl- 4-O-methyldopamine (101, Figure 8) and the well-known (E)-p-coumaric and (E)-ferulic acids, 4-hydroxybenzaldehyde, 2,4-dihydroxybenzaldehyde, 2-hydroxy-4-methoxybenzoic and syringic acids [85]. P. oleracea, commonly named purslane, is an annual, semi-succulent, trailing herbaceous plant used in folk medicine for its analgesic and wound-healing, anti-inﬂammatory properties [86,87]. N-(E)-feruloyl-4-O-methyldopamine (101), NTFT, 4-hydroxybenzaldehyde and 2,4-dihydroxybenzaldehyde were involved in the regulation in the inﬂammatory activity of the plant extract [83]. Biomolecules 2021, 11, 1765 13 of 43 clausine 2 0 6 9 13 of 43 clausine 2 0 6 9 Figure 8. The structures of compounds 99–114. Figure 8. The structures of compounds 99–114. Figure 8. The structures of compounds 99–114. Figure 8. The structures of compounds 99–114. NTCT, NCCT, NTFT and NCFT were isolated together with 13 megastigmanes, including a new megastigmane, nelumnucifoside A (102, Figure 8), and a new eudes- mane sesquiterpene, nelumnucifoside B (103, Figure 8), as well as 8 alkaloids and 11 ﬂavonoids from Nelumbo nucifera Gaertn. (Nymphaeaceae) [88]. This is a peren- nial aquatic herb commonly called lotus. This plant is widely diffused in Eastern Asia and used for food and medicine for a long time. The fruits, seeds, roots and leaves of N. nucifera are edible and have been not only used as food for a long time, but also used as antifebrile, sedative, antibacterial, antidiarrheal and hemostatic agents in folk medicine [89]. The other known compounds were identiﬁed as (E)-3-hydroxymegastigm-7-en-9-one, (−)-boscialin, (+)-dehydrovomifoliol, vomifoliol, 3-oxo-retro-α-ionol I, byzantionoside A, 5,6-epoxy-3-hydroxy-7-megastigmen-9-one, annuionone D, icariside B2, grasshopper ke- tone, 3S,5R-dihydroxy-6S,7-megastigmadien-9-one, (+)-epiloliolide, (6R,6aR)-roe-merine- Nβ-oxide, liriodenine, pronuciferin, oleracein E, quercetin, kaempherol, luteolin, quercetin 3-O-glucopyranoside, kaempherol 3-O-glucopyranoside, chrysoeriol 7-O-glucopyranoside, taxifolin, epitaxifolin, 5,7,3′,5′-tetrahydroxyﬂavanone, (−)-catechin and elephantorrhizol. NTCT and NCFT inhibited pancreatic lipase, while (6R,6aR)-roemerine-Nβ-oxide and Biomolecules 2021, 11, 1765 14 of 43 14 of 43 liriodenine showed a strong inhibition on adipocyte differentiation. Therefore, the extract of N. nucifera leaves has potential as an anti-obesity agent [88]. liriodenine showed a strong inhibition on adipocyte differentiation. Therefore, the extract of N. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources Its leaves and roots are used for coughs, asthma, dermatological diseases, viral hepatitis and gastrointestinal ailments [99], while the seeds are used for acute and chronic gastrointestinal inﬂammation and ulcers [100]. Several known compounds were also identiﬁed as wampetin, 8-geranyloxypsolaren, imperatorin, osthenol, isoimperatorin, O-demethylmurrayanine O-demethylmurrayanine, clausine D, methyl carbazole-3-carboxylate, murrayanine, O-methyllansinexanthotoxol, heraclenol, anisolactone, claulansine A, O-methylmukonidine, 3-formyl-9H-carbazole, claulansine F, claulansine C, 9H-carbazole-3-carboxylic acid, 1-methoxycarbazole-3-carboxylic acid, 4-methoxy-1-methyl-2(1H)-quinolinone, vanillic acid, 2,6-dimethoxy-p-benzoquinone, 4-hydroxybenzoic acid, N-phenethylcinnaamide, (E)-coniferaldehyde, claulansine J, 3-formyl-6-methoxycarbazole, tertmethoxyheraclenol, isogospherol, indicolactonediol, lucidafuranocoumarin B, mafaicheenamine C, syringaresinol, mafaicheenamine A, mukonine, dihydroalatamide, α-santalol, β-sitosterol, platydesmine and γ-fagarine. Clausemarin A (117), wampetin, 8-geranyloxypsolaren, imperatorin, osthenol, isoimperatorin and O-demethylmurrayanine showed strong inhibition of superoxide anion generation with IC50 values ranging from 1.9 to 8.4 µM, while compounds O-demethylmurrayanine, clausine D and murrayanine inhibited elastase release with IC50 values in the range from 2.0 to 6.9 µM [98]. Biomolecules 2021, 11, 1765 Biomolecules 2021 11 x FOR 15 of 43 16 of 42 15 of 43 16 of 42 Figure 9. The structures of compounds 115–137. NTCT and NTFT were isolated together with the well-known 4-hydroxyb hyde, N-p-coumarylserotonine (NTCS, 121, Figure 9) and N-p-coumaryl-tr (NTCTR, 122, Figure 9) from the stem of Zea mays, which is cultivated worldwid Figure 9. The structures of compounds 115–137. NTCT and NTFT were isolated together with the well-known 4-hydroxybenza N-p-coumarylserotonine (NTCS, 121, Figure 9) and N-p-coumaryl-tryptamine (NTC Figure 9) from the stem of Zea mays, which is cultivated worldwide as grain and f Figure 9. The structures of compounds 115–137. Figure 9. The structures of compounds 115–137. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources NTCT and NTFT were isolated together with the well-known 4-hydroxybenzalde- hyde, N-p-coumarylserotonine (NTCS, 121, Figure 9) and N-p-coumaryl-tryptamine (NTCTR, 122, Figure 9) from the stem of Zea mays, which is cultivated worldwide as grain and feed and its seeds oil stigma spike leaf and root have been used in Chinese NTCT and NTFT were isolated together with the well-known 4-hydroxybenzaldehyde, N-p-coumarylserotonine (NTCS, 121, Figure 9) and N-p-coumaryl-tryptamine (NTCTR, 122, Figure 9) from the stem of Zea mays, which is cultivated worldwide as grain and feed, and NTCT and NTFT were isolated together with the well-known 4-hydroxybenzalde- hyde, N-p-coumarylserotonine (NTCS, 121, Figure 9) and N-p-coumaryl-tryptamine (NTCTR, 122, Figure 9) from the stem of Zea mays, which is cultivated worldwide as grain and feed and its seeds oil stigma spike leaf and root have been used in Chinese NTCT and NTFT were isolated together with the well-known 4-hydroxybenzaldehyde, N-p-coumarylserotonine (NTCS, 121, Figure 9) and N-p-coumaryl-tryptamine (NTCTR, 122, Figure 9) from the stem of Zea mays, which is cultivated worldwide as grain and feed, and NTCT and NTFT were isolated together with the well-known 4-hydroxybenzalde- hyde, N-p-coumarylserotonine (NTCS, 121, Figure 9) and N-p-coumaryl-tryptamine (NTCTR, 122, Figure 9) from the stem of Zea mays, which is cultivated worldwide as grain d f d d d l k l f d h b d Ch NTCT and NTFT were isolated together with the well-known 4-hydroxybenzaldehyde, N-p-coumarylserotonine (NTCS, 121, Figure 9) and N-p-coumaryl-tryptamine (NTCTR, 122, Figure 9) from the stem of Zea mays, which is cultivated worldwide as grain and feed, and Biomolecules 2021, 11, 1765 16 of 43 its seeds, oil, stigma, spike, leaf and root have been used in Chinese traditional medicines. Z. mays chloroformic extract showed antiacetylcholinesterase activity [101]. its seeds, oil, stigma, spike, leaf and root have been used in Chinese traditional medicines. Z. mays chloroformic extract showed antiacetylcholinesterase activity [101]. NTCT and NCCT were isolated together with the already known methyl-10,10- dimethoxydecanoate, methyl-10-hydroxy-8E,12Z-octadecadienoate, methylcoriolate, trans-phytol, phytene-1,2-diol, phyton, (3S,5R,6S,7E,9R)-7-megastigmene-3,6,9-triol, (3S,5R, 6S,9R)-3,6,9-trihydroxymegastigman-7-ene, shikimic acid, p-coumaramide, tryptophan, thymidine, adenosine and deoxyadenosine from the aqueous methanol extract of Hosta longipes (Liliaceae) [102]. This is an edible plant widely distributed in Korea, China and Japan and has been used in traditional Korean medicine for treating cough, laryngopharyn- gitis, burns, swelling, snake bites and inﬂammation. Further studies on the chemical metabolites produced by S. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources proliferation in the two cancer cell lines as Hela and Siha, showing stronger activity than cisplatin in the cytotoxic assay [105]. the two cancer cell lines as Hela and Siha, showing stronger activity than cisplatin in the cytotoxic assay [105]. proliferation in the two cancer cell lines as Hela and Siha, showing stronger activity than cisplatin in the cytotoxic assay [105]. y y NTCT, NTFT, NTCO and NTFO, were isolated together with two C-methylated flavonoids, namely 5,6-dimethoxy-7-hydroxy-8-methyl-flavone and 5,6-dimethoxy-8-methyl-2-phenyl- 7H-1-benzopyran-7-one (136 and 137, Figure 9), and an α-pyrone, namely 4-methoxy-6- (2-hydroxy-4-phenylbutyl)-2H-pyran-2-one (138, Figure 10). They were also isolated with 13 known compounds, including ﬁve amides, from Talinum triangulare (Portulacaceae) [106]. This plant, probably native to tropical America, was introduced to Nigeria and other tropi- cal regions in Africa as a leaf vegetable. Now it is one of the most important vegetables in Nigeria known as the “waterleaf” [107]. However, its leaves were also used for the treatment of peptic ulcer, cuts, wounds and scabies, and the roots’ decoction for hyperten- sion [108,109]. The other known compounds were identiﬁed as cannabisin F, grossamide, aurantiamide, aurantiamide acetate, aurantiamide benzoate, indole-3-carboxylic acid, p-hydroxy benzoic acid, 3β-hydroxystigmast-5,22-dien-7-one and 3β-hydroxystigmast-5- en-7-one. Any compound showed cytotoxic activity against L5178Y mouse lymphoma cell line [106]. NTCT, NTFT, NTCO and NTFO, were isolated together with two C-methylated fla- vonoids, namely 5,6-dimethoxy-7-hydroxy-8-methyl-flavone and 5,6-dimethoxy-8-me- thyl-2-phenyl-7H-1-benzopyran-7-one (136 and 137, Figure 9), and an α-pyrone, namely 4-methoxy-6-(2-hydroxy-4-phenylbutyl)-2H-pyran-2-one (138, Figure 10). They were also isolated with 13 known compounds, including five amides, from Talinum triangulare (Por- tulacaceae) [106]. This plant, probably native to tropical America, was introduced to Ni- geria and other tropical regions in Africa as a leaf vegetable. Now it is one of the most important vegetables in Nigeria known as the “waterleaf” [107]. However, its leaves were also used for the treatment of peptic ulcer, cuts, wounds and scabies, and the roots’ de- coction for hypertension [108,109]. The other known compounds were identified as can- nabisin F, grossamide, aurantiamide, aurantiamide acetate, aurantiamide benzoate, in- dole-3-carboxylic acid, p-hydroxy benzoic acid, 3β-hydroxystigmast-5,22-dien-7-one and 3β-hydroxystigmast-5-en-7-one. Any compound showed cytotoxic activity against L5178Y mouse lymphoma cell line [106]. Figure 10. The structures of compounds 138–151. NTCT was isolated together with 9,10-dihydrophenanthrene-1,5-dihydroxy-3,4,7- trimethoxy-9,10-dihydrophenanthrene (139, Figure 10) and 24 known compounds from the whole plants of Dendrobium moniliforme (Orchidaceae) [110]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources melongena, in addition to the three alkylamides NTCT, NCCT and NTFT [76] reported above, showed that it also produced other interesting amides. In particular, N-trans-sinapoyloctopamine (NTSO), N-trans-caffeoyloctopamine (NTCAO), N-trans-feruloylnoradrenline (NTFA) and N-cis- feruloylnoradrenline (NCFA) (123–124, 126, Figure 9) were isolated for the ﬁrst time as naturally occurring compounds together with the already known 3-(4-hydroxyphenyl)- N-[2-(4-hydroxyphenyl)-2-methoxyethyl] acrylamide, 3-(4-hydroxy-3-methoxyphenyl)-N- [2-(4-hydroxyphenyl)-2-methoxyethyl] acrylamide and N-trans-p-coumaroylnoradrenline (NTCA, 127, Figure 9) [103]. NTFT, NTCT, NCFT and NTFO were isolated together with (3R)-3,7-dihydroxy- 8-methoxy-3-(4′-methoxybenzyl)-4-chromanone (128, Figure 9), four ﬂavonoids, three steroids, pinoresinol and lanost-9-en-3β-ol from the leaves of Dracaena cochinchinensis (Lour.) S. C. Chen (Asparagaceae). The four flavonoids and the three steroids were iden- tified as (2S)-4′, 7-dihydroxy-3′-methoxy-8-methylflavan (2S)-3′,7-dihydroxy-4′-methoxy-8- methylflavan, 7-hydroxy-3-(4′-methoxybenzyl)-4-chromanone and 2′,4′,4-trihydroxychalcone and (22E)-3β-acetoxystigmasta-5,22-diene, β-sitosterol and β-daucosterol, respectivelt [104]. y g p NTCT was isolated together with 5 phenolic glycosides, named sargentodosides A-E (129–133, Figure 9), 2 dihydronaphthalene lignans, named sargentodognans F and G (134 and 135, Figure 9) and 31 known phenolic compounds from the ethanolic ex- tract of Sargentodoxa cuneata (Oliv.) Rehd. Et Wils (Lardizabalaceae) [105]. This plant is diffused in south, east, central and southwest China, and its stems are used in Chi- nese folk medicine for the treatment of rheumatic arthritis, abdominal pain, acute ap- pendicitis, trauma, dysmenorrhea, amenorrhea and painful menstruation. The known compounds were identiﬁed as (+)-isolariciresinol-9′-O-β-D-glucopyranoside, slvadoraside, glehlinoside C7-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-N2,N3-bis(4-hydroxyphenethyl)- 6-methoxy-1,2-dihydro-naphthalene-2,3-dicarboxamide, sargentol, cuneataside C, osman- thuside H, crosatoside B, echipuroside A, 6-(β-D-glucopyranosyloxy)-2R,4-dihydroxy-2-[(4- hydroxyphenyl)methyl]-3(2H)-benzofuranone, 6-(β-D-glucopyranosyloxy)-2S,4-dihydroxy- 2-[(4-hydroxyphenyl)methyl]-3(2H)-benzofuranone, 1-O-α-rhamnopyranosyl-(1”→6′)-O- β-D-glucopyranosyl-2-methoxy-4-acetylphenol, 1-O-α-L-rhamnosyl(1”-6′)-β-D-glucopyra- nosyloxy-3,4,5-trimethoxybenzene, 4-O-β-D-glucopyranosyl-3-hydroxylbenzoic acid, protocatecheuic acid 3-O-β-D-glucoside, caffeic, protocatechuic, vanillic and 3-O-caffeoylq- uinic acids, catechin, (−)-epicatechin, dulcisﬂavan, cinchonains Ia, hydroxytyrosol, acid, calceolarioside B, 2-(4-hydroxyphenyl)ethyl-[6-O-(E)-caffeoryl]-O-β-D-glucopyranoside, salidroside, 2-(3,4-dihydroxyphenyl)ethyl-O-β-D-glucopyranoside, icariside D2, methyl 3-O-caffeoylquinate and procyanidin B-2 [105]. Catechin, (−)-epicatechin, dulcisﬂavan, cinchonains Ia, caffeic acid, 2-(4-hydroxyphenyl)ethyl-[6-O-(E)-caffeoryl]-O-β-D-glucopyr- anoside, 2-(3,4-dihydroxyphenyl)ethyl-O-β-D-glucopyranoside and methyl 3-O-caffeoylqu- inate showed antibacterial activities against Staphylococcus aureus ATCC 29213 with MIC values in the range of 2–516 µg/mL. Hydroxytyrosol showed the highest activity against the same bacterium with an MIC value of 2 µg/mL, while no compound exhibited an- timicrobial activities against C. albicans ATCC 10231. Sargentol, cinchonains Ia and 2-(3,4- dihydroxyphenyl)ethyl-O-β-D-glucopyranoside signiﬁcantly inhibited the proliferation in Biomolecules 2021, 11, 1765 Biomolecules 2021, 11, x FO 17 of 43 18 of 42 17 of 43 18 of 42 the two cancer cell lines as Hela and Siha, showing stronger activity than cisplatin in the cytotoxic assay [105]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources This plant is widely dis- tributed in China, India, Korea and Japan, and its constituents showed different biological activities including antitumor, anti-inflammatory, antiplatelet and anti-aggregation activ- ities [111,112]. The other known compounds were identified as ashircinol, (2R,3S)-3-hy- droxymethyl-9-methoxy-2-(4′-hydroxy-3′,5′-dimethoxyphenyl)-2,3,6,7-tetrahydrophe- nanthro[4,3-b]furan-5,11-diol, diospyrosin, aloifol I, moscatilin, 3,4′-dihydroxy-3′,4,5-tri- methoxybibenzyl, gigantol, 3,3′-dihydroxy-4,5-dimethoxybibenzyl, longicornuol A, pap- razine, N-trans-feruloyl 3′-O-methyldopamine, moupinamide, dihydroconiferyldihydro- Figure 10. The structures of compounds 138–151. NTCT was isolated together with 9,10-dihydrophenanthrene-1,5-dihydroxy-3,4,7- trimethoxy-9,10-dihydrophenanthrene (139, Figure 10) and 24 known compounds from the whole plants of Dendrobium moniliforme (Orchidaceae) [110]. This plant is widely distributed in China, India, Korea and Japan, and its constituents showed different bio- logical activities including antitumor, anti-inﬂammatory, antiplatelet and anti-aggregation activities [111,112]. The other known compounds were identiﬁed as ashircinol, (2R,3S)-3- hydroxymethyl-9-methoxy-2-(4′-hydroxy-3′,5′-dimethoxyphenyl)-2,3,6,7-tetrahydrophe- nanthro[4,3-b]furan-5,11-diol, diospyrosin, aloifol I, moscatilin, 3,4′-dihydroxy-3′,4,5-trime- thoxybibenzyl, gigantol, 3,3′-dihydroxy-4,5-dimethoxybibenzyl, longicornuol A, paprazine, N-trans-feruloyl 3′-O-methyldopamine, moupinamide, dihydroconiferyldihydro-p-couma- Figure 10. The structures of compounds 138–151. Figure 10. The structures of compounds 138–151. Figure 10. The structures of compounds 138–151. Figure 10. The structures of compounds 138–151. NTCT was isolated together with 9,10-dihydrophenanthrene-1,5-dihydroxy-3,4,7- trimethoxy-9,10-dihydrophenanthrene (139, Figure 10) and 24 known compounds from the whole plants of Dendrobium moniliforme (Orchidaceae) [110]. This plant is widely dis- tributed in China, India, Korea and Japan, and its constituents showed different biological activities including antitumor, anti-inflammatory, antiplatelet and anti-aggregation activ- ities [111,112]. The other known compounds were identified as ashircinol, (2R,3S)-3-hy- droxymethyl-9-methoxy-2-(4′-hydroxy-3′,5′-dimethoxyphenyl)-2,3,6,7-tetrahydrophe- nanthro[4,3-b]furan-5,11-diol, diospyrosin, aloifol I, moscatilin, 3,4′-dihydroxy-3′,4,5-tri- methoxybibenzyl, gigantol, 3,3′-dihydroxy-4,5-dimethoxybibenzyl, longicornuol A, pap- razine, N-trans-feruloyl 3′-O-methyldopamine, moupinamide, dihydroconiferyldihydro- NTCT was isolated together with 9,10-dihydrophenanthrene-1,5-dihydroxy-3,4,7- trimethoxy-9,10-dihydrophenanthrene (139, Figure 10) and 24 known compounds from the whole plants of Dendrobium moniliforme (Orchidaceae) [110]. This plant is widely distributed in China, India, Korea and Japan, and its constituents showed different bio- logical activities including antitumor, anti-inﬂammatory, antiplatelet and anti-aggregation activities [111,112]. The other known compounds were identiﬁed as ashircinol, (2R,3S)-3- hydroxymethyl-9-methoxy-2-(4′-hydroxy-3′,5′-dimethoxyphenyl)-2,3,6,7-tetrahydrophe- nanthro[4,3-b]furan-5,11-diol, diospyrosin, aloifol I, moscatilin, 3,4′-dihydroxy-3′,4,5-trime- thoxybibenzyl, gigantol, 3,3′-dihydroxy-4,5-dimethoxybibenzyl, longicornuol A, paprazine, N-trans-feruloyl 3′-O-methyldopamine, moupinamide, dihydroconiferyldihydro-p-couma- NTCT was isolated together with 9,10-dihydrophenanthrene-1,5-dihydroxy-3,4,7- trimethoxy-9,10-dihydrophenanthrene (139, Figure 10) and 24 known compounds from the whole plants of Dendrobium moniliforme (Orchidaceae) [110]. This plant is widely dis- tributed in China, India, Korea and Japan, and its constituents showed different biological activities including antitumor, anti-inflammatory, antiplatelet and anti-aggregation activ- ities [111,112]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources The other known compounds were identified as ashircinol, (2R,3S)-3-hy- droxymethyl-9-methoxy-2-(4′-hydroxy-3′,5′-dimethoxyphenyl)-2,3,6,7-tetrahydrophe- nanthro[4,3-b]furan-5,11-diol, diospyrosin, aloifol I, moscatilin, 3,4′-dihydroxy-3′,4,5-tri- methoxybibenzyl, gigantol, 3,3′-dihydroxy-4,5-dimethoxybibenzyl, longicornuol A, pap- razine, N-trans-feruloyl 3′-O-methyldopamine, moupinamide, dihydroconiferyldihydro- NTCT was isolated together with 9,10-dihydrophenanthrene-1,5-dihydroxy-3,4,7- trimethoxy-9,10-dihydrophenanthrene (139, Figure 10) and 24 known compounds from the whole plants of Dendrobium moniliforme (Orchidaceae) [110]. This plant is widely distributed in China, India, Korea and Japan, and its constituents showed different bio- logical activities including antitumor, anti-inﬂammatory, antiplatelet and anti-aggregation activities [111,112]. The other known compounds were identiﬁed as ashircinol, (2R,3S)-3- hydroxymethyl-9-methoxy-2-(4′-hydroxy-3′,5′-dimethoxyphenyl)-2,3,6,7-tetrahydrophe- nanthro[4,3-b]furan-5,11-diol, diospyrosin, aloifol I, moscatilin, 3,4′-dihydroxy-3′,4,5-trime- thoxybibenzyl, gigantol, 3,3′-dihydroxy-4,5-dimethoxybibenzyl, longicornuol A, paprazine, N-trans-feruloyl 3′-O-methyldopamine, moupinamide, dihydroconiferyldihydro-p-couma- Biomolecules 2021, 11, 1765 18 of 43 18 of 43 rate, dihydrosinapyl dihydro-p-coumarate, 3-isopropyl-5-acetoxycyclohexene-2-one-1, p-hydroxybenzaldehyde, vanillin, p-hydroxyphenylpropionic, vanillic and protocatechuic acids , (+)-syringaresinol, β-sitosterol and daucosterol [110]. rate, dihydrosinapyl dihydro-p-coumarate, 3-isopropyl-5-acetoxycyclohexene-2-one-1, p-hydroxybenzaldehyde, vanillin, p-hydroxyphenylpropionic, vanillic and protocatechuic acids , (+)-syringaresinol, β-sitosterol and daucosterol [110]. NTCT, NTFT and NCFT were isolated together with 12 known compounds from sweet potato (Ipomoea batatas) leaf. The other known compounds were identiﬁed as 3,4,5- tricaffeoylquinic (3,4,5-triCQA), 3,4-dicaffeoylquinic (3,4-diCQA), 3,5-dicaffeoylquinic (3,5- diCQA), 4,5-dicaffeoylquinic (4,5-diCQA), 4,5-feruloylcourmaoylquinic and caffeic acids, caffeic acid ethyl ester, 7-hydroxy-5-methoxycoumarin, quercetin-3-O-α-D-glucopyranoside, 7,3′-dimethylquercetin, rhamnetin and indole-3-carboxaldehyde. NTCT, NTFT, NCFT and 3,4,5-triCQA showed the strongest α-glucosidase inhibition, while 3,4,5-triCQA and diC- QAs were the dominant antioxidants. The results of a SAR study demonstrated that higher caffeoylation of quinic acid and lower methoxylation of ﬂavonols induced stronger antioxi- dant activity, while methylation and cis-conﬁguration of phenethyl cinnamides weaken the α-glucosidase inhibition [113]. NTFT, NTCAT and NTCT were isolated from the leaves Miliusa cuneata (Annonaceae) organic extract together with ﬁve oxoprotoberberine alkaloids, named miliusacunines A–E (140–144, Figure 10). The twig extract of the same plant allowed researchers to identify ﬁve known metabolites as 5-hydroxy-3,7-dimethoxy-3′,4′-methylenedioxyﬂavone, pachypodol, 4′-hydroxy-3,5,7,3′-tetramethoxyﬂavone, (+)-miliusol and (+)-syringaresinol [114]. This plant as well as others belonging to the same genus are distributed from the Indian subcontinent to Indochina, the Malaysia Peninsula and the southeast Asian islands, to New Guinea and northern Australia. Some species are used in traditional medicine as a tonic and aphrodisiac and for gastropathy. All the compounds were assayed for cytotoxic activity against KB and Vero cancer cell lines and for antimalarial activity against the Plasmodium falciparum. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources Miliusacunine A (138) showed in vitro antimalarial activity against the TM4 strain, with an IC50 value of 19.3 ±3.4 µM, while miliusacunine B (139) exhibited strong activity against the K1 strain, with an IC50 value of 10.8 ± 4.1 µM. No compound showed cytotoxic activity [114]. y y NTFT and NTCT were isolated together with 5 7-methoxyﬂavonols with pyrogallol B-ring moieties (145−149, Figure 10), a ﬁsetinidol glucoside (150, Figure 10), a benzyl glycoside (151, Figure 10), and 23 known compounds [115] from Atraphaxis frutescens (L.) K. Koch (Polygonaceae). This is a shrub found in the Mongolian Gobi [116] and its dried aerial parts are used in traditional Mongolian medicine for detoxiﬁcation and to treat lymph disorders, bacterial fevers, throat infections and eye diseases, including cataracts [117]. The known compounds were identiﬁed as europetin 3-O-α-L-rhamnopyranoside, myricitrin, ﬁsetinidol, gallocatechin, catechin, afzelechin, aromadendrin, epigallocatechin, epicate- chin, nikoenoside, emodin 8-O-β-D-glucopyranoside, emodin 8-O-(6′-O-malonyl)glucoside, torachrysone 8-O-β-D-(6′-O-malonyl) glucopyranoside, syringaresinol, dehydroconiferyl alcohol, 3,4,5-trimethoxyphenyl 1-O-β-D-glucopyranoside and methyl syringate [115]. Compounds containing either a pyrogallol or a catechol B-ring moiety showed potent radi- cal scavenging activity, while insect phenoloxidase and mushroom tyrosinase were, respec- tively, inhibited by phenylpropanoid amides and by the characteristic 7-methoxyﬂavonol- 3-O-rhamnopyranosides [115]. NCFT, NTCAT and NTCT were isolated together with 11 new octahydroxylated C21 steroids, named with lyciumsterols A–K (152–162, Figure 11), and 13 already known compounds from the root bark of Lycium chinense, a plant used in Chines folk medicine as described above. Lyciumsterols B, C and G (153, 154, and 157) showed protective effects on pancreatic islet cells but were dose dependent, while lyciumsterols G–I and K, (158–160 and 162) exhibited autophagy activation [118]. Biomolecules 2021, 11, 1765 19 of 43 p southeas f 19 of 43 p southeas Asia and the USA, is used in folk Chinese medicine to treat inflammation, dy of the endocrine system, chapped skin, warts, arthritis and neuralgia [120]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources 152, R1=H, R2=S1, R3=H, R4=βOH 153, R1=S1, R2=H, R3=H, R4=βOH 154, R1=S1, R2=H, R3=G1, R4=αOH 155, R1=H, R2=S1, R3=G2, R4=βOH 156, R1=H, R2=S1, R3=G2, R4=αOH 157, R1=S1, R2=H, R3=G2, R4=βOH 158, R1=S1, R2=H, R3=H, R4=βOH 159, R1=S1, R2=H, R3=G3, R4=αOH 160, R1=H, R2=S1, R3=G4, R4=βOH 161, R1=S1, R2=H, R3=G4, R4=βOH 162, R1=S1, R2=H, R3=G4, R4=βOH R4 R3O OH R2O OR1 OH OH O H OH O S1 O O H OMe O O H OMe OH O H OH O O H OMe O O H O O OMe OH O H N H O O OH HO OMe 166 N H O MeO HO MeO MeO OH 163 N H O HO OH OMe 164 NO2 N H OH O O 165 HO HO O OMe O β-D-Glc β-D-Glc-6 OH O O O OH O OH OH O OH 167 G3 G4 Figure 11. The structures of compounds 152–167. Figure 11. The structures of compounds 152–167. 152, R1=H, R2=S1, R3=H, R4=βOH 153, R1=S1, R2=H, R3=H, R4=βOH 154, R1=S1, R2=H, R3=G1, R4=αOH 155, R1=H, R2=S1, R3=G2, R4=βOH 156, R1=H, R2=S1, R3=G2, R4=αOH 157, R1=S1, R2=H, R3=G2, R4=βOH 158, R1=S1, R2=H, R3=H, R4=βOH 159, R1=S1, R2=H, R3=G3, R4=αOH 160, R1=H, R2=S1, R3=G4, R4=βOH 161, R1=S1, R2=H, R3=G4, R4=βOH 162, R1=S1, R2=H, R3=G4, R4=βOH R4 R3O OH R2O OR1 OH OH O H OH O S1 O O H OMe O O H OMe OH O H OH O O H OMe O O H O O O H G3 152, R1=H, R2=S1, R3=H, R4=βOH 153, R1=S1, R2=H, R3=H, R4=βOH 154, R1=S1, R2=H, R3=G1, R4=αOH 155, R1=H, R2=S1, R3=G2, R4=βOH 156, R1=H, R2=S1, R3=G2, R4=αOH 157, R1=S1, R2=H, R3=G2, R4=βOH 158, R1=S1, R2=H, R3=H, R4=βOH 159, R1=S1, R2=H, R3=G3, R4=αOH 160, R1=H, R2=S1, R3=G4, R4=βOH 161, R1=S1, R2=H, R3=G4, R4=βOH 162, R1=S1, R2=H, R3=G4, R4=βOH R4 R3O OH R2O OR1 OH OH O H OH O S1 O O H OMe O O H OMe OH G3 N H O MeO HO MeO MeO OH 163 N H O HO OH OMe 164 NO2 N H OH O O 165 N H 164 N H O O OH HO OMe 166 N H O O HO OMe 166 HO HO O OMe O β-D-Glc β-D-Glc-6 OH O O O OH O OH OH O OH 167 Figure 11. The structures of compounds 152–167. Figure 11. The structures of compounds 152–167. Figure 11. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources The structures of compounds 152–167 Figure 11. The structures of compounds 152–167. NTCT, NTFT, NTCAT and NCFT were isolated together with two new phenolic am- ides, characterized as (7R,8S)-7-(4-hydroxy-3,5-dimethoxyphenyl)-8-hydroxy methyl-10- NCCT and NTCT were isolated together with 13 already known compounds from Coixlachryma-jobi var. mayuen (Gramineae). The already known compounds were identiﬁed as (7R,8S)-3′-demethyl-dehydrodiconiferyl alcohol-3′-O-β-glucopyranoside, (7R,8S)-3′- demethyl-9′-butoxy-dehydrodiconiferyl-3′-O-β-glucopyranoside, adenosine 2-O-caffeoyl isocitricacid, pseudolaroside, 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one, 2-O-β- glucopyranosyl-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, 2-O-β-glucopyranosyl-4-hydroxy- 7-methoxy-2H-1,4-benzoxazin-3(4H)-one, 2-O-β-D-glucopyranosyl-7-hydroxy-2H-1,4-ben- zoxazin-3(4H)-one, p-coumaric acid and caffeic acid ethyl ester, p-coumaric acid and coixol [119]. C.-joby var. mayuen, which is an annual, tropical plant native of Asia, namely Biomolecules 2021, 11, 1765 20 of 43 20 of 43 from India to peninsular Malaysia, and it is now diffused in southeast Asia and the USA, is used in folk Chinese medicine to treat inﬂammation, dysfunctions of the endocrine system, chapped skin, warts, arthritis and neuralgia [120]. from India to peninsular Malaysia, and it is now diffused in southeast Asia and the USA, is used in folk Chinese medicine to treat inﬂammation, dysfunctions of the endocrine system, chapped skin, warts, arthritis and neuralgia [120]. NTCT, NTFT, NTCAT and NCFT were isolated together with two new phenolic amides, characterized as (7R,8S)-7-(4-hydroxy-3,5-dimethoxyphenyl)-8-hydroxy methyl- 10-[N-7”-(4”-hydrxyphenyl)ethyl]carbamoylethenyl-3′-methoxybenzodihydrofuran and cis-N-p-hydroxycinnamoyl-7′-methoxyethyltyramine (163 and 164, Figure 11), together with eight known compounds from Nicandra physaloides (Solanaceae) [121]. This is an annual herb native to Peru, but it is diffused in Yunnan, Guangxi, Guizhou and some other Chinese provinces, where it is used in traditional folk medicine as sedative, expectorant, antipyretic and as an antidote. Its leaf extracts induced decrease blood sugar but also showed antitumor and insect antifeedant properties [122–124]. The other known com- pounds were identiﬁed as trans-N-feruloyloctopamine (NTFO), trans-N-feruloyl-7′-methox- yltyramine, cannabisin D, grossamide K, trans-N-hydroxycinnamoyl-7′-methoxyltyramine, erythro-canabisine H and cannabisin E. NCFT, NTFO and NTCAT showed signiﬁcant pro- tective activities on 1-methyl-4-phenylpyridiniumion (MPP+)-induced damage in human dopaminergic neuroblastoma cells (SH-SY5Y). The cell protection mechanism of NCFT was due to its ability to inhibit apoptosis and inducing cytoprotective autophagy in Parkinson’s disease (PD) [121]. NTCT, the aristolochic acid II alanine amide (165, Figure 11) and other known com- pounds were isolated from Aristolochia maurorum (Aristolochiaceae) [125]. This latter is a perennial herb that widely grows in Jordan [126]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources The other known compounds were iden- tiﬁed as palmitic acid, β-sitosterol, E-ethyl-p-coumarate, Z-ethyl-p-coumarate, aristolochic acid IV methyl ester, aristolactam I, loliolide, (+)-dehydrovomifoliol, glycerol-1-palmitate, aristolochic acid I, E-p-coumaric acid, β-sitosterylglucoside, aristolochic acid IV, aristolochic acid III, esculetin, uracil, shepherdine and adenosine [125]. NTFT and NTCT were isolated together with phenolic amide, named cis-terrestriamide (166, Figure 11), and seven known compounds from the fruits’ organic extract of Tribulus terrestris (Zygophyllaceae) [127]. This is an annual creeping plant is widely diffused in tropi- cal regions, including Korea, China and Japan, and its fruits have been used in folk medicine to treat dizziness, headache, high blood pressure, menstrual irregularity, pruritus, eye prob- lems, edema, abdominal distention, sexual dysfunction and cardiovascular diseases [128]. The known compounds are essentially the alkylamides N-trans-cinnamoyltyramine (1), N-trans-feruloyloctopamine and N-(2-(4-hydroxyphenyl)-2-methoxyethyl)cinnamamide, terrestriamide and ferulamide [127]. [ ] NTCT, NTFT, NCCT, NCFT and a ﬂavonoid glucoside, named ruthenicunoid A (167 Figure 11), were isolated together with ﬁve known compounds from the fruits of Lycium ruthenicun Murr. (Solanacea) [129]. This plant is diffused in the northwest regions of China, and its edible fruits are used for the treatment of hypertension, ureteral stones, tinea, furuncle and gingival bleeding [130–132]. The other known compounds were identiﬁed as N1,N10-bis(dihydrocaffeoyl)spermidine, N-trans-feruloyl-3′-O-methyldopamine N-trans- feruloyloctopamine (NTFO) and N-cis-feruloyloctopamine (NCFO) [129]. Ruthenicunoid A (167) and N1,N10-bis(dihydrocaffeoyl)spermidine showed the concentration-dependent inhibition of SIRT1 (full-length human protein/cytokine/chemokine/growth factor) [129]. NTFT, NTCT and the benzophenone C-glucoside, named pseuduvarioside (168, Figure 12), were isolated together with four known compounds from the leaves and stems of Pseuduvaria fragrans Y. C. F. Su, Chaowasku and R.M.K. Saunders (Annonaceae) [133]. This species was collected in peninsular Thailand [134]. The other known compounds were identiﬁed as (−)-guaiol, (+)-isocorydine, cyathocaline and isoursoline. NTFT and NTCT were noncompetitive inhibitors of α-glucosidase [133]. NTCT, NTFT, NCCT, NCFT and a ﬂavonoid glucoside, named ruthenicunoid A (167 Figure 11), were isolated together with ﬁve known compounds from the fruits of Lycium ruthenicun Murr. (Solanacea) [129]. This plant is diffused in the northwest regions of China, and its edible fruits are used for the treatment of hypertension, ureteral stones, tinea, furuncle and gingival bleeding [130–132]. The other known compounds were identiﬁed as N1,N10-bis(dihydrocaffeoyl)spermidine, N-trans-feruloyl-3′-O-methyldopamine N-trans- feruloyloctopamine (NTFO) and N-cis-feruloyloctopamine (NCFO) [129]. Ruthenicunoid A (167) and N1,N10-bis(dihydrocaffeoyl)spermidine showed the concentration-dependent inhibition of SIRT1 (full-length human protein/cytokine/chemokine/growth factor) [129]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources NTFT, NTCT and the benzophenone C-glucoside, named pseuduvarioside (168, Figure 12), were isolated together with four known compounds from the leaves and stems of Pseuduvaria fragrans Y. C. F. Su, Chaowasku and R.M.K. Saunders (Annonaceae) [133]. This species was collected in peninsular Thailand [134]. The other known compounds were identiﬁed as (−)-guaiol, (+)-isocorydine, cyathocaline and isoursoline. NTFT and NTCT were noncompetitive inhibitors of α-glucosidase [133]. Biomolecules 2021, 11, 1765 Bi l l 2021 11 FO 21 of 43 22 f 42 21 of 43 22 f 42 Figure 12. The structures of compounds 168–182. Figure 12. The structures of compounds 168–182. Figure 12. The structures of compounds 168–182 Figure 12. The structures of compounds 168–182. NTCT and cadinane sesquiterpenoid glucoside, characterized as 2β,7,3-trihy- droxycalamenene 3-O-β-D-glucoside (170, Figure 12) were isolated together with five known compounds from the stem bark of Abelmoschus sagittifolius (Malvaceae) [136]. This plant is considered an edible food in Hainan Island of China and southeast Asian coun- tries and widely used in folk medicine for the treatment of phthisis, cough, constipation, NTFT and NTCT were isolated together with an isoindole alkaloid, named oleraisoin- dole (169, Figure 12), together with four known compounds, from Portulaca oleracea L. (Portulacaceae) [135]. The known compounds were identiﬁed as 7′-ethoxy-trans-feruloylty- ramine, N-trans-feruloyl-3-methoxytyramine, aurantiamide and ferulic acid methyl ester. Oleraisoindole (169) inhibited NO production in RAW 264.7 cells induced by LPS [135]. y p g p neurasthenia, carbuncle sore swollen poison, dizziness and lumbocrural and stomach pains. The already known compounds were identified as N-(p-trans-coumaroyl)-N-me- thyltyramine, cleomiscosin A, 9,12,13-trihydroxy-10,15-heptadecadienoic acid, cyto- chalasin B and marmesinin. All the isolated metabolites showed moderate cytotoxicity against Hela and HepG-2 human cancer cell lines [136]. NTCT and two new phenylpropanoid esters, named bobulretulates A (171 and 172, Figure 12) were isolated together with 10 known compounds from the whole plants of Bulbophyllum retusiusculum (Orchidaceae) [137]. This plant is widely diffused in China, Nepal, Sikkim, Bhutan, India, Burma, Laos and Vietnam. The other already known com- pounds were identified as paprazine, dihydro-feruloyltyramine, guaiacylglycerol, p y NTCT and cadinane sesquiterpenoid glucoside, characterized as 2β,7,3-trihydroxycala- menene 3-O-β-D-glucoside (170, Figure 12) were isolated together with ﬁve known com- pounds from the stem bark of Abelmoschus sagittifolius (Malvaceae) [136]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources This plant is con- sidered an edible food in Hainan Island of China and southeast Asian countries and widely used in folk medicine for the treatment of phthisis, cough, constipation, neurasthenia, carbuncle sore swollen poison, dizziness and lumbocrural and stomach pains. The already known compounds were identiﬁed as N-(p-trans-coumaroyl)-N-methyltyramine, cleomis- cosin A, 9,12,13-trihydroxy-10,15-heptadecadienoic acid, cytochalasin B and marmesinin. All the isolated metabolites showed moderate cytotoxicity against Hela and HepG-2 human cancer cell lines [136]. neurasthenia, carbuncle sore swollen poison, dizziness and lumbocrural and stomach pains. The already known compounds were identified as N-(p-trans-coumaroyl)-N-me- thyltyramine, cleomiscosin A, 9,12,13-trihydroxy-10,15-heptadecadienoic acid, cyto- chalasin B and marmesinin. All the isolated metabolites showed moderate cytotoxicity against Hela and HepG-2 human cancer cell lines [136]. NTCT and two new phenylpropanoid esters, named bobulretulates A (171 and 172, Figure 12) were isolated together with 10 known compounds from the whole plants of Bulbophyllum retusiusculum (Orchidaceae) [137]. This plant is widely diffused in China, Nepal, Sikkim, Bhutan, India, Burma, Laos and Vietnam. The other already known com- pounds were identified as paprazine, dihydro-feruloyltyramine, guaiacylglycerol, NTCT and cadinane sesquiterpenoid glucoside, characterized as 2β,7,3-trihydroxycala- menene 3-O-β-D-glucoside (170, Figure 12) were isolated together with ﬁve known com- pounds from the stem bark of Abelmoschus sagittifolius (Malvaceae) [136]. This plant is con- sidered an edible food in Hainan Island of China and southeast Asian countries and widely used in folk medicine for the treatment of phthisis, cough, constipation, neurasthenia, carbuncle sore swollen poison, dizziness and lumbocrural and stomach pains. The already known compounds were identiﬁed as N-(p-trans-coumaroyl)-N-methyltyramine, cleomis- cosin A, 9,12,13-trihydroxy-10,15-heptadecadienoic acid, cytochalasin B and marmesinin. All the isolated metabolites showed moderate cytotoxicity against Hela and HepG-2 human cancer cell lines [136]. Biomolecules 2021, 11, 1765 22 of 43 22 of 43 NTCT and two new phenylpropanoid esters, named bobulretulates A (171 and 172, Figure 12) were isolated together with 10 known compounds from the whole plants of Bulbophyllum retusiusculum (Orchidaceae) [137]. This plant is widely diffused in China, Nepal, Sikkim, Bhutan, India, Burma, Laos and Vietnam. The other already known com- pounds were identiﬁed as paprazine, dihydro-feruloyltyramine, guaiacylglycerol, erythro- guaiacylglycerol, 4-(2-hydroxyethyl)-2-methoxyphenyl-β-D-glucopyranoside, thymidine, uridine, roseoside, 6,9-dihydroxy-4,7-megastigmadien-3-one and β-sitosterol [137]. y y g g NTCT, NTFT and NTCAT were isolated together with 17 already known compounds, including three sterols, three phenols, four anthraquinones, one chromone, two stilbenes, three ﬂavonoids and one organic acid from Fallopia convolvulus (L.) A. Löve (Fallopia) [138]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources This is an annual herbaceous plant distributed in different Chinese districts, and its roots were used to treat inﬂammation, insomnia, infection and arthritis. The known compounds were identiﬁed as stigmast-4-en-3-one, stigmast-4-en-3,6-dione, stigmast-4-en-3β,6α-diol, ethyl-p-hydroxybenzoate, emodin-1,6-dimethylether, 7-hydroxy-2,5-dimethylchromone, physcion, citreorosein, trans-resveratrol, piceatannol, p-hydroxybenzaldehyde, protocate- chuic acid, rhein, tricin, luteolin, myricetin and succinic acid [138]. y NTCT, two lignanamides, named majusamides A and B (173 and 174, Figure 12), and two alkaloids, named chelidoniumine and tetrahydrocoptisine-N-oxide (175 and 176, Figure 12), were isolated together with ﬁve known hydroxycinnamic acid amides (HCCA) from Chelidonium majus (Papaveraceae) organic extract [139]. This plant is widely dif- fused in the south and northeast of China, including Inner Mongolia, Jilin, Heilongjiang, Liaoning, Henan and other places. The main active components of C. majus are alka- loids that exhibited analgesia, anti-inﬂammatory, anti-microbial, antineoplastic, insec- ticidal and antioxidant activity [140,141]. The already known compounds were identi- ﬁed as N-trans-feruloyldopamine, N-trans-feruloyl-3-methoxytyramin, (E)-3-(4-hydroxy-3- methoxybenzylidene)-4-(4-hydroxyphenyl)pyrolidin-2-one and ferulamide [139]. Among all the metabolites tested, only N-trans-feruloyldopamine and (E)-3-(4-hydroxy-3-methoxy- benzylidene)-4-(4-hydroxyphenyl) pyrrolidin-2-one showed moderate anti-inﬂammatory activity on the NO production in lipopolysaccharide (LPS)-induced macrophages’ activities with IC50 values of 25.3 ± 0.5 and 23.5 ± 1.7 µM, respectively [139]. 50 µ p y [ ] NTFT was isolated together with 5 aristolactam alkaloids named dasymaschalolac- tams A−E (177–181, Figure 12), dasymaschalolactone (182, Figure 12) and 18 other known compounds from the twig extract of Dasymaschalon dasymaschalum (Annonaceae). This plant is distributed worldwide in tropical countries in Asia (Thailand and the Malaysian peninsu- lar) and Africa [142]. The known compounds were identiﬁed as oldhamactam, velutinam, enterocarpam-III, grifﬁthinam, goniopedalin, taliscanine, duguevalline, desmethoxykanu- gin, 7,8-dimethoxy-5-hydroxyﬂavone, alpinetin, 8-hydroxynaringenin-4′-methyl ether, 7-methoxyisobenzofuran-1(3H)-one benzyl benzoate, 2-methoxybenzyl benzoate paprazine, (−)-zeylenol and (+)-crotepoxide 4-hydroxybenzaldehyde. NTFT and paprazine showed α-glucosidase inhibition with IC50 values of 4.5 and 24.7 µM, respectively [142]. NTCT, NTFT, ﬁve rearranged clerodane diterpenoids, named 4-epi-baenzigeride A, its 4-O-D-glucoside, 4,12-di-epi-baenzigeride A, tinobaenzins A and B (183, 187, 184–1864, Figure 13), along with four known compounds were isolated from Tinospora baenzigeri (Menispermacae) stem organic extract [143]. This plant is widely diffused in Asia, Africa, Australia and the Paciﬁc [143–145] and in Thailand its decotion is used in traditional medicine for antipyretic and antimalarial treatment as well as its root extract. The other already known compounds were identiﬁed as baenzigeroside B, (+)-lariciresinol, caruilig- nan D and the aglycone of breyniaionoside D. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources Only the last two compounds and NTCT showed hepatoprotective activity against N-acetyl-p-aminophenol (APAP)-induced HepG2 cell damage at 10 µM with 17.0%, 19.2% and 39.0% inhibition, respectively [143]. 23 of 43 Biomolecules 2021, 11, 1765 g y y O O O H H OR1H O O R2 183, R1=H, R2=αH 184, R1=H, R2=βH 187, R1=D-Glc, R2=αH O O O H H OH H O R1 R2 185, R1= O O OH R2=βH 186, R1= O OMe R2=αH HO HO MeO N O O R3 R2 R3 188, R1=Me, R2=COOH, R3=OH 189, R1=Me, R2=COOH, R3=H 190, R1=H, R2=COOMe, R3=H O O O NH2 MeO OH 191 O O OH O 192, R=OH 193, R=OAc OAc OH O O 194 O O OAc OH 195 HO NH O HO NH O 196 197 Figure 13. The structures of compounds 183–197. Figure 13. The structures of compounds 183–197. O O O H H OR1H O O R2 183, R1=H, R2=αH 184, R1=H, R2=βH 187, R1=D-Glc, R2=αH 185, R1= O R2=βH R2=αH MeO HO N O O R3 R2 R3 188, R1=Me, R2=COOH, R3=OH 189, R1=Me, R2=COOH, R3=H 190, R1=H, R2=COOMe, R3=H O O O NH2 MeO OH 191 H O O O O NH2 MeO OH 191 188, R1=Me, R2=COOH, R3=OH 189, R1=Me, R2=COOH, R3=H 190, R1=H, R2=COOMe, R3=H 191 191 O O OAc OH 195 195 196 197 Figure 13. The structures of compounds 183–197. Figure 13. The structures of compounds 183–197. NTCT, NTFT, 4 alkaloids named goniochelienic acids A and B, methyl goniochelien- ate and goniochelieninone (188–191, Figure 13), 4 styryllactones, named (−)- (4S,5S,6R,7S,8S)-goniochelienlactone, its 7-O-acetyl derivative, (+)-(7S,8S)-goni- ochelienbutenolide A and (−)-(7S,8R)-goniochelienbutenolide B (192–195, Figure 13), to- gether with 13 known compounds, were isolated from the twig and leaf extracts of Goni- othalamus cheliensis (Annonaceae) [150]. This large tree is distributed throughout the world, but it is present essentially in southeast Asia [151] and is used in folk medicine to treat fever, scabies, edema, rheumatism, tympanites and typhoid fever [151,152]. The other already known compounds were identified as 3-methyl-1H-benz[f]indole-4,9-dione, NTCT, NCCT, NTFT, NCFT and some of their derivatives as well as that of NCAT were identiﬁed in fruits, leaves and root barks of Lycium barbarum (Solanaceae) by UPLC- Q-Orbitrap-MS/MS [146]. They are widely used in traditional Chinese prescriptions and patent medicines [147–149]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources The other 131 known compounds were identiﬁed using the same method and among them, 98, 28 and 35 constituents were detected in L. barbarum fruits, leaves and root barks, respectively. Dicaffeoylspermidine/sperminidine derivatives were the most detected compounds (74/131) while six saponins and 5,6-dihydrosolasonine were reported for the ﬁrst time in this plant. The root bark extract possessed the strongest antioxidative and cytotoxic activity [146]. (−)-goniobutenolide B, 7-epi-(−)-goniobutenolide B, (+)-goniodiol, goniodiol-8-monoace- tate, (+)-7-O-acetylgoniodiol, 8-acetoxy goniofufurone, isoaltholactone, (+)-glaberide I, (−)- glaberide I, (+)-syringaresinol, (+)-medioresinol, (+)-episyringaresinol, (−)-syringaresinol, (−)-episyringaresinol, (−)-pinoresinol, griffithazanone A, cleistopholine, vanillic, p-hy- droxybenzoic, p-methylbenzoic and trans-ferulic acids, 4-hydroxy-3-methoxypropiophe- non, 3,5-dimethoxy-4-hydroxypropiophenone, p-hydroxybenzaldehyde, ethyl-4-hydro- zybenzoate, (−)-(3R)-mellein methyl ether, derrusnin, 5-hydroxy-7-methoxy-3′,4′-meth- ylene dioxy isoflavone, derrustone, robustone methyl ether, derrugenin, robustigenin and NTCT, NTFT, 4 alkaloids named goniochelienic acids A and B, methyl goniochelienate and goniochelieninone (188–191, Figure 13), 4 styryllactones, named (−)-(4S,5S,6R,7S,8S)- goniochelienlactone, its 7-O-acetyl derivative, (+)-(7S,8S)-goniochelienbutenolide A and (−)-(7S,8R)-goniochelienbutenolide B (192–195, Figure 13), together with 13 known com- pounds, were isolated from the twig and leaf extracts of Goniothalamus cheliensis (Annonaceae) [150]. This large tree is distributed throughout the world, but it is present es- sentially in southeast Asia [151] and is used in folk medicine to treat fever, scabies, edema, rheumatism, tympanites and typhoid fever [151,152]. The other already known com- pounds were identiﬁed as 3-methyl-1H-benz[f]indole-4,9-dione, (−)-goniobutenolide B, 7-epi-(−)-goniobutenolide B, (+)-goniodiol, goniodiol-8-monoacetate, (+)-7-O-acetylgoniodiol, Biomolecules 2021, 11, 1765 24 of 43 24 of 43 8-acetoxy goniofufurone, isoaltholactone, (+)-glaberide I, (−)-glaberide I, (+)-syringaresinol, (+)-medioresinol, (+)-episyringaresinol, (−)-syringaresinol, (−)-episyringaresinol, (−)-pin- oresinol, grifﬁthazanone A, cleistopholine, vanillic, p-hydroxybenzoic, p-methylbenzoic and trans-ferulic acids, 4-hydroxy-3-methoxypropiophenon, 3,5-dimethoxy-4-hydroxypro- piophenone, p-hydroxybenzaldehyde, ethyl-4-hydrozybenzoate, (−)-(3R)-mellein methyl ether, derrusnin, 5-hydroxy-7-methoxy-3′,4′-methylene dioxy isoﬂavone, derrustone, robustone methyl ether, derrugenin, robustigenin and methyl-BRM-5 [150]. Among all the compounds tested for cytotoxicity against human colorectal cancer cells (HCT-116), grifﬁthazanone A was the most potent with an IC50 value of 2.39 µM [150]. g p NTCT was isolated together with 14 alkaloids, including 2 indole alkaloids, 1 quino- line alkaloid, 2 pyridine alkaloids, 4 carbazole alkaloids and 3 amides from the aerial parts of Clausena lansium Lour. Skeels (Rutaceae). These metabolites were identiﬁed as 3-oxoindole and indole-3-carboxaldehyde, dictamine, murrayanine, claulansine G, clau- sine I, O-demethylmurrayanine, atanine, 4-methoxy-1H-quinolin-2-one and 4-methoxy- 1-methylquinolin-2-one. Among all the compounds assayed for their cytotoxic activity against Hela cancer cell line, four carbazole alkaloids, murrayanine, claulansine G, clausine I and O-demethylmurrayanine, showed weak cytotoxicity with IC50 values ranging from 69.31 to 138.32 µM [153]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources The structures of compounds 198–203. Figure 14. The structures of compounds 198–203. O O H H H HO O O O H HO O O O H HO O OH OH 198 199 200 O H 201 O HO2C H H H H H H 199 199 OH O O OH OH O H 201 O HO2C H H O O H HO O OH OH 198 200 O H H H 198 200 201 HO OH OH OH OMe OMe OH H 202 HO OH HO OMe OH O O HO OH HO O O O O OMe 203 203 Figure 14. The structures of compounds 198–203. Figure 14. The structures of compounds 198–203. NTCT, NTFT, NTFO and a previously undescribed cerebroside named eloundemno- side (204, Figure 15) were isolated together with 17 known compounds from the roots of Celtis adolphi-friderici Engl. (Cannabaceae) [162]. This semi-deciduous tree is diffused in the center region of Cameroon and known as “odou” by the Ewondo tribe, where its bark fruits and leaves are used in folk medicine to treat severe cough, fever, headache, tuber- culosis and sore eyes [163]. The other known compounds were identified as β-sitosterol, heptacosanoic vanilic azelaic, laceroic hydroxybenzoic and aspartic acids, 3-carboxalde- hyde, glycerol, 1-octadecanoate β-sitosterol-3-O-β-D-glucopyranoside, sapiol, indole and allantoin [162]. Heptacosanoic, vanilic and azakleic acids showed good antioxidant activ- NTCT, NTFT, NTST, a previously undescribed arylbenzofuran rhamnoside named aristolochiaside (203, Figure 14) and seven known compounds were isolated from Isotrema tadungense (Aristolochiaceae), from which the extract showed signiﬁcant cytotoxic activ- ity [161]. It is a plant essentially distributed in Vietnam. The already known compounds were identiﬁed as aristolactam AIIIa, aristololactam CII, grossamide, cannabisin D, melon- genamide, cannabisin F and N-trans-feruloyldopamine. Among the isolated compounds, aristolochiaside, aristolactam AIIIa and NTST showed strong and selective cytotoxicity on the HeLa human cancer cell line with IC50 values of 7.59 ± 1.03, 8.51 ± 1.73 and 9.77 ± 1.25 µM, respectively [161]. [ ] p , g ities with IC50 values of 22.2, 29.3 and 13.2 μM, respectively. Azelaic acid is also a strong inhibitor of lipoxygenase (IC50 value of 16.3 μM), while friedelin exhibited the highest in- hibition of urease with an IC50 value of 15.3 μM. However, all the compounds tested showed a moderate butyrylcholinesterase inhibition [162]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources NTCT was isolated together with speretin, 4-methoxyquinolin-2-one, pinoresinol, medioresinol, syringaresinol, N-benzoyl-L-phenylalaninol, L-sesamin, diosmetin, zhebeiresinol, vitexin and isoscopletin from the organic extract of Zanthoxylum nitidum (Roxb.) DC. (Rutaceae) leaves. Z. nitidium is widely used in traditional Chinese herbal medicines [154]. NTCT, NTCAT and two ceramides, named celtisamides A and B (196 and 197, Figure 13) were isolated together with platanic and betulinic acids, the (0.6:0.4) mixture of oleanolic and ursolic acids, friedelin, β-sitosterol, and β-sitosterol 3-O-β-D-glucoside and betulinic acid from the stem bark of Celtis tessmannii Rendle (Cannabaceae). p-Hydroxybenzoyl, p-coumaric acid anhydride, glucosyringic acid, cis-1-O-methyl-inositol and succinic acid were isolated from the root organic extract of the same plant [155]. C. tessmannii is used as analgesic and for the treatment of diarrhea, fever, inﬂammation of respiratory organs, tachycardia, anemia, gangrene, sexual weakness, insomnia, nervosity, muscles pain and malaria [156]. All the metabolites were tested for antiplasmodium and cytotoxic activities. cis-1-O-Methylinositol (IC50 = 14.3 µM) showed the strongest inhibition of urease, while succinic acid (IC50 = 12.9 µM) exhibited the best inhibition against lipoxygenase. Succinic acid (IC50 = 9.5 µM) showed the best DPPH radical scavenging activity, while betulinic acid exhibited a strong (IC50 values ranging from 1.87-2.34 µg/mL) against chloroquine- sensitive (Pf 3D7), and chloroquine-resistant (Pf Dd2 and Pf INDO) strains of Plasmodium falciparum [155]. NTCT, NTFT and NTFO were isolated together with four new steroidal sapogenins, named dracaenogenins C–F (198–201, Figure 14), a new conjugated chalcone-stilbene, 3′′-methoxycochinchinenene H (202, Figure 14), and eight known compounds from the stems of Dracaena usambarensis Engl. (Asparagaceae) [157]. The organic extracts of this tree showed anticancer [158], anti-inﬂammatory [159] and antimicrobial properties [158] and antiestrogenic, antioxidative, and bacteriostatic activities [160]. 3′′-Methoxycochinchinenene H (202), 4,4′-dihydroxy-3′-methoxychalcone and grossamide tested at 100 µM were sub- stantially more potent than ibuprofen, inhibiting the release of all the cytokines, IL-1β, IL-2, GM-CSF and TNF-α from 0.06% to 58.04% compared to LPS control. Trans-resveratrol signiﬁcantly reduced the GM-CSF (6.11% of LPS control) and TNF-α (18.35% of LPS control) release [157]. 25 of 43 26 of 42 Biomolecules 2021, 11, 1765 Biomolecules 2021 11 x FOR O O H H H HO O O O H HO O O O H HO O OH OH 198 199 200 O H 201 O HO2C HO OH OH OH OMe OMe OH H 202 O O HO OH HO O O O O OMe 203 H H H H H H Figure 14. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources NTFT, NTCAT, NTCT and two previously undescribed phenolic imidates, named fistuloimidates A and B (205 and 206, Figure 15), were isolated together with persicoimi- date, N-coumaroyltyrosine, isorhamnetin-3-O-galactopyranoside and 1-O-(4-hy- droxybenzoyl)-β-D-glucopyranose from the extract of the previously described A. fistu- losum [164]. Fistuloimidate A (205) and 1-O-(4-hydroxybenzoyl)-β-D-glucopyranose showed antibacterial activity against E. coli with MIC values of 2000 and 1000 μg/mL, re- spectively, while fistuloimidate B (206) showed the same activity against both E. coli and S. aureus with MIC values of 7.8 and 3.9 μg/mL respectively. Persicoimidate and N-cou- maroyltyrosine showed the same activity against S. aureus with MIC values for both com- p y NTCT, NTFT, NTFO and a previously undescribed cerebroside named eloundemno- side (204, Figure 15) were isolated together with 17 known compounds from the roots of Celtis adolphi-friderici Engl. (Cannabaceae) [162]. This semi-deciduous tree is diffused in the center region of Cameroon and known as “odou” by the Ewondo tribe, where its bark fruits and leaves are used in folk medicine to treat severe cough, fever, headache, tuberculosis and sore eyes [163]. The other known compounds were identiﬁed as β-sitosterol, heptacosanoic vanilic azelaic, laceroic hydroxybenzoic and aspartic acids, 3-carboxaldehyde, glycerol, 1-octadecanoate β-sitosterol-3-O-β-D-glucopyranoside, sapiol, indole and allantoin [162]. Heptacosanoic, vanilic and azakleic acids showed good antioxidant activities with IC50 values of 22.2, 29.3 and 13.2 µM, respectively. Azelaic acid is also a strong inhibitor of lipoxygenase (IC50 value of 16.3 µM), while friedelin exhibited the highest inhibition of ure- ase with an IC50 value of 15.3 µM. However, all the compounds tested showed a moderate butyrylcholinesterase inhibition [162]. Biomolecules 2021, 11, 1765 26 of 43 ytotoxic p y [ ] Figure 15. The structures of compounds 204–208. Figure 15. The structures of compounds 204–208. s 204 208 Figure 15. The structures of compounds 204–208. Figure 15. The structures of compounds 204–208. NCCT, NTFT, NTCT, 2 previously undescribed tetrahydroprotoberberine, named 7R,14S-cis-tetrahydrocoptisine N-oxides and 7R,14R-trans-tetrahydrocoptisine N-oxide (207 and 208, Figure 15), and 11 known compounds were isolated from the aerial parts of Chelidonium majus L. (Papaveraceae) [165]. The known compounds were identified as im- patien B, spallidamine, oxychelerythrine, dihydrosanguinarine, N-demethyloxysangui- narine, chelidonine, isochelidonine, 4-[formyl-5-methoxymethyl-1H-pyrol-1-yl] buta- noate, noroxyhydrastinine, 3,4-dehydrotheaspirone and loliolide. 7R,14R-trans-Tetrahy- drocoptisine N-oxide (208), N-demethyloxysanguinarine, chelidonine, isochelidonine, NTCT, 4-[formyl-5-methoxymethyl-1H-pyrol-1-yl] butanoate and 3,4-dehydrotheaspi- rone inhibited the nitric oxide production in LPS-induced RAW 264.7 macrophages with the IC50 values ranging from 1.1 to 31.9 μM [165]. ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of ramine. 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of 3. Structure and Biological Activity of Cinnamoyltyramine Alkylamides and of Co-Metabolites Isolated from the Same Natural Sources NTFT, NTCAT, NTCT and two previously undescribed phenolic imidates, named ﬁs- tuloimidates A and B (205 and 206, Figure 15), were isolated together with persicoimidate, N-coumaroyltyrosine, isorhamnetin-3-O-galactopyranoside and 1-O-(4-hydroxybenzoyl)- β-D-glucopyranose from the extract of the previously described A. ﬁstulosum [164]. Fistuloimidate A (205) and 1-O-(4-hydroxybenzoyl)-β-D-glucopyranose showed antibacte- rial activity against E. coli with MIC values of 2000 and 1000 µg/mL, respectively, while ﬁstuloimidate B (206) showed the same activity against both E. coli and S. aureus with MIC values of 7.8 and 3.9 µg/mL respectively. Persicoimidate and N-coumaroyltyrosine showed the same activity against S. aureus with MIC values for both compounds of 250 µg/mL. Among all the compounds tested against the breast cancer cell line MCF-7, persicoimidate and isorhamnetin 3-O-galactopyranoside showed low cytotoxic effects in a dose-dependent manner with IC50 values of 94.4 ± 5.1 and 94.1 ± 1.8 µg/mL, respectively [164]. 4. Conclusions The sources and biological activities of both E- and Z-diastereomers of p-coumaroyl- , caffeoyl-, feruloyl-, 5-hydroxyferuloyl- serotonine-, sinapoyl- and tryptamine-tyramine alkylamides and other related alkylamides described in the text are summarized in Table 1, while those of the co-metabolites isolated from the same sources are reported in Table 2. Among the alkylamides, NTCT is that produced by several plants belonging to different species followed by NTFT and NCFT. Some promising activities were also reported for them suggesting their potential use in different fields. However, further studies are needed to determine their mode of actions as well as suitable formulations should be pre- pared for their practical applications. g p y NCCT, NTFT, NTCT, 2 previously undescribed tetrahydroprotoberberine, named 7R,14S-cis-tetrahydrocoptisine N-oxides and 7R,14R-trans-tetrahydrocoptisine N-oxide (207 and 208, Figure 15), and 11 known compounds were isolated from the aerial parts of Chelidonium majus L. (Papaveraceae) [165]. The known compounds were identiﬁed as impa- tien B, spallidamine, oxychelerythrine, dihydrosanguinarine, N-demethyloxysanguinarine, chelidonine, isochelidonine, 4-[formyl-5-methoxymethyl-1H-pyrol-1-yl] butanoate, norox- yhydrastinine, 3,4-dehydrotheaspirone and loliolide. 7R,14R-trans-Tetrahydrocoptisine N-oxide (208), N-demethyloxysanguinarine, chelidonine, isochelidonine, NTCT, 4-[formyl- 5-methoxymethyl-1H-pyrol-1-yl] butanoate and 3,4-dehydrotheaspirone inhibited the nitric oxide production in LPS-induced RAW 264.7 macrophages with the IC50 values ranging from 1.1 to 31.9 µM [165]. N-trans-p-Coumaroyltyramine (NTCT, paprazine, 4, Figure 2) Table 1. Alkyl 4. Conclusions e Source Biological Activity References ne (1, Scheme 3) A. yunnanensis No activity [95] CFT, 2, Figure 2) C. annuum var. grossum “ [13,14] C. annuum “ [29] P. suberosa “ [32,34] The sources and biological activities of both E- and Z-diastereomers of p-coumaroyl-, caffeoyl-, feruloyl-, 5-hydroxyferuloyl- serotonine-, sinapoyl- and tryptamine-tyramine alkylamides and other related alkylamides described in the text are summarized in Table 1, while those of the co-metabolites isolated from the same sources are reported in Table 2. Among the alkylamides, NTCT is that produced by several plants belonging to different species followed by NTFT and NCFT. Some promising activities were also reported for them suggesting their potential use in different ﬁelds. However, further studies are needed to determine their mode of actions as well as suitable formulations should be prepared for their practical applications. 27 of 43 Biomolecules 2021, 11, 1765 Table 1. Alkylamides, their sources and biological activities. Alkylamide Source Biological Activity References N-trans-Cinnamoyltyramine (1, Scheme 3) A. yunnanensis No activity [95] N-cis-Feruloyltyramine (NCFT, 2, Figure 2) C. annuum var. grossum “ [13,14] C. annuum “ [29] P. suberosa “ [32,34] A. elegans “ [38] P. longifolia var. pendula “ [42] P. hyrcanicum “ [82] N. nucifera Inhibition of pancreatic lipase [88] D. cochinchinensis No activity [104] I. batatas Inhibition of α-glucosidase [113] L. chinense No activity [118] N. physaloides Inhibition of apoptosis and cytoprotective [121] L. ruthenicun No activity [129] L. barbarum “ [146] N-trans-p-Coumaroyltyramine (NTCT, paprazine, 4, Figure 2) C. annuum var. grossum “ [13,14] S. melongena “ [15] A. chinense Inhibition of thromboxane and prostaglandin synthetase [23] A. triloba No activity [26] I. maitlandii “ [27] C. annuum “ [29,30] A. mollissima “ [28] P. suberosa Anticancer activity and inhibition of protein tyrosine kinases [32–35] C. chinensis Inhibition of acetylcholinesterase [36,37] A. elegans No activity [38] P. sanctum Antibiotic [41] P. longifolia var. pendula No activity [42] S. tupiniquinorum “ [43] P. duclouxii “ [44] B. vulgaris “ [45] C. asiaticum var. sinicum “ [52] T. sinensis “ [54] P. nigrum “ [58] C. gaudichaudianus “ [59] D. opposita Antidiabetic [64] A. ﬁstulosum No activity [67] C. annum “ [75] S. melongena Antidiabetic [76,103] P. hyrcanicum Antiprotozoal [80] Table 1. Alkylamides, their sources and biological activities. Biomolecules 2021, 11, 1765 28 of 43 Table 1. Cont. Source Biological Activity References L. chinense Moderate radical scavenging, anti-inﬂammatory and antidiabetic [83,97,118] P. Table 1. Alkyl 4. Conclusions oleracea No activity [85,135] N. nucifera Inhibition of pancreatic lipase [88] S. buddleifolium No activity [90] C. lansium “ [98] Z. mays “ [101] H. longipes “ [102] D. cochinchinensis “ [104] S. cuneata “ [105] T. triangulare “ [106] D. moniliforme “ [110] I. batatas Inhibition of α-glucosidase [113] M. cuneata No activity [114] A. frutescens “ [115] C.-jobi var. mayuen “ [119] N. physaloides “ [121] A. maurorum “ [125] T. terrestris “ [127] L. ruthenicun “ [129] P. fragrans Inhibition of α-glucosidase [133] A. sagittifolius Moderate cytotoxicity [136] B. retusiusculum No activity [137] F. convolvulus “ [138] C. majus “ [139] D. dasymaschalum Inhibition of α-glucosidase [142] T. baenzigeri Hepatoprotective activity [143] L. barbarum No activity [146] G. cheliensis “ [150] C. lansium “ [153] Z. nitidum “ [154] C. tessmannii “ [155] D. usambarensis “ [156] I. tadungense “ [161] C. adolphi-friderici “ [162] A. ﬁstulosum “ [164] C. majus Inhibition of NO production in RAW 264.7 cells [165] C. annuum var. grossum No activity [13,14] S. melongena “ [15] F. indica “ [25] Alkylamide 29 of 43 Biomolecules 2021, 11, 1765 Table 1. Cont. Table 1. Cont. Table 1. Cont. Source Biological Activity References A. triloba “ [26] C. annuum “ [29] P. suberosa “ [32,34] A. elegans “ [38] P. sanctum “ [41] P. longifolia var. pendula “ [42] S. tupiniquinorum “ [43] P. duclouxii “ [44] C. gaudichaudianus “ [59] A. ﬁstulosum Radical scavenging [67] S. melongena Antidiabetic [76,103] P. hyrcanicum Antiprotozoal [80] L. chinense Moderate radical scavenging, anti-inﬂammatory and antidiabetic [83,97] P. oleracea Anti-inﬂammatory [85,135] N. nucifera No activity [88] S. buddleifolium “ [90] P. ﬂaviﬂorum “ [92] Z. mays “ [101] D. cochinchinensis “ [104] T. triangulare “ [106] I. batatas Inhibition of α-glucosidase [113] M. cuneata No activity [114] A. frutescens “ [115] N. physaloides “ [121] T. terrestris “ [127] L. ruthenicun “ [129] P. fragrans Inhibition of α-glucosidase [133] F. convolvulus No activity [138] D. dasymaschalum Inhibition of α-glucosidase [142] T. baenzigeri No activity [143] L. barbarum “ [146] G. cheliensis “ [150] D. usambarensis “ [157] I. tadungense “ [161] C. adolphi-friderici “ [162] A. ﬁstulosum “ [164] C. majus “ [165] Capsicum annuum var. grossum “ [13,14] Solanum melongena L. “ [15] T. triangulare “ [106] 30 of 43 Biomolecules 2021, 11, 1765 Table 1. Cont. Table 1. Alkyl 4. Conclusions Alkylamide Source Biological Activity References N-trans-Feruloyloctopamine (NTFO, 7, Figure 2) Capsicum annuum var. grossum “ [13,14] Solanum melongena L. “ [15] D. cochinchinensis “ [104] T. triangulare “ [106] N. physaloides Cytoprotective [121] L. ruthenicun No activity [129] C. adolphi-friderici “ [162] N-cis-p-Cumaroyltyramine (NCCT, 11, Figure 2) A. chinense Inhibition of prostaglandin and thromboxane synthetase [23] A. mollissima No activity [28] C. annuum “ [29] A. elegans “ [38] P. duclouxii “ [44] D. opposita “ [64] S. melongena Antidiabetic [76,103] N. nucifera No activity [88] C. lansium “ [98] H. longipes “ [102] C.-jobi var. mayuen “ [119] L. ruthenicun “ [129] L. barbarum “ [146] C. majus “ [165] N-trans-Sinapoyltyramine (NTST, 52, Figure 4) P. longifolia var. pendula “ [42] I. tadungense “ [161] N-trans-Caffeoyltyramine (NTCAT, 59, Figure 5) C. asiaticum var. sinicum “ [52] C. asiaticum “ [75] P. hyrcanicum Antiprotozoal [80] L. chinense Moderate radical scavengingNF-κB inhibitory [83,97,118] S. buddleifolium No activity [90] M. cuneata “ [114] N. physaloides Cytoprotective [121] F. convolvulus No activity [138] C. tessmannii “ [155] A. ﬁstulosum “ [164] 3′-Methoxy-NTFT (86, Figure 7) A. ﬁstulosum Radical scavenging [67] 4′-O-Methyl-TNCT (88, Figure 7) C. annuum No activity [75] 4′-O-Methyl-TNCAT (89, Figure 7) C. annuum “ [75] Table 1. Cont. Table 1. Cont. 31 of 43 Biomolecules 2021, 11, 1765 Table 1. Cont. Alkylamide Source Biological Activity References N-cis-feruloyloctopamine (NCFO, 91, Figure 7) L. chinense Moderate radical scavenging, anti-inﬂammatory and antidiabetic [83] L. ruthenicun No activity [129] D. usambarensis “ [157] N-trans-p-oumaroylserotonine (NTCS, 121, Figure 9) Z. mays “ [101] N-trans.-p-coumaroyltryptamine (NTCTR, 122, Figure 9) “ “ “ N-trans-sinapoyloctopamine (NTSO, 123, Figure 9) S. melongena “ [103] N-trans-caffeoyloctopamine (NTCAO, 124, Figure 9) “ “ “ N-trans-feruloylnoradrenline (NTFA, 125, Figure 9) “ “ “ N-cis-feruloylnoradrenline (NCFA 126, Figure 9) “ “ “ N-trans-p-coumaroylnoradrenline (NTCA, 127, Figure 9) “ “ “ Table 2. Co-metabolites, their sources, and biological activities. Metabolite Source Biological Activity References Grossamide (3, Figure 2) Capsicum annuum var. grossum No activity [1,14] Hordatin A (8, Figure 2) H. vulgare Antifungal [17] Hordatin B (9, Figure 2) “ “ “ Hordatin M (10, Figure 2) “ “ “ Lunularic acid (12, Figure 2) A. chinense Inhibition of thromboxane and prostaglandin synthetase [23] Rhapontigenin (13, Figure 2) R. Table 1. Alkyl 4. Conclusions rhabarbarum “ [24] Piceatannol, (14, Figure 2) “ “ “ Rhaponticin (15, Figure 2) “ “ “ Piceatannol glucoside (16, Figure 2) “ “ “ Mandolin S (17, Figure 2) A. mollissima No activity [29,30] Mandolin R (18, Figure 2) “ “ “ Mandolin U (19, Figure 2) “ “ “ Mandolin W (20, Figure 3) “ “ “ Mandolin X (21, Figure 3) “ “ “ Canusesnol A (22, Figure 3) C. annuum Cytotoxic [31] Canusesnol B (23, Figure 3) “ No activity “ Canusesnol C (24, Figure 3) “ “ “ Canusesnol D (25, Figure 3) “ “ “ Canusesnol E (26, Figure 3) “ “ “ Canusesnol F (27, Figure 3) “ “ “ Canusesnol G (28, Figure 3) “ “ “ Table 1. Cont. Alkylamide Source Biological Activity References N-cis-feruloyloctopamine (NCFO, 91, Figure 7) L. chinense Moderate radical scavenging, anti-inﬂammatory and antidiabetic [83] L. ruthenicun No activity [129] D. usambarensis “ [157] N-trans-p-oumaroylserotonine (NTCS, 121, Figure 9) Z. mays “ [101] N-trans.-p-coumaroyltryptamine (NTCTR, 122, Figure 9) “ “ “ N-trans-sinapoyloctopamine (NTSO, 123, Figure 9) S. melongena “ [103] N-trans-caffeoyloctopamine (NTCAO, 124, Figure 9) “ “ “ N-trans-feruloylnoradrenline (NTFA, 125, Figure 9) “ “ “ N-cis-feruloylnoradrenline (NCFA 126, Figure 9) “ “ “ N-trans-p-coumaroylnoradrenline (NTCA, 127, Figure 9) “ “ “ Table 1. Cont. Table 2. Co-metabolites, their sources, and biological activities. Metabolite Source Biological Activity References Grossamide (3, Figure 2) Capsicum annuum var. grossum No activity [1,14] Hordatin A (8, Figure 2) H. vulgare Antifungal [17] Hordatin B (9, Figure 2) “ “ “ Hordatin M (10, Figure 2) “ “ “ Lunularic acid (12, Figure 2) A. chinense Inhibition of thromboxane and prostaglandin synthetase [23] Rhapontigenin (13, Figure 2) R. rhabarbarum “ [24] Piceatannol, (14, Figure 2) “ “ “ Rhaponticin (15, Figure 2) “ “ “ Piceatannol glucoside (16, Figure 2) “ “ “ Mandolin S (17, Figure 2) A. mollissima No activity [29,30] Mandolin R (18, Figure 2) “ “ “ Mandolin U (19, Figure 2) “ “ “ Mandolin W (20, Figure 3) “ “ “ Mandolin X (21, Figure 3) “ “ “ Canusesnol A (22, Figure 3) C. Table 1. Alkyl 4. Conclusions annuum Cytotoxic [31] Canusesnol B (23, Figure 3) “ No activity “ Canusesnol C (24, Figure 3) “ “ “ Canusesnol D (25, Figure 3) “ “ “ Canusesnol E (26, Figure 3) “ “ “ Canusesnol F (27, Figure 3) “ “ “ Canusesnol G (28, Figure 3) “ “ “ Table 2. Co-metabolites, their sources, and biological activities. Biomolecules 2021, 11, 1765 32 of 43 Table 2. Cont. Metabolite Source Biological Activity References Canusesnol H (29, Figure 3) “ “ “ Canusesnol I (30, Figure 3) “ “ “ Canusesnol J (31, Figure 3) “ “ “ Aristolactam E (32, Figure 3) A. elegans “ [38] Aristolactam-AIIIa-6-O-β-D-glucoside (33, Figure 3) “ “ “ Aristoquinoline A (34, Figure 3) “ “ “ Aristoquinoline B (35, Figure 3) “ “ “ Aristoquinoline C (36, Figure 3) “ “ “ Aristogin F (37, Figure 3) “ “ “ 2-Oxo-12-(3′,4′- methylenedioxyphenyl)dodecane (38, Figure 4) P. sanctum Antibiotic [41] 2-Oxo-14-(3′,4′- methylenedioxyphenyl)tetradecane (39, Figure 4) “ “ “ (40, Figure 4) “ No activity “ 2-Oxo-18-(3′,4′- methylenedioxyphenyl)octadecane (41, Figure 4) “ “ “ 2-Oxo-14-(3’,4’-methylenedioxyphenyl)- trans-13-tetradecene (42, Figure 4) “ “ “ 2-Oxo-16-(3′,4′-methylenedioxyphenyl)- trans-15-hexadecene (43, Figure 4) “ Antibiotic “ 2-Oxo-18-(3′,4′-methylenedioxyphenyl)- trans-17-octadecene (44, Figure 4) “ No activity “ 2-Oxo-16-phenyl-trans-3-hexadecene (45, Figure 4) “ “ “ Methyl [6-(10-phenyldecanyl)tetrahydropyran-2- yl]acetate (46, Figure 4) “ “ “ Methyl 2-(6-tridecyltetrahydro-2H-pyran-2- yl)acetate (47, Figure 4) “ “ “ Methyl 2-(5-tetradecyltetrahydro-2- furanyl)acetate (48, Figure 4) “ “ “ 2-Oxo-14-(3′,4′-methylenedioxyphenyl)- trans-3-tetradecene (49, Figure 4) “ “ “ 2-Oxo-16-(3′,4′-methylenedioxyphenyl)- trans-3-hexadecene (50, Figure 4) “ “ “ 2-Oxo-16-phenyl-3-hexadecane (51, Figure 4) “ “ “ Table 2. Cont. Table 2. Cont. Biomolecules 2021, 11, 1765 33 of 43 Table 2. Cont. Metabolite Source Biological Activity References Lignan 53 (Figure 5) P. duclouxii No activity [44] Lignan 54 (Figure 5) “ “ “ Lignan 55 (Figure 5) “ Anticancer “ Lignan 56 (Figure 5) “ “ “ Lignan 57 (Figure 5) “ “ “ Lignan 58 (Figure 5) “ Anti-inﬂammatory “ Asiaticumine A (60, Figure 5) C. asiaticum var. sinicum No activity [52] Asiaticumine B (61, Figure 5) “ “ “ 4-Methyl-heptadec-6-enoic acid ethyl ester and (62 Figure 5) T. sinensis Antileishmanial [54] 3-Hydroxy-2,9,11-trimethoxy-5,6-dihydro isoquino[3,2-a] isoquinolinylium (63 Figure 5) “ “ “ 1-Nitrosoimino-2,4,5-trimethoxybenzene (64, Figure 5) P. sarmentosum Cytotoxic [58] Alkaloid 65 (Figure 5) C. Table 1. Alkyl 4. Conclusions gaudichaudianus No activity [59] Alkaloid 66 (Figure 5) “ “ “ Alkaloid 67 (Figure 5) “ “ “ Alkaloid 68 (Figure 5) “ “ “ Eryciboside A (69, Figure 6) E. hainanesis No activity [60] Eryciboside B (70, Figure 6) “ Hepatoprotective “ Eryciboside C (71, Figure 6) “ No activity “ Eryciboside D (72, Figure 6) “ “ “ Eryciboside E (73, Figure 6) “ “ “ Eryciboside F (74, Figure 6) “ Hepatoprotective “ Eryciboside G (75, Figure 6) “ No activity “ Eryciboside H (76, Figure 6) “ “ “ Eryciboside I (77, Figure 6) “ “ “ Eryciboside J (78, Figure 6) “ “ “ Eryciboside K (79, Figure 6) “ “ “ Eryciboside L (80, Figure 6) “ Hepatoprotective “ Chlorogenic acid derivative (81, Figure 6) “ No activity “ Chlorogenic acid derivative (82, Figure 6) “ “ “ Chlorogenic acid derivative (83, Figure 6) “ “ “ Chlorogenic acid derivative (84, Figure 6) “ “ “ Biscoumarin (85, Figure 6) “ Hepatoprotective “ Kaempferol (87, Figure 7) A. ﬁstulosum No activity [67] N-trans-3,4- dimethoxycinnamoyldopamine (90, Figure 7) P. hyrcanicum “ [82] Neolignanamide (92, Figure 7) L. chinense Moderate radical scavenging [83] Neolignanamide (93, Figure 7) “ “ “ Neolignanamide (94, Figure 7) “ “ “ Table 2. Cont. Table 2. Cont. Biomolecules 2021, 11, 1765 34 of 43 Table 2. Cont. Metabolite Source Biological Activity References Neolignanamide (95, Figure 7) “ “ “ Neolignanamide (96, Figure 7) “ “ “ Neolignanamide (97, Figure 7) “ “ “ Neolignanamide (98, Figure 7) “ “ “ Lignanamide (99, Figure 7) “ “ “ Portulacaldehyde (100, Figure 7) P. oleracea No activity [85] N-(E)-Feruloyl-4-O-methyldopamine (101, Figure 8) “ Anti-inﬂammatory “ Nelumnucifoside A (102, Figure 8) N. nucifera No activity [88] Nelumnucifoside B (103, Figure 8) “ “ “ Flaviﬂoramide A (104, Figure 8) P. ﬂaviﬂorum “ [92] Flaviﬂoramide B (105, Figure 8) “ “ “ Aristoyunnolin I (106, Figure 8) A. yunnanensis “ [95] Aristoyunnolin I (107, Figure 8) “ “ “ Custonolide (108, Figure 8) “ Moderate cytotoxicity “ Claulamine C (109, Figure 8) C. Table 1. Alkyl 4. Conclusions lansium No activity [98] Claulamine D (110, Figure 8) “ “ “ Claulamine E (111, Figure 8) “ “ “ Clausenaline B (112, Figure 8) “ “ “ Clausenaline C (113, Figure 8) “ “ “ Clausenaline D (114, Figure 8) “ “ “ Clausenaline E (115, Figure 9) “ “ “ Clausenaline F (116, Figure 9) “ “ “ Clausemarins A (117, Figure 9) “ Anti-inﬂammatory “ Clausemarin B (118, Figure 9) “ No activity “ Clausemarin C (119, Figure 9) “ “ “ Clausemarin D (120, Figure 9) “ “ “ (3R)-3,7-dihydroxy-8-methoxy-3-(4′- methoxybenzyl)-4-chromanone (128, Figure 9) D. cochinchinensis “ [104] Sargentodoside A (129, Figure 9) S. cuneata “ [105] Sargentodoside B (130, Figure 9) “ “ “ Sargentodoside C (131, Figure 9) “ “ “ Sargentodoside D (132, Figure 9) “ “ “ Sargentodoside E (133, Figure 9) “ “ “ Sargentodognan F (134, Figure 9) “ “ “ Sargentodognan G (135, Figure 9) “ “ “ 5,6-Dimethoxy-7-hydroxy-8-methyl- ﬂavone (136, Figure 9) T. triangulare “ [106] 5,6-Dimethoxy-8-methyl-2-phenyl-7H-1- benzopyran-7-one (137, Figure 9) “ “ “ Table 2. Cont. Table 2. Cont. Biomolecules 2021, 11, 1765 35 of 43 Table 2. Cont. Metabolite Source Biological Activity References 4-Methoxy-6-(2-hydroxy-4-phenylbutyl)- 2H-pyran-2-one (138, Figure 10) “ “ “ 9,10-Dihydrophenanthrene-1,5-dihydroxy- 3,4,7-trimethoxy-9,10- dihydrophenanthrene (139, Figure 10) D. moniliforme “ [110] Miliusacunine A (140, Figure 10) M. cuneata Antimalaria [114] Miliusacunine B (141, Figure 10) “ “ “ Miliusacunine C (142, Figure 10) “ No activity “ Miliusacunine D (143, Figure 10) “ “ “ Miliusacunine E (144, Figure 10) “ “ “ 7-Methoxyﬂavonol (145, Figure 10) A. frutescens Radical scavenging [115] 7-Methoxyﬂavonol (146, Figure 10) “ Inhibition mushroom tyrosinase “ 7-Methoxyﬂavonol (147, Figure 10) “ “ “ 7-Methoxyﬂavonol (148, Figure 10) “ “ “ 7-Methoxyﬂavonol (149, Figure 10) “ Radical scavenging “ Fisetinidol glucoside (150, Figure 10) “ “ “ Benzyl glycoside (151, Figure 11) “ “ “ Lyciumsterol A (152, Figure 11) L. Table 1. Alkyl 4. Conclusions chinense No activity [118] Lyciumsterol B (153, Figure 11) “ Protective effects on pancreatic islet cells “ Lyciumsterol C (154, Figure 11) “ “ “ Lyciumsterol D (155, Figure 11) “ No activity “ Lyciumsterol E (156, Figure 11) “ “ “ Lyciumsterol F (157, Figure 11) “ Protective effects on pancreatic islet cells “ Lyciumsterol G (158, Figure 11) “ Protective effects on pancreatic islet cells and autophagy activation “ Lyciumsterol H (159, Figure 11) “ No activity “ Lyciumsterol I (160, Figure 11) “ Autophagy activation “ Lyciumsterol J (161, Figure 11) “ No activity “ Lyciumsterol K (162, Figure 10) “ Autophagy activation “ (7R, 8S)-7-(4-Hydroxy-3,5- dimethoxyphenyl)-8-hydroxy me-thyl-10-[N-7”-(4”- hydrxyphenyl)ethyl]carbamoylethenyl-3′- methoxybenzodihydrofuran (163, Figure 11) N. physaloides No activity [121] cis-N-p-Hydroxycinnamoyl-7′- methoxyethyltyramine (164, Figure 11) “ “ “ Aristolochic acid II alanine amide (163, Figure 11) A. maurorum “ [125] cis-Terrestriamide (165, Figure 11) T. terrestris “ [127] Table 2. Cont. Biomolecules 2021, 11, 1765 36 of 43 Table 2. Cont. Metabolite Source Biological Activity References Ruthenicunoid A (166 Figure 11) L. ruthenicun Inhibition of SIRT1 [129] Pseuduvarioside (167, Figure 12) P. fragrans No activity [133] Oleraisoindole (168, Figure 12) P. oleracea Inhibited NO production in RAW 264.7 cells [135] 2β,7,3-Trihydroxycalamenene 3-O-β-D-glucoside (170, Figure 12) A. sagittifolius Moderate cytotoxicity [136] Bobulretulate A (171 Figure 12) B. retusiusculum No activity [137] Bobulretulate B (172, Figure 12) “ “ “ Majusamide A (173, Figure 12) C. majus “ [139] Majusamide B (174, Figure 12) “ “ “ Chelidoniumine (175, Figure 12) “ “ “ Tetrahydrocoptisine-N-oxide (176, Figure 12) “ “ “ Dasymaschalolactam A (177, Figure 12) D. dasymaschalum “ [142] Dasymaschalolactam B (178, Figure 12) “ “ “ Dasymaschalolactam C (179, Figure 12) “ “ “ Dasymaschalolactam D (180, Figure 12) “ “ “ Dasymaschalolactam E (181, Figure 12) “ “ “ Dasymaschalolactone (182, Figure 12) “ “ “ 4-epi-Baenzigeride A (183, Figure 13) T. baenzigeri “ [143] 4,12-di-epi-Baenzigeride A (184, Figure 13) “ “ “ Tinobaenzin A (185, Figure 13) “ “ “ Tinobaenzin B (186, Figure 13) “ “ “ 4-O-D-glucoside (187, Figure 13) “ “ “ Goniochelienic acid A (188, Figure 13) G. Table 1. Alkyl 4. Conclusions cheliensis “ [150] Goniochelienic acid B (189, Figure 13) “ “ “ Methyl goniochelienate (190, Figure 13) “ “ “ Goniochelieninone (191, Figure 13) “ “ “ (−)-(4S,5S,6R,7S,8S)-goniochelienlactone (192, Figure 13) “ “ “ 7-O-Acetyl derivative of 192 (193, Figure 13) “ “ “ (+)-(7S,8S)-Goniochelienbutenolide A (194, Figure 13) “ “ “ (−)-(7S,8R)-Goniochelienbutenolide B (195, Figure 13) “ “ “ Celtisamide A (196, Figure 13) C. tessmannii “ [155] Celtisamide B (197, Figure 13) “ “ “ Dracaenogenin C (198, Figure 14) D. usambarensis “ [157] Dracaenogenin D (199, Figure 14) “ “ “ Dracaenogenin E (200, Figure 14) “ “ “ Dracaenogenin F (201, Figure 14) “ “ “ Table 2. Cont. Biomolecules 2021, 11, 1765 37 of 43 Table 2. Cont. Metabolite Source Biological Activity References 3′′-Methoxycochinchinenene H (202, Figure 14) “ Anti-inﬂammatory “ Aristolochiaside (203, Figure 14) I. tadungense Cytotoxic [161] Eloundemnoside (204, Figure 15) C. adolphi-friderici Moderate butyrylcholinesterase inhibition [162] Fistuloimidate A (205, Figure 15) A. ﬁstulosum Antibiotic [164] Fistuloimidate B (206, Figure 15) “ “ “ 7R,14S-cis-Tetrahydrocoptisine N-oxides and (207, Figure 15) C. majus No activity [165] 7R,14R-trans-Tetrahydrocoptisine N-oxide (208, Figure 15) “ Inhibited NO production in RAW 264.7 cells “ Table 2. Cont. Author Contributions: Conceptualization, A.E.; writing—original draft preparation, A.E.; writing— review and editing, M.M. and A.E. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. 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https://openalex.org/W2721034113	https://hal.science/hal-01580200v2/document	English	null	Core–Shell Microgel-Based Surface Coatings with Linear Thermoresponse	Langmuir	2,017	cc-by	6,990	Core-Shell Microgel-Based Surface Coatings with Linear Thermoresponse Marian Cors, Oliver Wrede, Anne-Caroline Genix, Dario Anselmetti, Julian Oberdisse, Thomas Hellweg To cite this version: Marian Cors, Oliver Wrede, Anne-Caroline Genix, Dario Anselmetti, Julian Oberdisse, et al.. Core- Shell Microgel-Based Surface Coatings with Linear Thermoresponse. Langmuir, 2017, 33 (27), pp.6804-6811. 10.1021/acs.langmuir.7b01199. hal-01580200v2 Core-Shell Microgel-Based Surface Coatings with Linear Thermoresponse Marian Cors, Oliver Wrede, Anne-Caroline Genix, Dario Anselmetti, Julian Oberdisse, Thomas Hellweg To cite this version: Marian Cors, Oliver Wrede, Anne-Caroline Genix, Dario Anselmetti, Julian Oberdisse, et al.. Core- Shell Microgel-Based Surface Coatings with Linear Thermoresponse. Langmuir, 2017, 33 (27), pp.6804-6811. 10.1021/acs.langmuir.7b01199. hal-01580200v2 Core-shell microgel based surface coatings with linear thermo-response Marian Cors1, 2, Oliver Wrede1, Anne-Caroline Genix2, Dario Anselmetti3, Julian Oberdisse2, Thomas Hellweg1 1 Department of Physical and Biophysical Chemistry, Bielefeld University, Universitaetsstr. 25, 33615 Bielefeld, Germany 2 Laboratoire Charles Coulomb (L2C), CNRS, Université de Montpellier, 34095 Montpellier, France 3Department for Experimental Biophysics and Applied Nanoscience, Bielefeld University, Universitaetsstr. 25, 33615 Bielefeld, Germany HAL Id: hal-01580200 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. 1 1 Department of Physical and Biophysical Chemistry, Bielefeld University, Universitaetsstr. 25, 33615 Bielefeld, Germany 2 Laboratoire Charles Coulomb (L2C), CNRS, Université de Montpellier, 34095 Montpellier, France 3Department for Experimental Biophysics and Applied Nanoscience, Bielefeld University, Universitaetsstr. 25, 33615 Bielefeld, Germany Introduction Acrylamide-based microgels are colloidal particles comprising a gel network of solvated polymer chains.1 As a function of external stimuli, and in particular temperature, these particles show a reversible shrinking and swelling behavior granting them the name “smart microgels”.2–5 The most prominent examples are microgels based on poly(N-isopropylacrylamide) (pNIPAM)6,7, which have a volume phase transition temperature of ca. 32–33 °C in water, corresponding to the lower critical solution temperature (LCST) in the phase diagram.8 However, also other monomers lead to the formation of microgels with thermo-responsive behavior, with various phase transition temperatures.9–14 By copolymerizing them statistically, the phase transition temperature can be tuned.11,15–20 Moreover, by designing complex particle architectures4,21–30, i.e. microgel particles with compartments of different monomeric composition or composition gradients, particle properties are expected to be varied, and stimuli-response controlled. Furthermore, also the properties of single adsorbed microgels and of microgel coatings have been studied.31–39 A core-shell architecture, where the core and the shell have a different phase transition temperature is particularly promising. Zeiser et al. have recently shown that for core-shell particles with a shell of smaller LCST than the core, a qualitatively different bulk swelling behavior is present, including a linear swelling over a broad range of temperatures.26 Beside the scientific interest in this volume phase transition, growing attention arises from potential applications of these materials. Prominent examples are their use as surface coatings for smart cultivation of vertebrate cells and as etalons.34,40–43 In this context, using different particle architectures might be advantageous, as it would allow making use of progressive swelling/shrinking as a function of temperature, as opposed to a sudden collapse. However, it remains to be shown that core-shell microgels made of poly(N-isopropylmethacrylamide) (pNIPMAM) and poly(N-n-propylacrylamide) (pNNPAM) still exhibit a linear swelling response in the adsorbed state. Hence, in this article, we focus on core- shell microgels made of a large pNIPMAM core having a high LCST of 44°C, surrounded by a shell of pNNPAM having an inferior LCST of 21°C, similar in architecture to those described by Zeiser et al26. The magnitude of the response of these core-shell microgels is mainly controlled by the cross-linker content of the core if the shell composition is chosen to be constant. Therefore, in this work we systematically vary the core crosslinker content (CCC) of the pNIPMAM cores. Abstract We study the swelling and shrinking behavior of core-shell microgels adsorbed on silicon wafers. In these systems, the core is made of cross-linked poly(N-isopropylmethacrylamide) and the shell consists of cross-linked poly(N-n-propylacrylamide). In suspension, these particles exhibit an extended linear swelling behavior in the temperature interval between the lower critical solution temperatures of the two polymers. Using ellipsometry and AFM, we show that this linear response is also observed in the adsorbed state. 2 Experimental Section Synthesis: The core monomer N-isopropylmethacrylamide (NIPMAM, purity > 97 %), the initiator ammonium peroxodisulfate (APS, >98 %), the crosslinker N,N’-methylenebisacrylamide (BIS, 99 %), the ionic surfactant sodium dodecyl sulfate (SDS, >99 %) were purchased from Sigma-Aldrich and used as received. The shell monomer N-n-propylacrylamide was synthesized following a classical Schotten- Baumann reaction described by Hirano et al.44, using acryloyl chloride (97 %, Aldrich, USA), triethylamine (99 %, Grüssing, Germany), propylamine (99 %, Fluka, USA) and methylene chloride (p. A.) as solvent. For the synthesis of pNIPMAM cores with different crosslinker contents, a 250 mL three- necked flask under gentle nitrogen flow with a mechanical stirrer on the top opening is used. This flask was placed in a heating oil bath with magnetic stirrer. The two side openings were equipped with a septum with a cannula as nitrogen inlet and a reflux condenser with bubble counter, respectively. The crosslinker contents, SDS concentration and radical starter specified as mol% with respect to the amount of monomer are listed in Table 1 for all samples. The crosslinker was dissolved in water (140 mL) first and subsequently heated to 70 °C. Its concentration in the core is given as CCC (core crosslinker content) throughout this article. After purging the solution for 1 h with nitrogen the monomer followed by surfactant was added as powder and after 5 minutes the reaction was initiated by injecting 1 mL of an aqueous solution of the radical starter. After rinsing the neck of the flask and initiation the volume was 150 mL. The total reaction took 5 h, followed by continued stirring at room temperature overnight, and purification. The purification was done at room temperature by centrifugation at 48,384 g for 45 minutes, decantation of the supernatant and redispersion of the precipitate with water. The redispersion was dome with a vortexer (MS 3 basic, IKA-Werke GmbH & CO. KG, Staufen, Germany) for typically 1 minute at maximum speed. This procedure was repeated five times. Shell synthesis was done with a similar protocol, also at high temperature and thus in the collapsed state, in the same setup with a 100 mL flask, by precipitating NNPAM on previously synthesized and purified pNIPMAM cores resulting in a pNIPMAM-core pNNPAM-shell microgel system. The total suspension volume was 50 mL. The shell had always the same nominal composition. Introduction Both the swelling properties of the cores and of the core-shell microgel particles are studied in suspension by photon correlation spectroscopy (PCS) first. Then the particles are deposited on a silicon wafer by spin-coating, and the thickness of the layer and individual height profiles are studied by AFM and ellipsometry in the dry state, followed by an investigation of the swelling properties when the layers are covered with water. 3 Experimental Section The amount of crosslinker BIS was 1.90 mol% with respect to the NNPAM feed and the added amount of the core was 0.15 wt% with respect to the solvent. The concentration of SDS was 1.1 mmol per Liter (2.75 mol%). The core-shell particles were again purified by the same purification procedure as the core particles. Table 1: pNIPMAM-core crosslinker (BIS) content. Constant amounts of pNIPMAM (12.4 mmol, 1.5628 g ± 0.0004 g), SDS (3 mol%, 1.0730 g ± 0.0010 g) and radical starter (3.3 mol%, 0.0924 g ± 0.0001 g) were used. BIS / mol% BIS / g 5.00 0.0947 7.50 0.1421 4 4 10.00 0.1894 12.50 0.2368 15.00 0.2841 The resulting molecular architecture is depicted in Scheme 1. Scheme 1: Core-shell architecture obtained by precipitating and cross-linking of NNPAM (red) on the pNIPMAM core (black). The collapsed core particles are used as seeds in the precipitation polymerization of pNNPAM. The shell synthesis is done at 70 °C, which is largely above both LCSTs of the two polymers. The resulting molecular architecture is depicted in Scheme 1. The resulting molecular architecture is depicted in Scheme 1. Scheme 1: Core-shell architecture obtained by precipitating and cross-linking of NNPAM (red) on the pNIPMAM core (black). The collapsed core particles are used as seeds in the precipitation polymerization of pNNPAM. The shell synthesis is done at 70 °C, which is largely above both LCSTs of the two polymers. Photon correlation spectroscopy (PCS): PCS experiments were performed using two different setups. Angular dependent PCS experiments were done using a classical goniometer setup (ALV GmbH, Langen, Germany) equipped with an Ar+-ion laser (Stabilite 2017, Spectra Physics, Santa Clara, USA) emitting at 514.5 nm. The sample was placed in a decalin matching bath kept at constant temperature using a thermostat (Haake DC50, Thermo Fisher Scientific, Waltham, USA). The scattered light was analyzed using a multiple τ hardware correlator (ALV 5000 Fast, ALV GmbH, Langen, Germany). The obtained correlation functions were analyzed using numerical Laplace inversion by means of CONTIN 45,46. This method provides the intensity-weighted relaxation rate Γ of the observed dynamics in the sample. For pure diffusional modes the dispersion Γ ~ q2 is expected and the diffusion coefficient can be obtained from the corresponding plot. The expected dispersion is found for both the core and for the core-shell particles. Experimental Section Hence, the temperature-dependent measurements were performed at a fixed angle of = 60 ° in a partly home-made setup. The PCS-setup had a He-Ne laser (HNL210L-EC, 632.8 nm, Thorlabs, Newton, USA) as light source, a multiple tau digital correlator (ALV-6010, ALV GmbH, Langen, Germany) and a detector (SO-SIPD, ALV GmbH, Langen, Germany). The sample was placed in a decalin matching bath. For temperature control a refrigerated bath (Haake C25P, Thermo Fisher Scientific, Waltham, USA) with a controller (Phoenix II, Thermo Fisher Scientific, Waltham, USA) was used. 5 Spin coating: Microgel monolayers on silicon wafers (Sigert Wafer, diameter = 50.8 mm, Aachen, Germany) were obtained by spin coating (LabSpin6, SÜSS micro Tec, Garching, Germany), after plasma cleaning (Zepto B, Diener Electronics, Ebhausen, Germany) with an O2-plasma for 15 min at 100 W. The calculated arithmetical mean deviation roughness as well as the root mean square roughness was below 1nm. For microgel layers with a high surface coverage first, a dilute purified microgel dispersion (0.5 wt%) was put on the whole silicon wafer for 45 min, followed by spinning for 300 s at a rotation speed of 1000 rpm. For well-separated microgel particles the same procedure was used without waiting 45 min before spinning the wafer. Spin coating: Microgel monolayers on silicon wafers (Sigert Wafer, diameter = 50.8 mm, Aachen, Germany) were obtained by spin coating (LabSpin6, SÜSS micro Tec, Garching, Germany), after plasma cleaning (Zepto B, Diener Electronics, Ebhausen, Germany) with an O2-plasma for 15 min at 100 W. The calculated arithmetical mean deviation roughness as well as the root mean square roughness was below 1nm. For microgel layers with a high surface coverage first, a dilute purified microgel dispersion (0.5 wt%) was put on the whole silicon wafer for 45 min, followed by spinning for 300 s at a rotation speed of 1000 rpm. For well-separated microgel particles the same procedure was used without waiting 45 min before spinning the wafer. Ellipsometry: For ellipsometry measurements a single-wavelength ellipsometer (SE 400adv, SENTECH Instruments GmbH, Berlin, Germany) was used. The method of ellipsometry was described elsewhere.47,48 Temperature control and liquid cell are custom-made products from SENTECH. The accessible temperature range was 15 °C to 55 °C. The measurements and modeling were done with the software “SE400advanced” from SENTECH. Experimental Section For modeling, a first guess of the microgel layer thickness was used based on the layer thickness determined by AFM measurements, and the initial refractive index was estimated based on literature to 1.35.31 Ellipsometry was already used to characterize the swelling behavior of adsorbed microgels in our previous work from Schmidt et al.31 and in literature.49 AFM: AFM measurements were performed on a FlexAFM (Nanosurf AG, Liestal, Switzerland) with an Easyscan 2 controller (Nanosurf AG, Liestal, Switzerland). To avoid background vibrations, the AFM was placed on a sample stage (Flex, Nanosurf AG) on top of a marble plate which lies on tennis balls. All measurements where performed in tapping (“Phase Contrast”) mode. We used standard silicon cantilevers with 30 nm aluminum reflex coating from BudgetSensors® (Tap300Al-G, BudgetSensors®, Sofia, Bulgaria). The cantilevers had a force constant of 40 N/m and a resonance frequency of approximately 290 kHz. The scan rate was between 1 Hz and 0.33 Hz with 256 or 512 points per line. The setpoint was 55 % of the maximal deflection and the free vibration amplitude was approximately 200 mV. The thickness of the dry microgel monolayer was determined as the average height over an area of 25 µm2 using the software of the AFM (Nanosurf Easyscan 2, Nanosurf AG, Liestal, Swiss). For investigation of the individual height profiles of single microgels, we analyzed the recorded raw data with the open source data evaluation program for scanning probe microscopy Gwyddion.50 After background correction we extracted the profile of at least 20 microgels and calculated the mean profile. Determination of the amount of water of dried microgels: The water content of dried microgels can be determined by Karl-Fischer titration.51 The measurements were done using a Karl-Fischer coulometer (684 KF Coulometer, Metrohm AG, Herisau, Swiss). The titration solution was CombiCoulomat fritless (Meck KGaA, Darmstadt, Germany). The microgels were dried for 24 h at 70 °C in a drying oven (U30, Memmert GmbH + Co. KG, Schwabach, W - Germany) and after storing them for 24 h at ambient they 6 were dissolved in methanol (Methanol AnalaR NORMAPUR® ACS, Reag. Ph. Eur. zur Analyse, VWR International, Radnor, USA) resulting in a solution of 1 wt%. The measurements was done with a sample volume of 100 µL. The water content of the solvent was measured and subtracted. Results and Discussion After microgel synthesis, the particles have been characterized in aqueous suspension first. Then they have been deposited on silicon wafers by spin-coating, and their swelling properties in the dry and wet state have been investigated by ellipsometry. Moreover, AFM has been used to measure the height profile of individual dry microgel particles. Synthesis and properties in suspension The microgel cores were produced following the synthesis protocol outlined in Section 2 and the parameters are summarized in Table 1. The key parameter is the core crosslinker content (CCC), which allows fine tuning of the global swelling properties of the core-shell particles in suspension.26 We have first checked that the cores alone have the expected swelling properties of pNIPMAM, see Supporting Information Figure S1 for details. Then, we have measured the hydrodynamic radius RH as a function of temperature of the series of core-shell microgels by PCS. These results are shown in Figure 1. They agree nicely with previous findings by Zeiser et al26 and thereby validate the synthesis. Figure 1: Swelling curves of core-shell microgels as a function of temperature with different core crosslinker contents (CCC) (5.0 mol% CCC (□), 7.5 mol% CCC (○), 10.0 mol% CCC (△), 12.5 mol% CCC (▽) and 15.0 mol% CCC (◇)). a) Hydrodynamic radius RH. The linear swelling zone is indicated by the vertical dashes. An example of the error of PCS data estimated to be 5% is indicated. b) Same data normalized with respect to the high-temperature average (T = 45 C° – 50 °C) of the hydrodynamic radius RHmin. Inset: Zoom of the linear region. Figure 1: Swelling curves of core-shell microgels as a function of temperature with different core crosslinker contents (CCC) (5.0 mol% CCC (□), 7.5 mol% CCC (○), 10.0 mol% CCC (△), 12.5 mol% CCC (▽) and 15.0 mol% CCC (◇)). a) Hydrodynamic radius RH. The linear swelling zone is indicated by the vertical dashes. An example of the error of PCS data estimated to be 5% is indicated. b) Same data normalized with respect to the high-temperature average (T = 45 C° – 50 °C) of the hydrodynamic radius RHmin. Inset: Zoom of the linear region. The original swelling curves shown in Figure 1a display the hydrodynamic radius RH as a function of temperature, for microgel suspensions of various CCC as given in the legend. Three swelling regimes The original swelling curves shown in Figure 1a display the hydrodynamic radius RH as a function of temperature, for microgel suspensions of various CCC as given in the legend. Three swelling regimes 7 are found. Below T = 20 °C, the entire particles are swollen, and the total hydrodynamic radius is typically greater than 200 nm. Synthesis and properties in suspension Note that due to the different formulations with the various crosslinker concentrations, the total swollen sizes of the core-shell microgels are not identical. At the VPTT of pNNPAM (23 °C), the system starts to collapse, resulting in a size transition step. The region of interest in the intermediate temperature range, between 23 °C and 44 °C, shows a swelling behavior where RH changes linearly with temperature. Instead of some average transition temperature with a steep collapse, the swelling extends thus over the entire temperature range. At high temperature, finally, both core and shell are collapsed. A second size transition step at the VPTT of pNIPMAM (44 °C) does not occur because the particle size is already compressed to the range of the collapsed core (compare Figure S1). As the microgels do not have the same size from the start, it is instructive to normalize the size by the collapsed radius RHmin taken as the high-temperature average (T = 45 – 50 °C). The result, RH/RHmin, is shown in Figure 1b, where the high temperature regime now superimposes by construction. This representation illustrates that the swelling is higher for the samples with lower CCC. The slope in the linear regime connecting the collapsed and the swollen state can therefore be tuned by the core crosslinker content. A zoom of the linear region is given in the inset of Figure 1b for different crosslinker concentrations. The relative value of the slope is found to decrease from 2.25 %/°C to 1.22 %/°C, with reasonable accuracy (± 0.06 %). These values are in line with the total relative decrease of about 30 % in radius, over a temperature range of some 15 degrees. The change in slope corresponds to a diminished sensitivity of swelling to temperature by a factor of 1.8, when the CCC is changed from 5 to 15 mol%. Finally, the swelling is sometimes expressed in terms of volume change which is given by (RH/RHmin)3. This leads to a qualitatively similar figure as Figure 1b, with the linear domain slightly curved, and the low-temperature values giving the maximum volumetric swelling. In our case, the maximum swelling varies from about six to eight with CCC. Spin coating and characterization of dry surfaces Well-separated core-shell microgel particles have been deposited by spin coating on silicon wafers, and studied by AFM. The height profiles of individual particles in the dry state can be determined by image treatment of the AFM results, and averaged over several particles, typically 20. In the SI (Figure S3), sample measurements are shown for illustration. 8 Figure 2: a) Average height profiles for dry core-shell microgels determined from AFM height scans, for different core crosslinker contents as indicated in the legend. b) Schematic drawing of a microgel in suspension and an adsorbed microgel. Figure 2: a) Average height profiles for dry core-shell microgels determined from AFM height scans, for different core crosslinker contents as indicated in the legend. b) Schematic drawing of a microgel in suspension and an adsorbed microgel. In Figure 2a, the average height profiles for microgels with different CCC are shown. One should pay attention to the different X- and Z-scales: all microgel particles are squeezed and adopt a rather flat, pancake-like shape. With increasing CCC, the particles become less flat: an increase in CCC by a factor of three from 5 to 15 mol% results in an increase in height by a factor of three as well, but even particles with the highest degree of crosslinking have an aspect ratio of ca. four. Figure 2b shows a schematic drawing of an adsorbed microgel particle. The total volume of these pancakes can be determined by integration, i.e. adding up the volume of hollow cylinders delimited by the height profile. If this volume is taken as the microgel volume (with water) then the total polymer volume (without water) can be determined by subtraction of the volume of the water present in the “dry” sample, determined by Karl- Fischer-Titration. Then the water contents can be determined by comparing the polymer volume (without water) to the swollen (low-T) volume in water estimated from PCS. The calculated volumes are given in Table 2. From the ratio of the swollen to the dry volume, a water content of ca. 75-95 % in the swollen state can be deduced. This is approximately four times higher compared to the water content of dried microgels (ΦH2O,dry) of about 20 % residual water. The water content in the dried state was determined by Karl-Fischer-Titration. Spin coating and characterization of dry surfaces Table 2: Volume of dry core-shell microgels estimated with the average height profiles determined by AFM (Figure 3), comparison to volumes in suspension (PCS), estimated water fractions in suspension, in the swollen state (ΦH2O,sw) and measured water contents in the dried state (ΦH2O,dry) by Karl-Fischer-Titration for different core crosslinker contents (CCC). Core crosslinker content / mol% Vdry. / nm3 Vcollapsed / nm3 Vswollen / nm3 ΦH2O,dry ΦH2O, sw 5 1.19·107 5.13·106 3.88·107 18 % 74 % 7.5 3.61·106 6.71·106 4.46·107 17 % 93 % 10 4.84·106 8.58·106 5.44·107 22 % 93 % 9 12.5 9.17·106 5.42·106 3.88·107 19 % 80 % 15 7.66 ·106 1.15·107 6.95·107 22 % 91% As a next step, successful deposition of microgel particles with a high surface coverage was obtained by spin coating on silicon wafers, following the protocol given in Section 2. The optimization of system parameters, and in particular the introduction of a waiting time of 45 minutes before spinning, as well as the appropriate concentration, were essential to promote the formation of homogeneous layers. In Figure 3, an AFM height profile scan over a lateral size of 5 µm of a dry microgel particle layer at room temperature (22±1 °C) is presented. It shows that the core-shell particles cover the surface, forming a dense monolayer. The mean height of an area of 25 µm2 was determined and is given in Table 3. To verify homogeneity of the surface coating, different magnitudes up to an image with 50 µm lateral size are shown in the SI (Figure S2). Figure 3: Zoom of an AFM height scan of a monolayer of dried core-shell microgels with 5 mol% core crosslinker content. The lateral image size is 5 µm and the average height is 30.8 nm. Figure 3: Zoom of an AFM height scan of a monolayer of dried core-shell microgels with 5 mol% core crosslinker content. The lateral image size is 5 µm and the average height is 30.8 nm. Given that the geometry in Figure 3 is not a laterally homogeneous flat layer due to the density fluctuations caused by the array of squeezed spherical microgel particles and the presence of holes, the average thickness of the dry layers determined by AFM has been confirmed by ellipsometry. The ellipsometry measurements yielded also the refractive index of the microgel particles. Both thickness measurements are compared in Table 3. Spin coating and characterization of dry surfaces Table 3: Thickness of dried layers of core-shell microgel particles deposited on a wafer, measured by AFM and ellipsometry. The thickness was deduced by the mean thickness of an area of 25 µm2 for AFM and ca. 1 mm2 for ellipsometry. The refractive index was determined by ellipsometry. The error of thicknesses determined by AFM is estimated to be 5 % based on multiple measurements. 10 Core crosslinker content (mol%) Thickness AFM (nm) Thickness by ellipsometry (nm) Refractive index by ellipsometry 5 30.8 36.0±0.8 1.48±0.02 7.5 46.8 41.1±0.9 1.51±0.02 10 59.6 51.8±1.2 1.50±0.02 12.5 71.8 56.0±1.2 1.49±0.02 15 74.5 64.0±1.4 1.46±0.01 The general tendency of increase in thickness with CCC is well captured by both methods, and the results agree at least semi-quantitatively. The difference in thickness determined by AFM and ellipsometry may be due to surface roughness. While AFM measures the detailed topography of the microgel surface, and average height can be easily computed, single wavelength ellipsometry averages over the layer. Then surface roughness like the one due to the individual microgel particles contributes, and may result in an inaccuracy in thickness.52 Comparison of swelling in water at surfaces and in bulk suspension The thicknesses in water of the core-shell microgel layers obtained by spin coating on silicon wafers have also been assessed with ellipsometry. The raw results are plotted as a function of temperature in Figure 4a, for three different CCC as given in the legend. At low temperature, the total thicknesses are a bit smaller than twice the hydrodynamic radii in suspension (cf. Figure 1a), which can be understood from the probable fuzzy nature of the particle surface, not dense enough to be picked up by ellipsometry. Moreover, particles are probably slightly deformed upon contact with the wafer. The ellipsometric thickness is found to be about 70% to 85% of the hydrodynamic diameter, which seems to be compatible with such a scenario. Furthermore, as the initial microgel particles differ in size due to the varying compositions, their thicknesses d is normalized to the unswollen, high-temperature (45 °C – 50 °C) thickness dmin in Figure 4b, in analogy to Figure 1b. Figure 4: Ellipsometric measurements in water of the a) layer thickness d of core-shell microgels deposited on a silicon wafer, for different core crosslinker contents as indicated in the legend, as a function of temperature. In b), Figure 4: Ellipsometric measurements in water of the a) layer thickness d of core-shell microgels deposited on a silicon wafer, for different core crosslinker contents as indicated in the legend, as a function of temperature. In b), 11 the curves are normalized to the high temperature average (45-50 °C) dmin, allowing a comparison of the swelling properties. The data clearly reveal the linear change in thickness of these layers. The inset shows a zoom of the linear region with the respective linear fits. The main result shown in Figure 4 is the similarity of the swelling behavior in the adsorbed state compared to results found in suspension. In the intermediate T-range from 25 °C to 37 °C the layer thickness changes linearly. The resulting slopes are again found to depend on the CCC, and are reported in the inset of Figure 4b. They vary approximately by a factor 1.3, as the crosslinker content is increased from 5 to 15mol%. This agrees qualitatively with the results in suspension, where, however, a larger variation by a factor of 1.8 was found. Comparison of swelling in water at surfaces and in bulk suspension The change in swelling is thus reduced, which might be due to a hindered swelling of particles due to van der Waals interactions with the surface. In order to pursue the comparison between surface and bulk swelling properties in water, the corresponding normalized swelling curves obtained either by PCS (bulk) or ellipsometry (surface) are plotted for three different CCCs in Figure 5. As the curves are normalized to the high-temperature minimum hydrodynamic radius RHmin and ellipsometric thickness dmin, respectively, they are naturally identical in the high-T range. In the intermediate-T range, where the linear swelling laws are observed, they superimpose almost equally well for all CCC. Again the above discussed lower change in slope of the adsorbed particles (Ellipsometry) compared to the particles in suspension (PCS) is observed, resulting in a small change from Fig 5a to 5c of the slope of the particles in suspension with respect to slope of the adsorbed ones. The evolution of the slopes with CCC has already been discussed with Figure 1 for bulk microgel particles: higher CCC leads to lower swelling. At the lowest temperatures, finally, the flattening and hindrance due to adsorption leads to the abovementioned reduced size of deposited microgels with respect to the freely suspended ones. Altogether, Figure 5 provides convincing evidence that free microgel particles in suspension and those adsorbed on wafers present the same linear swelling behavior, independently of the crosslinker content of the core. In order to pursue the comparison between surface and bulk swelling properties in water, the corresponding normalized swelling curves obtained either by PCS (bulk) or ellipsometry (surface) are plotted for three different CCCs in Figure 5. As the curves are normalized to the high-temperature minimum hydrodynamic radius RHmin and ellipsometric thickness dmin, respectively, they are naturally identical in the high-T range. In the intermediate-T range, where the linear swelling laws are observed, 12 12 Figure 5: Swelling curves of hydrodynamic radius RH or ellipsometric thickness d normalized to their high- temperature average (45 °C – 50 °C) of core-shell microgel particles in bulk (PCS, RH/RHmin) and on surfaces (ellipsometry, d/dmin), for different core crosslinker contents a) 5 mol% CCC b) 10 mol% CCC c) 15 mol% CCC). Comparison of swelling in water at surfaces and in bulk suspension Figure 5: Swelling curves of hydrodynamic radius RH or ellipsometric thickness d normalized to their high- temperature average (45 °C – 50 °C) of core-shell microgel particles in bulk (PCS, RH/RHmin) and on surfaces (ellipsometry, d/dmin), for different core crosslinker contents a) 5 mol% CCC b) 10 mol% CCC c) 15 mol% CCC). Summary and Conclusion Such as system is expected to show completely different swelling curves, as well as modified mechanical properties. 53,54. As a promising perspective, one may explore the system where the LCST contrast is inverted. As the temperature is increased in such an inverse system, the core made of pNNPAM shrinks first (above 22°C), while the shell made of pNIPMAM is still in good solvent conditions. Such as system is expected to show completely different swelling curves, as well as modified mechanical properties. Acknowledgements: The authors are thankful for support by the joint ANR/DFG CoreShellGel project, grant ANR-14-CE35-0008-01 of the French Agence Nationale de la Recherche, and grant HE2995/5-1 by Deutsche Forschungsgemeinschaft. We also thank Dagmar Wiechoczek for the help with the Karl- Fischer titration. Summary and Conclusion In the present contribution, we have prepared surface coatings using core-shell microgels with core and shell made of two distinct polymers with a significant contrast in LCST (ΔLCST = 23 °C). The shell has the inferior LCST, thus it shrinks first as temperature is increased, and compresses the core. Inversely, starting from the high synthesis temperature and decreasing T, the core tends to swell first, but is hindered by the still collapsed shell, which leads to a peculiar – linear – swelling behavior. The slope of the evolution of the radius with temperature has been successfully correlated with the crosslinker density of the core: softer, less crosslinked cores have a stronger response. We have then deposited the microgel particles by spin-coating. The key result of this article is that the same particles respond also in a linear manner in size to temperature changes when they are adsorbed to a hard surface. In particular, we have shown by combinations of PCS, ellipsometry and AFM, that an extended linear behavior very similar to the one of the respective suspended particles persists, with a convincing overlap of the linear regions when expressed as relative swelling. Moreover, the average shape of individually adsorbed dry microgel particles has been measured by AFM. The total particle height could again be correlated with the core crosslinker density, the weaker microgels being flatter. The CCC is thus again shown to be the most important system parameter. Moreover, a volume analysis using the AFM height profiles allows an estimation of the average dry volume, and thus of the water content in suspension, in both the swollen and the collapsed state. Such layers with a linear change in thickness might allow to tune, e. g., the interaction with vertebrate cells. Such a behavior might be of special interest in the context of stem cell differentiation since it can be expected that the Young modulus of the layers changes linearly and can be adjusted by the correct choice of the temperature. Moreover, such layers are certainly advantageous in microgel based etalons 13 53,54. 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https://openalex.org/W4389673735	https://www.e3s-conferences.org/articles/e3sconf/pdf/2023/97/e3sconf_bft2023_11014.pdf	English	null	Genetic study of the metabolic syndrome in the Moroccan population: A scoping review	E3S web of conferences	2,023	cc-by	5,047	Hamid Najeh1, Bouchra Rherissi1, Sayeh Ezzikouri2, Ahmed Belmouden1, and Smail Chadli1,3 Hamid Najeh1, Bouchra Rherissi1, Sayeh Ezzikouri2, Ahmed Belmouden1, and Sm Chadli1,3 1Cellular Biology and Molecular Genetics Laboratory, Sciences Faculty, Ibn Zohr University, Agadir, Morocco,Postcode 80000. 2Virology Unit, Viral Hepatitis Laboratory, Pasteur institute, Casablanca, Morocco,Postcode20360 3Sciences of Health and Environment Laboratory; Team of Biotechnology, Environment and Health; Higher Institute of Nursing Professions and Health Techniques, (ISPITS) Agadir, Morocco,Postcode 80000 Abstract. Complications of metabolic syndrome include cardiovascular disease and type 2 diabetes mellitus for different ethnic populations, which represent a growing public health burden. The identification of genetic factors contributing to the metabolic syndrome is of great interest for the prevention and treatment of cardiovascular diseases in Morocco. This scoping review summarizes the available data on genetic variants associated with metabolic syndrome in the Moroccan population. Electronic searches of PubMed and EMBASE databases were conducted to identify all studies published from January 2000 to 2022, on genetic susceptibility to metabolic syndrome in the Moroccan population. The studies included in this review met the pre-specified inclusion criteria. Studies included in this review matched the requirements for inclusion. Five research targeted genetic variations as their main subject. Data were narratively summarized since the studies were high degree of heterogeneity. There was a total of thirteen polymorphisms in the eight metabolic syndrome susceptibility genes that had different effects and were linked to characteristics in the Moroccan population. There is a clear need to improve our understanding of the genetic causes of the metabolic syndrome. This is the first review to comprehensively and rigorously summarizes the available data on the genetic determinants of the metabolic syndrome, a major contributor to the cardiovascular diseases burden of the Moroccan population. 1 Introduction After controlling many of the world's formerly infectious diseases, non-communicable diseases (NCDs) have become the leading cause of morbidity and mortality not only in developed countries, but also in underdeveloped countries (Coates et al. 2020). Among all these NCDs, metabolic syndrome has been the real burden worldwide (Saklayen, 2018). * Corresponding author: najehhami@yahoo.fr © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/e3sconf/202346011014 https://doi.org/10.1051/e3sconf/202346011014 E3S Web of Conferences 460, 11014 (2023) BFT-2023 E3S Web of Conferences 460, 11014 (2023) BFT-2023 https://doi.org/10.1051/e3sconf/202346011014 Metabolic syndrome (MetS) is a combination of metabolic risk factors for cardiovascular disease and type 2 diabetes. The principal components of MetS include central obesity, dyslipidemia (increased triglycerides (TG), decreased high-density lipoprotein cholesterol (HDL-C)), increased blood pressure, and increased fasting blood glucose. The presence of three of these risk factors represents a positive diagnosis of MetS (Alberti et al., 2009). The morbidity of MetS is increasing worldwide, making MetS a huge public health burden for many countries (Alberti et al., 2005; Grundy, 2008; Ekelund et al., 2009). MetS is widely estimated to affect a considerable proportion of the world's population, with a reported incidence of 20-25% in the adult population of developed countries (Saklayen, 2018). In Morocco, the burden of noncommunicable diseases is high, causing over 75% of all deaths; cardiovascular disease (CVD), diabetes and cancers are among the principal causes of death (57%) (Cahdli et al., 2018). CVD is frequently the leading cause of death worldwide and therefore further studies are important to define the role played by the MetS in this pathology, in order to reduce its heavy negative impact on public health and economic systems. (Fahed et al., 2022). The prevalence of MetS has been estimated in Morocco to be 40.0% (48.0% in women and 31.9% in men) as recently reported by Pengpid & Peltzer (Pengpid and Peltzer, 2020). The pathogenesis of MetS incorporates several genetic and epigenetic factors (Fathi Dizaji, 2018) that can be linked to insulin tolerance and chronic low-level inflammation. In addition, certain lifestyles such as diet and physical activity, coupled with genetic factors, clearly interact as major contributors to the development of MetS (Fahed et al., 2022). Untreated, MetS is significantly linked to an elevated risk of cardio-vascular disease and type 2 diabetes (Pekgor et al., 2019). Recently, there has been much interest in a potential genetic contribution to different mechanisms of MetS. Genome-wide linked studies (GWAS catalog) have found many genetic loci involved in different components of MetS (240 associations and 23 studies) (GWAS, n.d). To identify the genetic factors which contribute to the MetS is a great need for the prevention and reduction of the incidence of cardio-vascular disease in Morocco. This systematic review reports the existing data on genetic variants that are linked with the MetS in the Moroccan population. * Corresponding author: najehhami@yahoo.fr © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 460, 11014 (2023) BFT-2023 2 Applied Methods for Systematic Review The present review is reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) (Liberati et al., 2009). An extensive search was conducted on PubMed/Medline and Scopus databases for articles that were published from January 2000 to 2022, using keywords related to MetS (MetS; Morocco; Genetics). Studies were selected based on an initial screening of titles and abstracts. A comprehensive review was performed to exclude research that couldn’t meet the eligibility criteria. Inclusion criteria: MetS; Moroccan Genetics and Exclusion criteria: Publications that did not report genetic variants - Case reports - Book section - Review or meta-analysis articles. The search strategy shows the general pattern of the literature search for MetS papers in this systematic review and their inclusion in the final report (Figure 1). 2 2 https://doi.org/10.1051/e3sconf/202346011014 E3S Web of Conferences 460, 11014 (2023) BFT-2023 Fig. 1. The flow diagram explains the process of article selection 3 Literature review and Study Characteristics Our study strategy (Figure 1 and Methods section) identified five studies that met our inclusion criteria (Table 1) (Ajjemami et al., 2014, 2015; El Yaagoubi et al., 2017; Morjane et al., 2017; Elkhattabi et al.,2018). All studies used the International Diabetes Federation (IDF) definition of MetS. IDF demands as a prerequisite the presence of abdominal obesity in addition to two further criteria: Elevated TG levels or taking lipid-lowering drugs, HDL- C decrease or treatment, high blood pressure ≥ 130/85mmHg or taking antihypertensive drugs, elevated fasting blood glucose ≥ 100 mg/dL or treatment for type 2 diabetes. Table1. Genetic polymorphisms reported with metabolic syndrome in Morocco population. Reference Population (Patients/Controls) Used definition Gene Polymorphism Alleles Records identified from: Pubmed (n = 25) Scopus (n = 11) Records removed before screening: Duplicate records removed (n = 14) Records screened (n = 12) Reports excluded: No genotyping Book section Case report (n= 7) Studies included in review (n = 5) Identification of studies via databases Identification Screening Included Fig. 1. The flow diagram explains the process of article selection Records identified from: Pubmed (n = 25) Scopus (n = 11) Records removed before screening: Duplicate records removed (n = 14) Records screened (n = 12) Reports excluded: No genotyping Book section Case report (n= 7) Studies included in review (n = 5) Identification of studies via databases Identification Screening Included Fig. 1. The flow diagram explains the process of article selection 3 Literature review and Study Characteristics Our study strategy (Figure 1 and Methods section) identified five studies that met our inclusion criteria (Table 1) (Ajjemami et al., 2014, 2015; El Yaagoubi et al., 2017; Morjane et al., 2017; Elkhattabi et al.,2018). All studies used the International Diabetes Federation (IDF) definition of MetS. IDF demands as a prerequisite the presence of abdominal obesity in addition to two further criteria: Elevated TG levels or taking lipid-lowering drugs, HDL- C decrease or treatment, high blood pressure ≥ 130/85mmHg or taking antihypertensive drugs, elevated fasting blood glucose ≥ 100 mg/dL or treatment for type 2 diabetes. e1. Genetic polymorphisms reported with metabolic syndrome in Morocco population. Table1. Genetic polymorphisms reported with metabolic syndrome in Morocco population. Reference Population (Patients/Controls) Used definition Gene Polymorphism Alleles Ajjemami et al., 2014 176P/105C IDF APOC3 SstI (3238C> G) (rs5128 C/G Ajjemami et al., 2015 176P/105C IDF APOA5 rs2266788 T/C rs662799 T/C rRs3135506 C/G rs2075291 G > T 177P/139C IDF ADCY5 rs11708067 A/G 3 https://doi.org/10.1051/e3sconf/202346011014 E3S Web of Conferences 460, 11014 (2023) BFT-2023 Lakbakbi El Yaagoubi et al., 2017 APOC 3 rs5128 (C33238C>G) C/G DUSP9 rs5945326 A/G G6PC2 rs560887 A/G PROX1 rs340874 A/G UBE2E2 rs7612463 A/C Morjane et al., 2017 104P/137C IDF HNF1A rs1169288 A/C rs2464196 G/A rs735396 T/C Elkhattabi et al., 2018 50P/50C IDF HNF1A rs1169288 -- rs2464196 -- 4 Results This scoping review summarizes the existing data on genetic variants influencing the risk of developing MetS in the Moroccan population (Table 2). This scoping review summarizes the existing data on genetic variants influencing the risk of developing MetS in the Moroccan population (Table 2). Table 2. Genotypic and allelic distribution for candidate SNPs in Moroccan patients with MetS and control subjects. Gene Polymorphisme Alleles model genotype OR [95% CI] P-value ADCY5 rs11708067 A/G AA vs AG 0.51 (0.28– 0.95) 0.034 APOA5 rs 2266788 T/C -- -- -- -- rs662799 (−1131) T/C Codominant T//C 10.13 (4.65- 22.06) <0.0001 Dominant T//C – C//C 7.82 (3.79- 16.14) <0.0001 rs3135506 (c.56) C/G Codominant C//G 2.13 (1.05- 4.31) 0.035 Dominant C//G – G//G 2.07 (1.07- 4.03) 0.032 rs 2075291 (c.553) G > T Haplotype CCGT 3.223 (1.43; 7.25) 0.00278 CGGT 8.234 (1.6; 42.5) 0.00534 APOC3 SstI (3238C> G) C/G Codominant [CC vs CG] 4.21 [1.66- 10.68] 0.0008 Dominant [CC vs CG+GG] 3.83 [1.59- 9.19] 0.0010 APOC3 rs5128 (C3238C>G) C/G Codominant [CC vs CG] 4.39 (1.66– 11.56) 0,003 Dominant [CC vs CG+GG] 3.73 (1.48– 9.41) 0,005 DUSP9 rs5945326 A/G AA vs AG 0.32 (0.17– 0.62) 0.001 G6PC2 rs560887 A/G -- -- -- -- HNF1A rs1169288 A/C Codominant AC 2.15 (1.16- 3.97) 0.021 Dominant AA 1.77 (1.15- 2.72) 0.008 Table 2. Genotypic and allelic distribution for candidate SNPs in Moroccan patients with MetS an control subjects. 4 https://doi.org/10.1051/e3sconf/202346011014 E3S Web of Conferences 460, 11014 (2023) BFT-2023 Log additive model AA 2.23 (1.26- 3.96) 0.006 Model allelic AA 2.05 (1.36- 3.14) 0.0005 rs2464196 G/A Codominant GA 1.81 (0.93- 3.53) 0.040 Dominant GG 2.06 (1.11- 3.83) 0.020 Log additive model GG 1.63 (1.11- 2.41) 0.012 Model allelic GG 1.52 (1.05- 2.20) 0.0272 rs735396 T/C -- -- -- -- PROX1 rs340874 A/G Codominant (AA vs GG) 2.81 (1.09– 7.27) 0.033 Recessive (AA+AC VS CC) 2.55 (1.04– 6.25) 0.041 UBE2E2 rs7612463 A/C -- -- -- -- 4.1 Genetic association with MetS Two genetic variations, rs5945326 in the DUSP9 gene and rs11708067 in the ADCY5 gene, were 5 https://doi.org/10.1051/e3sconf/202346011014 E3S Web of Conferences 460, 11014 (2023) BFT-2023 positively correlated with preventing the emergence of MetS. SNPs rs7612463 (UBE2E2) and rs560887 (G6PC2) were not observed to be associated with MetS risk. positively correlated with preventing the emergence of MetS. SNPs rs7612463 (UBE2E2) and rs560887 (G6PC2) were not observed to be associated with MetS risk. and rs560887 (G6PC2) were not observed to be associated with MetS risk. Polymorphism in Hepatocyte-Nuclear-Factor1-alpha (HNF1A) gene such as rs1169288 and rs2464196, genotype and allele data are presented in Table 2 The rs1169288, rs2464196, and rs735396 genotypes showed significant relationship with MetS (Morjane et al., 2017). In addition, all four haplotypes (CAC, AAC, AAT, AGT) of these SNPs and rs735396 were also significantly associated with MetS. In contrast, there was no association between rs735396 genotype and MetS. Carriers of the CC and AC genotypes of the HNF1A rs1169288 gene in the co-dominant state had an increased risk (2.54% and 2.15%, respectively) of developing MetS, compared with AA genotype subjects. The same was true for carriers of the AA and GA genotypes of the HNF1A rs2464196 gene in the co-dominant state, who had a 2.64% and 1.81% increased risk of developing MetS, respectively, compared with AA genotype subjects. Association analysis of metabolic traits with the three HNFA1 variants shows that there is an association between rs 1169288 and all four traits (holding circumference; systolic blood pressure; impaired fasting glucose; triglycerides) and between rs2464196 and the triglycerides trait, for the rs 735396 trait there is an association with both traits (total cholesterol; HDL). The gene for apolipoprotein A5 (APOA5) is located on a short arm of human chromosome 11 at region (11q23) and is part of the (APOA1/APOC3/APOA4/APOA5) gene cluster (Pennacchio and al., 2001). Genetic variation in the human APOA5 locus correlates with changes in plasma lipoprotein levels (Van Dijk and al., 2004; Hubacek, 2005; Talmud and al., 2002), and a common APOA5 polymorphism is significantly linked with increased risk of MetS (Yamada and al., 2007; Niculescu and al., 2007). Many single-nucleotide polymorphisms in the APOA5 gene have been linked to the development of MetS, and thus cardio-vascular disease and T2D in different ethnic groups, according to genome-wide association studies (GWAS). 4.1 Genetic association with MetS In a group of 176 patients and 105 controls from the Moroccan population, the Apolipoprotein A5 (APOA5) gene polymorphism (-1131T>C; C.56C>G; c.553G>T; and C.1259T>C) was investigated (Ajjemami et al., 2015). This study demonstrated a strong association between the APOA5 -1131 T > C polymorphism and MetS in both co-dominant and dominant models. MetS patients and bystanders with the APO A5 variation, c.56 G, had elevated waist circumference and triglyceride levels when compared to those without the variation. The risk of developing MetS was 10.13% higher for those with the APOA5 -1131C genotype in the co-dominant state versus to those with the TT genotype, and in the dominant state, individuals with the CC and TC genotype had a 7.82% higher risk to acquire MetS than those with the TT genotype. g yp The codominant and dominant models both demonstrated a significant correlation for the APO A5 c.56 C>G polymorphism, with p-values of 0.035 and 0.032, respectively. In the codominant state, carriers of the C>G genotype have a 2.13% increased risk of developing MetS compared to people with the CC genotype, and in the dominant state, carriers of the CG and GG genotype had a 2.07% higher risk of developing MetS compared to people with the CC genotype. No significant correlation between the c.553 G>T and C.1259 T>C polymorphisms of the APOA5 gene and the MetS was discovered in any of the genetic models. In the population of Morocco, the SstI polymorphism is investigated. Two models, codominant and dominant, were used to examine the relationship between the susceptibility to the APOC3 3238C>G polymorphism and the MetS. Nevertheless, for all of the APOC3 SNPs 3238C> G, no correlation was discovered in the codominant-two recessive mode (Ajjemami et al. 2014). Moreover, compared to MetS patients and controls without this mutation, the APOC3 3238G polymorphism was related with heightened levels of TG and LDL (El Yaagoubi et al., 2017). Comparisons of genotype and allelic frequency distributions of the rs5128 (APOC3), rs7612463 (UBE2E2 gene), rs560887 (G6PC2 gene), rs340874 (PROX1 gene), rs5945326 (DUSP9 gene) and rs11708067 (ADCY5 gene) polymorphisms between MetS and non-MetS subjects according to the codominant, dominant, and recessive models are shown in Table 2. Elevated risk of MetS was significantly associated with the single nucleotide polymorphisms rs5128 (APOC3) and rs340874 (PROX1). 4.1 Genetic association with MetS The patterns of linkage between SNPs at the APOA5 locus and TG levels differed between 6 E3S Web of Conferences 460, 11014 (2023) BFT-2023 https://doi.org/10.1051/e3sconf/202346011014 populations; for example, the “CC” genotype of rs2266788 was strongly correlated with increased TG levels and decreased HDL cholesterol levels in Europeans but not in Moroccans (Kraja et al., 2011). The human APOC3 gene encoding ApoC-III is situated on chromosome 11 region 11q23.3 within the APOA5-APOA4-APOC3-APOA1 gene cluster. The rs5128 polymorphism in the 3′untranslated region (UTR) of the APO C3 gene was first identified in 1983 (Rees and al., 1983). The studies which we find in this review found an association between the rs5128 variant and MetS in the population of Morocco. A cross-sectional survey of the Northeast Chinese population found that subjects with the GC genotype of rs5128 had a more advanced risk of MetS than persons with the GG or CC genotypes of rs5128 (Wu et al., 2016). We found that rs5128 was associated with triglyceride and LDL cholesterol concentrations (Ajjemami et al., 2014), but TG associations were absent in the study by El Yaagoubi et al., 2017, but several studies found APOC3 as rs5128 to be linked with increased plasma TG levels (Liu et al., 2010; Qi et al., 2007). A recent meta-analysis of several populations (Indian and US Indians, London Indians, Singaporeans, UK Indians, Singaporean Chinese, London Europeans, UK Europeans, Mexican Americans in San Antonio and multi-ethnic people in Oklahoma) found a major relationship between rs5128 and elevated plasma TG levels and a 3% increase in rs5128 (Goyal et al., 2021). Similarly, a meta-analysis of 19 published studies finds that individuals carrying the rs5128 polymorphism in the APOC3 allele are more likely to develop coronary heart disease (Li et al., 2016). 5 Conclusion This analysis has several limitations. First, it contains only a few research studies with modest sample sizes. Second, ethnic differences are not accounted for in the available research included in this review; therefore, the same SNPs may have different effects by ethnicity (or be associated with one ethnicity but not another). It is also critical to note that the fact that there are different ethnic groups within the Moroccan population makes it difficult to define the population precisely. This suggests that several ethnic-related characteristics, including Linkage Disequilibrium patterns and allelic frequencies, have not been considered in most of the literature. In spite of these shortcomings, this is the first review to completely and rigorously summarize the existing data on the genetic factors of the MetS, a major contributor to the CVD burden of the Moroccan population. As a whole, the impact of genetic variations on the Moroccan MetS was inconsistent, and further research is needed to improve our understanding of the genetic causes of the MetS. The authors declare that they have no conflict of interest. 4.1 Genetic association with MetS In the current study, the APOA5 SNP rs662799 was found to be significant in predicting MetS modulation in a Moroccan population. In addition, the APOA5 rs662799 SNP was found to have a genome-wide significant association with high blood pressure, fasting blood sugar, and HDL. Although the APO A5 rs662799 polymorphism has been widely associated with diabetes risk. The APOA5 rs662799 SNP has been linked to an increased risk of Mets in Greeks (Vasilopoulos and al., 2011), Tunisians (Hechmi and al., 2020) Pakistanis (Fiaz and al., 2019), Caucasians (Maasz et al., 2007) Japanese (Yamada et al., 2007), Taiwanese (Lin and al., 2017; Hsu et al., 2008), Hong Kongese (Ong and al., 2011), Chinese (Xu and al., 2013), and Koreans (Kim et al., 2016; Oh et al., 2020). However, some studies on Caucasian (Grallert and al., 2007; Fallah and al., 2016) and Hispanic (Mattei and al., 2009) populations have shown conflicting results compared to the Moroccan population. Several meta-analyses have also found that the APOA5 rs662799 SNP is linked with an increased risk of MetS in Asians but not in Europeans (Grallert and al., 2007; Mattei and al. 2009; Liu and al. 2012; Xu and al., 2013; Fallah and al. 2016; Kim et al., 2016; Oh and al., 2020). The association between MetS and APOA5 rs3135506 has been obscured. In the population of Morocco, we found a significant linked between the c.56 C>G polymorphism and Increased TG levels and waist circumference. Several studies in other populations have found an relationship between allele “G” of the ApoA5 c.56 C>G polymorphism and TG, Roma population samples (Sumegi et al. 2017), and, in addition, in studies by Maász et al. 2007, the minor G allele at SNP rs3135506 was linked with approximately 50% greater risk of MetS in Caucasian populations (Grallert and al., 2007). 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W3193346044.txt	https://pure.rug.nl/ws/files/201704967/Research_and_Practice_in_Thrombosis_and_Haemostasis_2021_Piersma_Wichers_Convenience_and_satisfaction_in_direct_oral.pdf	en		Convenience and satisfaction in direct oral anticoagulant–treated patients with atrial fibrillation	Research and practice in thrombosis and haemostasis	2,021	cc-by	4,964	University of Groningen Convenience and satisfaction in direct oral anticoagulant-treated patients with atrial fibrillation Piersma-Wichers, Margriet; Elling, Tessa; de Vries-Bots, Anne M. B.; Kooistra, Hilde A. M.; Meijer, Karina Published in: Research and practice in thrombosis and haemostasis DOI: 10.1002/rth2.12577 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2021 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Piersma-Wichers, M., Elling, T., de Vries-Bots, A. M. B., Kooistra, H. A. M., & Meijer, K. (2021). Convenience and satisfaction in direct oral anticoagulant-treated patients with atrial fibrillation. Research and practice in thrombosis and haemostasis, 5(6), Article 12577. https://doi.org/10.1002/rth2.12577 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverneamendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 21-05-2024 Received: 19 February 2021 \| Revised: 18 June 2021 \| Accepted: 12 July 2021 DOI: 10.1002/rth2.12577 ORIGINAL ARTICLE Convenience and satisfaction in direct oral anticoagulant– treated patients with atrial fibrillation Margriet Piersma-Wichers MD1,2 \| Tessa Elling MD1 \| Anne M. B. de Vries-Bots MPharm3 \| Hilde A. M. Kooistra MD, PhD1 \| Karina Meijer MD, PhD1 1 Department of Haematology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands 2 Certe Thrombosis Service Groningen, Groningen, the Netherlands 3 Community Pharmacy Medical Center Hoogezand-Sappemeer, Hoogezand, the Netherlands Correspondence Margriet Piersma-Wichers, MD, University Medical Center Groningen (AA24), Hanzeplein 1, 9713GZ, Groningen, The Netherlands. Email: m.piersma@umcg.nl Handling Editor: Dr Suzanne Cannegieter Abstract Background: Direct oral anticoagulants (DOACs) are the preferred anticoagulants for thromboprophylaxis in atrial fibrillation. We aimed to identify determinants of quality of life related to DOAC treatment to optimize DOAC treatment convenience and satisfaction. Methods: We conducted a cross-sectional study in DOAC users. DOAC treatment– related convenience and satisfaction were measured by Perception of Anticoagulant Treatment Questionnaire. Higher scores are more favorable (range, 0-100). Patient- reported outcome measures and drug- and organization-related factors were collected. Multiple regression analyses were used to evaluate the association between these factors (ie, exposure variables) and DOAC treatment–related convenience and treatment satisfaction (ie, outcome variables). Results: Of 1598 patients invited, 1035 responded, and 962 were included. The median convenience score was 98.1 (94.2-100.0), mean satisfaction score 66.5± 14.9. Twenty-four percent felt not well informed at the start of DOAC; 6.9% did not know who to turn to with questions. Multiple regression analyses showed that lacking sense of security, the predefined composite of receiving insufficient information at start of DOAC and/or not knowing who to turn to with questions was associated with lower convenience (regression coefficient, −1.29; 95% confidence interval [CI], −2.16 to −0.41). Bleeding, gastrointestinal complaints, and lower medication adherence were also associated with lower convenience. Missing sense of security (regression coefficient −6.59; 95% CI, −8.94 to −4.24) and bleeding without consultation were associated with lower treatment satisfaction. Conclusions: Accessible interventions to improve DOAC care could be providing more instruction at treatment initiation and ensuring that patients know who to contact in case of problems. KEYWORDS anticoagulants, atrial fibrillation, medication adherence, patient reported outcome measures, quality of life This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH). Res Pract Thromb Haemost. 2021;5:e12577. https://doi.org/10.1002/rth2.12577 wileyonlinelibrary.com/journal/rth2 \| 1 of 7 2 of 7 \| PIERSMA-WICHERS et al. Essentials • Direct oral anticoagulants (DOACs) are the preferred anticoagulants for thromboprophylaxis in atrial fibrillation. • Underexposed factors associated with DOAC-related quality of life were evaluated. • Treatment convenience and satisfaction with a DOAC were, respectively, high and fairly high. • More instructions at initiation of a DOAC and contact information may improve anticoagulation care. 1 \| I NTRO D U C TI O N September 2018, we conducted a cross-sectional study in all patients with AF who were registered at Certe Thrombosis Service be- For individuals of European descent, the lifetime risk of developing tween January 1, 2014, and June 8, 2018. Without any selection, the atrial fibrillation (AF) is ≈25%.1 AF increases the risk of thrombo- patients were signed up for registration at the Thrombosis Service embolic stroke four to five times, 2 and therefore lifelong throm- by the community pharmacists when they started a DOAC, accord- boprophylaxis is indicated for patients with additional risk factors ing to a regional transmural protocol. The intention of this registry (CHA 2DS2-Vasc≥1).3 For most patient groups, direct oral anticoagu- was to facilitate and monitor the annual kidney function check. lants (DOACs) have gradually become the preferred anticoagulant This study (University Medical Centre Groningen [UMCG] RR drugs over vitamin K antagonists (VKAs). In the second half of 2019, number 201899276) was assessed by the Medical Ethics Review DOACs were used by 307 000 patients in the Netherlands.4 Board of the UMCG, which concluded that a formal review process The main advantages of DOACs over VKAs are no need of frequent laboratory monitoring and fewer food and drug interactions. was not needed under Dutch law (WMO;METc2018/213). All participants provided written informed consent. This has made anticoagulant care less complicated and probably more convenient. Previous observational research confirmed that patients treated with DOACs scored significantly higher on treat- 2.2 \| Participants and methods ment satisfaction than patients using VKAs.5 On the other hand, some patients preferred International Normalized Ratio monitoring Patients were eligible to participate in our study when they used a to no monitoring. The benefits were related to reassurance, routine DOAC for the indication AF and were included in the DOAC regis- feedback on the effect of the anticoagulants, and contact with the try at Certe Thrombosis Service. There were no exclusion criteria. physician.6 Without these, patients might experience insufficient In September 2018 we sent all eligible patients an information let- medical support in case of side effects, bleeding complications, and/ ter and two questionnaires by mail. We asked that they return the or a medical intervention. These uncertainties and other patient- questionnaires with a completed informed consent. After 1 month, related outcome measures that could lower convenience and satis- nonresponders received a reminder. faction of DOAC use could compromise medication adherence. The latter is determinative for an effective and safe anticoagulant treatment.7 Moreover, anticoagulant treatment not only aims to prolong 2.3 \| Study outcomes life expectancy but also to improve quality of life by preventing ischemic complications such as cerebral vascular events.8 Optimizing We focused on patient-reported outcome measures. The primary treatment satisfaction and convenience could lower treatment bur- outcome of our study was the anticoagulation-related quality of life, den and in this way increase its benefit. expressed in treatment convenience and satisfaction score. This was We hypothesized that factors as side effects, intake regimen, assessed using the validated Perception of Anticoagulant Treatment handling around interventions, and patient information at the start Questionnaire (PACT- Q)9 (Supporting Information). The conveni- and during DOAC use might influence the patients’ experienced an- ence score was based on questions about intake of the tablets/ ticoagulation care–related quality of life. capsules, handling around interventions, dependence on others in connection with the anticoagulant treatment, and questions con- 2 \| M E TH O D S cerning limitations in daily life activities and physical complaints. The satisfaction score reflects on self-reliance and physical well-being with the anticoagulant treatment. The last item of the questionnaire The data that support the findings of this study are available upon concerns satisfaction in general. Higher scores indicate higher de- reasonable request. gree of convenience and satisfaction. The maximum score for both components is 100. The secondary outcome of our study was DOAC 2.1 \| Study aims and design nonadherence. To collect data on patient-, drug-, and organization-related factors, we used a questionnaire that was created within our clinically The aim of our study was to identify possibly underexposed fac- experienced team (Supporting Information). For the primary analyses tors associated with DOAC-related quality of life. Therefore, in we combined the answers of two questions (“Did the patient receive \| PIERSMA-WICHERS et al. 3 of 7 sufficient information at the start of DOAC?” and “Did they know items within a ≥50% completed dimension were replaced. Thereafter, who to turn to with questions?”) into a composite variable “sense of multivariate imputation by chained equations was performed to im- security.” Sense of security was scored positive/present if both ques- pute the patient-, drug-, and organization-related variables. With an tions were answered yes. Furthermore, we collected patient-reported iteration number of 10, five imputed data sets were generated in outcome measures from the previously mentioned questionnaire on which the pooled results were used to estimate regression parame- bleeding and thrombotic events, type of DOAC, use of an antithrom- ters. The data were assumed to be missing at random, based on the botic in the past, and gastrointestinal complaints during DOAC use. intermittent pattern of the missing values. Differences between the We defined self-reported adherence if a patient indicated that they original and imputed data set were evaluated. For all statistical analy- never forget the DOAC; all other options were classified as nonadher- ses, P values <.05 were considered statistically significant. ence. We have chosen this arbitrary cutoff point because we believe that the actual frequency of forgetting doses of medication is likely to be underreported by patients and therefore less informative. The neighborhood socioeconomic status (SES) score was retrieved from the Netherlands Institute for Social Research. The SES 3 \| R E S U LT S 3.1 \| Patient flow and characteristics score is based on income, education, and occupation of the inhabitants and expressed as a Z score with a normal distribution. A higher The flow of participants is outlined in Figure 1. One thousand thirty- score represents a higher SES. five patients responded to our invitation (65%). From this group we had to exclude 73 patients for various reasons as shown in Figure 1. 2.4 \| Statistical analysis We included 962 patients. The patient characteristics are summarized in Table 1. Fifty-seven percent were men. The mean age was 72.6 years (±9.7). The SES Statistical analyses were performed with SPSS Statistics version 24 scores ranged from −3.71 to 2.59. Responders were comparable with (IBM, Armonk, NY, USA). Continuous data were reported as means nonresponders regarding sex, age, and SES (Supporting Information). (standard deviation) for data with a normal distribution or medians The most often used DOAC was dabigatran, the first approved (interquartile range) for nonnormal distributed data. The Students’ DOAC in the Netherlands. Before start of a DOAC, 41.2% of the independent samples t test or Mann Whitney U test was used to patients used VKA and/or had antiplatelet therapy (APT). Sixty-nine compare continuous variables, as appropriate. Categorical data were patients (7.3%) had discontinued their DOAC before completing the compared using the chi-square test. Multiple regression analyses were used to evaluate the associ- questionnaires. Eighty patients (8.4%) reported a bleeding during DOAC use. Gastrointestinal complaints were reported by 12% of the ation between the patient-, drug-, and organization-related factors patients. Twenty-four percent of all patients felt not well informed, and Perception of Anticoagulant Treatment Questionnaire (PACT-Q ) and 6.9% of all patients did not know who to turn to with questions. scores and self-reported DOAC nonadherence, respectively. The In our study population, 21.6% of all patients reported nonadher- multiple linear regression model (ie, PACT-Q scores) were adjusted ence; this was the same with naïve DOAC users (22.5%) as with pa- for sex, age, and SES. tients who used VKAs and/or had APT before (20.5%) (P = .46). A prespecified sensitivity analysis was performed, excluding the patients with an affirmative answer to the questionnaire item, “Have you stopped taking DOAC in the meantime?”. Post hoc imputation 3.2 \| Primary Outcome was performed to substitute missing data. The missing items on the PACT-Q questionnaire were replaced by the mean of nonmissing Table 2. shows which patient-, drug-, and organization-related fac- items, stratified by dimension (ie, treatment satisfaction and conve- tors were associated with convenience. The analysis was based nience), as recommended in the PACT-Q manual.10 Only the missing on 796 patients because of missing values for the other patients. F I G U R E 1 Patient flowchart. DOAC, direct oral anticoagulant 4 of 7 \| PIERSMA-WICHERS et al. TA B L E 1 Patient characteristics DOAC use (n = 962) Data available Male sex, n (%) 547 (56.9) Age, y, mean ± SD 72.6 ± 9.7 SES, a median (IQR) −0.8 (−1.5 to 0.3) DOAC type, n (%) Factor RC (95% CI) n = 962 Convenience (constant) 96.58 (95.25 to 97.91) n = 962 Negative sense of security −1.29 (−2.16 to −0.41) n = 953 Bleeding n = 962 P value .004 No bleeding Reference Dabigatran 384 (39.9) Without consultation −0.40 (−2.67 to 1.86) .73 Rivaroxaban 160 (16.6) With evaluation by physician −3.43 (−5.49 to −1.37) .001 Apixaban 350 (36.4) With hospitalization −0.39 (−3.32 to 2.54) .80 Edoxaban 23 (2.4) Thrombotic event 1.05 (−1.70 to 3.80) .45 Multiple (serial) 36 (3.7) Gastrointestinal side effects −1.92 (−3.07 to −0.76) .001 Unknown 9 (0.9) Previous use of VKA and/or APT −0.76 (−1.52 to 0.01) .053 Antithrombotic use before DOAC, n (%) 379 (41.2) VKA 176 (19.1) APT 188 (20.4) VKA +APT 15 (1.6) n = 921 DOAC frequency Once daily Reference Twice daily −0.20 (−1.17 to 0.77) .69 −1.42 (−2.32, −0.51) .002 DOAC nonadherence Stopped DOAC, n (%) 69 (7.3) n = 939 Bleeding during DOAC, n (%) 80 (8.4) n = 954 Without intervention 30 (3.1) With evaluation by physician 35 (37) With hospitalization 15 (1.6) Thrombotic event, n (%) 21 (2.2) n = 953 Gastrointestinal complaints, n (%) 112 (12.0) n = 936 Sense of security present, n (%) 700 (74.5) n = 940 Well informed 716 (76.0) n = 942 Know who to turn to 886 (93.1) n = 952 Forget to take DOAC never, n (%) 747 (78.4) n = 953 Convenience, median (IQR) 98.1 (94.2-100.0) n = 866 Satisfaction, mean ± SD 66.7 ± 15.1 n = 879 Abbreviations: APT, antiplatelet therapy; DOAC, direct oral anticoagulant; IQR, interquartile range; SD, standard deviation; SES, socioeconomic status; VKA, vitamin K antagonist. a TA B L E 2 Factors associated with the PACT-Q Convenience score According to the Netherlands Institute for Social Research. Intervention at dentist No intervention Reference Intervention with DOAC adjustment −1.39 (−3.06 to 0.28) .10 Intervention without DOAC adjustment −0.44 (−1.29 to 0.40) 0.31 Abbreviations: APT, antiplatelet therapy; CI, confidence interval; DOAC, direct oral anticoagulant; PACT-Q , Perception of Anticoagulant Treatment Questionnaire; RC, regression coefficient; VKA, vitamin K antagonist. n = 796, R square =0.090, analysis adjusted for sex, age, and socioeconomic status. Table 3 shows the results of the multiple regression analysis of satisfaction (806 patients included in the analysis). The characteristics of the included and excluded patients are available in the Supporting Information. The mean satisfaction score was fairly high, at 66.5 ± 14.9 (Table 1). We found a clear decrease of satisfaction with a negative sense of security. Patients who experienced a bleeding without consultation also had a significantly lower satisfaction The differences between the included and excluded patients in the score. After imputation, by which 131 additional patients could be analysis are available in the Supporting Information. The median included in the analysis, DOAC use twice daily had the same point convenience score was high at 98.1 interquartile range, 94.2-100) estimate, but became significant. The other results were not sub- (Table 1). If patients had a negative sense of security and/or were stantially different (Supporting Information). evaluated by a physician because of bleeding, this was associated with a significantly lower convenience score. We saw also a lower convenience score in patients with gastro- For both convenience and satisfaction, the sensitivity analyses, excluding the patients who stopped DOACs before completing the questionnaire, did not yield substantially different results. intestinal side effects and in nonadherent patients. All other factors had no significant correlation with treatment convenience. After imputation, by which 146 additional patients could be included in the 3.3 \| Secondary outcome analysis, the remaining number of excluded patients was only 20. The results after imputation were highly comparable with the main analy- Figure 2 shows the correlation (765 patients included in the sis. Only the association of previous use of a VKA and/or APT became analysis) between nonadherence; convenience; satisfaction; and significant but had the same point estimate (Supporting Information). patient, drug-, and organization-related factors. The differences \| PIERSMA-WICHERS et al. TA B L E 3 Factors associated with the PACT-Q Treatment Satisfaction score Factor RC (95% CI) Treatment satisfaction (constant) 67.13 (63.58 to 70.68) Negative sense of security −6.59 (−8.94, −4.24) 5 of 7 consultation was associated with lower satisfaction. Although there P value is no consensus about a cutoff for a clinical relevant improvement or decline, it is possible to determine this on the basis of calculating absolute risk differences. By means of this calculation a decline of five or more points was set as a relevant decline, as suggested by <.001 van Miert et al.11 When applying this method for the convenience score, no individual factors were associated with a relevant decline. Bleeding However, the variables “negative sense of security” and “bleeding No bleeding Reference Without consultation −6.44 (−12.41 to −0.46) .04 without consultation” were associated with a relevant lower treat- With evaluation by physician −1.54 (−7.00 to 3.92) .58 ment satisfaction score. With hospitalization −3.37 (−11.28 to 4.53) .40 Thrombotic event 1.70 (−5.27 to 8.66) .63 Gastrointestinal side effects −1.98 (−5.03 to 1.07) .20 Previous use of VKA or APT −0.09 (−2.15 to 1.97) .93 DOAC frequency Once daily Twice daily DOAC nonadherence We found a number of associations between DOAC nonadherence on the one hand and convenience; satisfaction; and patient-, drug-, and organization-related factors on the other hand. Use of a DOAC with twice-daily administration was correlated to nonadherence, as was a higher SES score. In contrast, higher convenience, knowing who to turn to with questions, and age >67 years were as- Reference 2.14 (−0.45 to 4.72) .11 −1.94 (−4.36 to 0.49) .12 Intervention at dentist sociated with better adherence. We used patient-reported outcome measures to focus on different aspects of anticoagulation treatment in relation to convenience, satisfaction, and nonadherence to DOAC treatment trying to find determinants of anticoagulant care–related quality of life. No intervention Reference With DOAC adjustment −1.64 (−6.13 to 2.86) .48 Without DOAC adjustment 0.60 (−1.66 to 2.86) .60 APT, antiplatelet therapy; CI, confidence interval; DOAC, direct oral anticoagulant; PACT-Q , Perception of Anticoagulant Treatment Questionnaire; RC, regression coefficient; VKA, vitamin K antagonist. n = 806, R square =0.068, analysis adjusted for, sex, age, and socioeconomic status. Patient reported outcome measures are useful tools and are increasingly being used to obtain data on patients’ perceptions of their received health care.12 The outcomes of treatment convenience and satisfaction in our study were very similar to the results of an observational study of Benzimra et al, 5 who also used the PACT-Q questionnaires. In our study, a bleeding was correlated to lower treatment convenience and satisfaction. Others have shown that bleeding impacts quality of life negatively.13 Although the between the included and excluded patients are available in the measuring methods do not quite match with our methods, it seems Supporting Information. Patients who knew who to turn to for that especially the clinically relevant nonmajor and minor bleeds questions reported to be significantly more adherent. Also, higher have a long-lasting impact on quality of life. The occurrence of gas- convenience and age >67 years were associated with adherence. A trointestinal complaints in our study was similar to the findings in twice-daily regimen and higher SES were associated with report- the ReLy study, in which the safety and efficacy of dabigatran and ing nonadherence. Patients who underwent a dental intervention warfarin were compared in patients with AF.14 These symptoms without a necessary DOAC adjustment reported to be significantly were associated with a decrease in treatment convenience, and more nonadherent than patients without an intervention. Men re- although this is quite plausible, to our knowledge this was never ported more often to be nonadherent than women. After imputa- published before. tion, by which an additional 172 patients could be included in the After the introduction of DOACs, much concern arose about analysis, the results were not substantially different (Supporting medication nonadherence. Because of the short half-lives of DOACs, Information). nonadherence can quickly result in subtherapeutic anticoagulant levels and increase the risk of thromboembolic events.15 The de- 4 \| DISCUSSION terminants associated with nonadherence in our study partly agree with the questionnaire results of Toorop et al,16 namely, a younger age and a twice-daily dosing regimen. The correlation between Most participants indicated a high convenience and a fairly high knowing who to turn to with questions and nonadherence has not satisfaction score with their DOAC treatment. However, we found been investigated elsewhere. several patient-, drug-, and organization-related factors that were Our study had no restrictive inclusion and exclusion criteria, so associated with lower scores. A negative sense of security was as- the setting can be considered as real life and therefore represen- sociated with lower treatment convenience and even stronger with tative for daily clinical practice. The sample size was large, and the lower satisfaction. Also, a bleeding with evaluation by a physician, response rate of our questionnaires was high (65%). Responders gastrointestinal side effects, and DOAC nonadherence were as- were comparable with nonresponders regarding sex, age, and sociated with lower convenience. In addition, a bleeding without SES. Unfortunately, we are not further informed about the 6 of 7 \| PIERSMA-WICHERS et al. F I G U R E 2 Factors associated with self-reported DOAC nonadherence. APT, antiplatelet therapy; DOAC, direct oral anticoagulant; GI, gastrointestinal; ref, reference; VKA, vitamin K antagonist nonresponders, and it is possible that patients who had already a small part of the patients’ treatment convenience and satisfaction, stopped their DOAC were underrepresented in our sample. For as the R square is relatively low. this reason, persistence was not an outcome in our study. Because of different definitions for nonadherence in the literature our work is only partially comparable to previous studies. Moreover, 5 \| CO N C LU S I O N we realize that self-reported adherence may be overestimated because of socially desirable answers. As validated questionnaires Based on our findings, several factors are associated with DOAC- and definitions were lacking for variables that we were interested related quality of life. Improving the information at treatment initia- in, we had to use a new, as yet not validated, questionnaire. tion and providing contact information that can be used in case of Our study identified a number of determinants correlated to the patients’ experienced quality of life related to DOAC treatment. problems might be accessible factors for improvement of treatment convenience and satisfaction. Most of these are fixed, but others might be influenceable. The latter can help health care providers to improve the quality of anticoagula- AC K N OW L E D G M E N T S tion care. An obvious solution could be providing information cards The authors gratefully acknowledge all employees of Certe and access information, possibly in a broader educational context of Thrombosis Service for their contribution in identifying potential a management coagulation service for all patients on DOAC therapy. participants. Explanation of the risks of non-adherence can also receive attention in an Anticoagulation Monitoring Service.17 Specific medication R E L AT I O N S H I P D I S C LO S U R E S intake instructions and/or comedication can possibly relieve gas- MP-W reports travel support from LEO pharma; travel and confer- trointestinal complaints and improve convenience, but also DOAC ence support from Pfizer; and financial support of a randomized rotation can be considered. Maybe the choice of a once-daily instead controlled trial pilot study by the Federatie van Nederlandse of a twice-daily medication regimen can contribute to improvement Trombosediensten, The Netherlands, outside the submitted work. of adherence. Further research is required. TE has nothing to disclose. AMBdV-B reports financial compensa- Furthermore, fixed factors are important because they can be tion for training employees by Bayer, outside the submitted work. used to focus more on certain subgroups of patients. However, we HAMK reports travel support and financial support for print- realize that the identified determinants in our analyses explain only ing PhD thesis from Bayer Healthcare, and financial support for \| PIERSMA-WICHERS et al. printing PhD thesis from CSL Behring; Federatie van Nederlandse Trombosediensten; University Medical Center Groningen/GUIDE; Pfizer; Foundation for the Promotion of Research and Education in Haemostasis, Thrombosis, and Rheology Groningen; University of Groningen; and Dutch Heart Foundation, outside the submitted work. KM reports travel support from Baxter; grants, travel support, and speaker fees from Bayer; grants and speaker fees from Sanquin; grants from Pfizer; speaker fees from Boehringer Ingelheim; speaker fees from BMS; speaker fees from Aspen; and consulting fees from Uniqure, all outside the submitted work. AU T H O R C O N T R I B U T I O N S All authors were involved in the design of the study. MP-W collected the data and was responsible for data handling. TE supported the statistical analysis. AMBdV-B stimulated the community pharmacists to sign up the patients for registration. MP-W prepared the manuscript. All authors discussed the results, revised the manuscript critically, and approved submission of the manuscript. DATA AVA I L A B I L I T Y S TAT E M E N T The data that support the findings of this study are available upon reasonable request. K. Thedinga (k.thedinga@umcg.nl) will serve as an additional point contact for data access. REFERENCES 1. Lloyd-Jones DM, Wang TJ, Leip EP, et al. Lifetime risk for development of atrial fibrillation: The Framingham Heart Study. Circulation. 2004;110(9):1042-1046. 2. Wolf PA, Abbott RD, Kannel WB. Atrial fibrillation as an independent risk factor for stroke: The Framingham Study. Stroke. 1991;22(8):983-988. 3. Camm AJ, Lip GYH, De Caterina R, et al. 2012 focused update of the ESC Guidelines for the management of atrial fibrillation: an update of the 2010 ESC Guidelines for the management of atrial fibrillation. Developed with the special contribution of the European Heart Rhythm Association. Eur Heart J. 2012;33(21):2719-2747. 4. Stichting Farmacaeutische Kengetallen. I. ISBN/EAN 978-90- 830805-0 -5. 2020 August. 5. Benzimra M, Bonnamour B, Duracinsky M, et al. Real-life experience of quality of life, treatment satisfaction, and adherence in patients receiving oral anticoagulants for atrial fibrillation. Patient Prefer Adherence. 2018;12:79-87. 6. Weernink MGM, Vaanholt MCW, Groothuis-Oudshoorn CGM, von Birgelen C. IJzerman MJ, van Til JA. Patients’ priorities for oral anticoagulation therapy in non-valvular atrial fibrillation: a multi-criteria decision analysis. Am J Cardiovasc Drugs [Internet]. 2018;18(6):493-502. 7 of 7 7. Borne RT, O’Donnell C, Turakhia MP, et al. Adherence and outcomes to direct oral anticoagulants among patients with atrial fibrillation: findings from the Veterans Health Administration. BMC Cardiovasc Disord. 2017;17(1):1-7. 8. Chinitz JS, Castellano JM, Kovacic JC, Fuster V. Atrial fibrillation, stroke, and quality of life. Ann N Y Acad Sci. 2012;1254(1):140-150. 9. Prins MH, Guillemin I, Gilet H, et al. Scoring and psychometric validation of the Perception of Anticoagulant Treatment Questionnaire (PACT-Q ©). Health and Quality of Life Outcomes. 2009;7(30):1–12. http://dx.doi.org/10.1186/1477-7525-7-3 0 10. Mouchet J, Carita P, Gilet HMH. Perception of Anticoagulant Treatment Questionnaire, information booklet, 4th ed. MAPI Research Trust; 2014. pp. 19. https://mapi-trust.org/ 11. van Miert JHA, Kooistra HAM, Veeger NJGM, Westerterp A, Piersma-Wichers M, Meijer K. Quality of life after switching from well-controlled vitamin K antagonist to direct oral anticoagulant: Little to GAInN. Thromb Res. 2019;2020(190):69-75. 12. Kingsley C, Patel S. Patient-reported outcome measures and patient-reported experience measures. BJA Education. 2017;17(4): 137–144. http://dx.doi.org/10.1093/bjaed/mkw060 13. Wang K, Li H, Kwong WJ, et al. Impact of spontaneous extracranial bleeding events on health state utility in patients with atrial fibrillation: Results from the ENGAGE AF-TIMI 48 trial. J Am Heart Assoc. 2017;6(8):1-7. 14. Connolly SJ, Ezekowitz MD, Yusuf S, et al. Dabigatran versus Warfarin in Patients with Atrial Fibrillation. New England Journal of Medicine. 2009;361(12):1139–1151. http://dx.doi.org/10.1056/ nejmoa 0905561 15. Conway SE, Hwang AY, Ponte CD, Gums JG. Laboratory and clinical monitoring of direct acting oral anticoagulants: what clinicians need to know. Pharmacotherapy. 2017;37(2):236-248. 16. Toorop MMA, Rein N, Nierman MC, et al. Self-reported therapy adherence and predictors for nonadherence in patients who switched from vitamin K antagonists to direct oral anticoagulants. Res Pract Thromb Haemost. 2020;4(4):586-593. 17. Clark NP. Role of the anticoagulant monitoring service in 2018: beyond warfarin. Hematol (United States). 2018;2018(1):348-352. S U P P O R T I N G I N FO R M AT I O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Piersma-Wichers M, Elling T, de Vries-Bots AMB, Kooistra HAM, Meijer K. Convenience and satisfaction in direct oral anticoagulant–treated patients with atrial fibrillation. Res Pract Thromb Haemost. 2021;5:e12577. https://doi.org/10.1002/rth2.12577
https://openalex.org/W2902835978	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0206686&type=printable	English	null	Network assessment of demethylation treatment in melanoma: Differential transcriptome-methylome and antigen profile signatures	PloS one	2,018	cc-by	13,515	RESEARCH ARTICLE Editor: Roger Chammas, Universidade de Sao Paulo, BRAZIL Editor: Roger Chammas, Universidade de Sao Paulo, BRAZIL Editor: Roger Chammas, Universidade de Sao Paulo, BRAZIL Received: June 20, 2018 Accepted: October 17, 2018 Published: November 28, 2018 Abstract Citation: Jiang Z, Cinti C, Taranta M, Mattioli E, Schena E, Singh S, et al. (2018) Network assessment of demethylation treatment in melanoma: Differential transcriptome-methylome and antigen profile signatures. PLoS ONE 13(11): e0206686. https://doi.org/10.1371/journal. pone.0206686 Background In melanoma, like in other cancers, both genetic alterations and epigenetic underlie the met- astatic process. These effects are usually measured by changes in both methylome and transcriptome profiles, whose cross-correlation remains uncertain. We aimed to assess at systems scale the significance of epigenetic treatment in melanoma cells with different met- astatic potential. ☯These authors contributed equally to this work. * ecapobianco@med.miami.edu Zhijie Jiang1☯, Caterina Cinti2☯, Monia Taranta2, Elisabetta Mattioli3,4, Elisa Schena3,5, Sakshi Singh2, Rimpi Khurana1, Giovanna Lattanzi3,4, Nicholas F. Tsinoremas1,6, Enrico CapobiancoID1* Zhijie Jiang1☯, Caterina Cinti2☯, Monia Taranta2, Elisabetta Mattioli3,4, Elisa Schena3,5, Sakshi Singh2, Rimpi Khurana1, Giovanna Lattanzi3,4, Nicholas F. Tsinoremas1,6, Enrico CapobiancoID1* 1 Center for Computational Science, University of Miami, Miami, FL, United States of America, 2 Institute of Clinical Physiology, CNR, Siena, Italy, 3 CNR Institute of Molecular Genetics, Bologna, Italy, 4 IRCCS Rizzoli Orthopedic Institute, Bologna, Italy, 5 Endocrinology Unit, Department of Medical & Surgical Sciences, Alma Mater Studiorum University of Bologna, S Orsola-Malpighi Hospital, Bologna, Italy, 6 Department of Medicine, University of Miami, Miami, FL, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. ☯These authors contributed equally to this work. * ecapobianco@med.miami.edu * ecapobianco@med.miami.edu Methods and findings Treatment by DAC demethylation with 5-Aza-2’-deoxycytidine of two melanoma cell lines endowed with different metastatic potential, SKMEL-2 and HS294T, was performed and high-throughput coupled RNA-Seq and RRBS-Seq experiments delivered differential pro- files (DiP) of both transcriptomes and methylomes. Methylation levels measured at both TSS and gene body were studied to inspect correlated patterns with wide-spectrum tran- script abundance levels quantified in both protein coding and non-coding RNA (ncRNA) regions. The DiP were then mapped onto standard bio-annotation sources (pathways, bio- logical processes) and network configurations were obtained. The prioritized associations for target identification purposes were expected to elucidate the reprogramming dynamics induced by the epigenetic therapy. The interactomic connectivity maps of each cell line were formed to support the analysis of epigenetically re-activated genes. i.e. those supposedly silenced by melanoma. In particular, modular protein interaction networks (PIN) were used, evidencing a limited number of shared annotations, with an example being MAPK13 (cas- cade of cellular responses evoked by extracellular stimuli). This gene is also a target Copyright: © 2018 Jiang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Network assessment of demethylation treatment in melanoma: Differential transcriptome-methylome and antigen profile signatures Zhijie Jiang1☯, Caterina Cinti2☯, Monia Taranta2, Elisabetta Mattioli3,4, Elisa Schena3,5, Sakshi Singh2, Rimpi Khurana1, Giovanna Lattanzi3,4, Nicholas F. Tsinoremas1,6, Enrico CapobiancoID1* Conclusion Demethylation treatment strongly affects early melanoma progression by re-activating many genes. This evidence suggests that the efficacy of this type of therapeutic intervention is potentially high at the pre-metastatic stages. The biomarkers that can be assessed through antigens seem informative depending on the metastatic conditions, and networks help to elucidate the assessment of possible targets actionability. Network assessment of demethylation treatment effects in melanoma associated to the PANDAR ncRNA, therapeutically relevant because of its aberrant expres- sion observed in various cancers. Overall, the non-metastatic SKMEL-2 map reveals post- treatment re-activation of a richer pathway landscape, involving cadherins and integrins as signatures of cell adhesion and proliferation. Relatively more lncRNAs were also annotated, indicating more complex regulation patterns in view of target identification. Finally, the anti- gen maps matched to DiP display other differential signatures with respect to the metastatic potential of the cell lines. In particular, as demethylated melanomas show connected targets that grow with the increased metastatic potential, also the potential target actionability seems to depend to some degree on the metastatic state. However, caution is required when assessing the direct influence of re-activated genes over the identified targets. In light of the stronger treatment effects observed in non-metastatic conditions, some limitations likely refer to in silico data integration tools and resources available for the analysis of tumor antigens. Competing interests: The authors have declared that no competing interests exist. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Istituto Toscano Tumori, 2013 Oncology Competition, http://www.ittumori.it/IttSanitaSrty/jsp/start_ENG. jsp?lang=ENG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 November 28, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Network assessment of demethylation treatment effects in melanoma investigated in relation to the silencing effects exerted over gene expression levels. This type of regulation affects especially the CpG islands, as these are proximal to gene promoter regions. Generally speaking, both hyper- and hypo-methylation states are relevant to cancer conditions through chromosomal instability, re-activation of retrotransposons, and oncogenic expression levels. Tumor suppressor genes are usually silenced due to hyper-methylation affecting cell cycle, invasion and DNA repair, and involving protein coding and non-coding genomic regions [4]. However, a general hypo-methylation has also been observed in comparison with normal cells [5,6]. Additionally, the study of resistance acquisition mechanisms is increasingly observed under the epigenetic lens in view of personalized treatments. In such regards, DNA methylation inhibition (5-Aza types) is in principle emerging as a treatment option in some cancers (i.e. CRC, see [7]) due to induced activation of the immune response and co-activation of both tumor suppressors and DNA repair genes. Melanoma is a malignant tumor of melanocytes whose incidence is increasing worldwide, therefore attracting major interest in the current research [8]. With metastasis, both genetic and epigenetic alterations become relevant. Of interest in this work is to build the coupled pro- file of transcriptome and methylome features from two different melanoma cell lines, i.e. two of different metastatic potential (one non-metastatic, SKMEL-2, and the other metastatic, HS294T). The main characteristic is that these melanoma cell lines are subject to the same epi- genetic treatment. Since we measured both transcriptome and methylome profiles, it is natural to ask whether these are possibly correlated. This relationship is quite controversial in light of the literature results. Machine learning algorithms are designed for automatically learning tasks or functions, offering the advantage of managing heterogeneity of data and scalability of methods. For instance, they can perform automated actions within networks, directed to the discovery of differential behaviors, to the detection of anomalies, to measuring the correlation between patterns. We aim to use networks to reconcile the various experimental, computa- tional and annotation evidences under a system’s configuration allowing the identification of melanoma targets. Ultimately, target validation may lead to understanding biological mecha- nisms relevant to melanoma, and to therapeutic intervention. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Introduction Cancer genomics deals with a wealth of ‘signals’ and ‘marks’ routinely detected through quan- titative measurements obtained at wide genome spectrum. Naturally enough, a first level of complexity involves genomics, due to the diversity of biotypes (protein coding genes, non-cod- ing RNAs) and the computational methods that need to be specifically used to retrieve them. The expected outcome of such process is a reference “signal space’ as a product of the synergy between encoding bases, transmission mechanisms, cellular networking, all factors spanning a multitude of possible states and their communication patterns [1]. The perturbations to such systems come from factors altering the internally generated signal dynamics and enabling aberrant patterns. Measuring the induced effects at systems scale and establishing the signifi- cant impacts of such influencers from multiple perturbation sources are key steps. A first aspect is the multiscale localization, by which genomic information is heterogeneously distrib- uted across scales (from bases to megabases), and different patterns are observable but can be measured only through different resolution-sensitive tools. Another aspect involves the role of causative, correlative and confounding factors that need rigorous assessment for inferring any valuable association between observed signals and patterns. More complexity appears in a cancer-contextualized way, and is highly heterogeneous. Each cancer is bringing the signature of general hallmarks but also specific features. Next Gen- eration Sequencing protocols and their ad hoc methodological pipelines have clearly indicated that a variety of molecular landscapes and profiles (expressional and mutational) exist, with melanoma as an example [2]. Additionally, the centrality of large-scale transcriptome studies and mutational approaches targeted to capture non-coding RNA evidences boosted the prom- ised impact of epigenetic features [3]. As an example, DNA methylation has been often PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 2 / 26 Melanoma cell state transitions Despite primary tumors cause widespread cells dissemination, only a relatively limited fraction of them form metastasis in the end [17]. Cell adhesion mechanisms are crucially involved in basic cellular processes and their changes are implicated in cancer through the loss of control of cell proliferation and the start of metastatic dynamics. Changes in cell adhesive properties specifically induce plasticity, which plays a central role for metastatic phenotype development. Cadherins and Integrins are examples of adhesion molecules. Cadherins [18] exert functions at intercellular level, mainly communicating by cell connections through calcium ion-dependent binding. Instead, at an intracellular level, cadherins bind to catenin molecules establishing links with the cytoskeleton. Their specific role in melanoma is illustrated, among other refer- ences, in [19]. Their role in tumor progression is illustrated in [20]. Correlation between reduced levels of E-type cadherin and reduced survival has been studied recently in various melanoma types [21]. Here, it was also investigated in vitro transcription restoration following 5-aza-dC treatment (DNMT1 silencing), indicating an inverse correlation with a potential therapeutic role for promoter demethylation re-activating cadherin expression. With regards to the role of integrins, and their effects on migration and invasion potential, it is known that melanoma cell lines with different metastatic power exhibit heterogeneity [22]. In general, integrins function like ‘radars’, i.e. transmembrane cell-surface receptors, by bridging between the extracellular matrix and the cytoskeleton when environmental changes are detected. They are involved in multiple key processes, such as differentiation, adhesion, migration, proliferation and survival [23]. Finally, the interplay between integrins and growth factors presents clear opportunities for therapeutic targeting [24]. A study showed that BRAF (V600E) is associated with specific methylation changes, thus driving effects over target genes and activating growth signaling [25]. RASSF1A is another example of tumor suppressor gene involved in cell cycle and apoptosis, and representing a good candidate marker of tumor progression. Furthermore, it often results hyper-methylated, while deregulated in expression levels. Re-expression can be induced by 5-aza through demethylation of its promoter. Another study focused on the reprogramming phase between proliferative (SOX10/MITF) and invasive (AP-1/TEAD) melanoma cells (from biopsies) through the integration between transcriptomic and epigenetic modifications and methylation profiles [26]. These last authors inferred a functional network representing cell state transition, probably driven by the tumor microenvironment, and explaining the observed transcriptional reprogramming. It is important to stress that changes in network dynamics may facilitate the identification of cancer pre-transition states. Network assessment of demethylation treatment effects in melanoma that reasoning in predictive terms from methylation to expression levels remains an uncertain task, mostly supported by methylated promoters in correspondence with gene silencing, and contrary to non-methylated promoters. To bypass this limitation, research focused on how networks may identify ‘epi-oncomarkers’ [12], and ‘epigenetic modules’ [13]. These ideas were used in pan-cancer studies [14]. Here, the observed correlative or causative dynamics call for further investigation, and single cancers already represent highly complex systems. For instance, melanoma involves multiple signaling pathways, cell cycle regulators and apoptotic control mechanisms whose dynamics can be studied at a genomic network scale [15]. Interest- ingly, while the genetic predisposition explains no more than 10% of melanoma cases, and BRAF is the most frequently mutated gene (in up to 70% of cases), the role of epigenetics and epigenetically driven regulatory networks in both initiation and progression of melanoma remain to be clarified. This holds with regard to the associations between epigenetic marks and non-coding RNAs, and the co-regulations or mediations thus induced [16]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 The complexity of epigenetic regulation Epigenetic modifications involve heritable and reversible changes in gene expression levels not referred to DNA sequence alterations, but rather to DNA methylation and histone modifica- tions. DNA methylation is known to induce transcriptional inactivation or silencing in geno- mic regions (including non-coding). Silencing may also affect the expression levels of key transcriptional regulators, and exert cascading effects to downstream targets. Significant changes are especially expected in the presence of cancer. Hyper-methylation is considered responsible for transcriptional quiescence and suppression of expression. In turn, microsatel- lite instability and higher mutational frequency may be triggered. These activities are sup- ported by the chromatin structure close to the gene promoters. By affecting DNA methylation, the transcriptional activity states get perturbed. Methylation within gene promoters can turn off their potential of suppressing tumorigenesis, cell adhesion, differentiation and growth. All such processes play roles in tumor initiation, metastasis and thus progression. Regarding pro- gression and response to drug treatment, it is important to quantify epigenetic drivers of clonal heterogeneity [9]. Differentially methylated genes (DMG) and differentially expressed genes (DEG) can reveal cross-correlative patterns for their differential profiles, here indicated by DiP. Increased meth- ylation levels tend to be associated with decreased expression levels at gene promoters, and such correlation seems harder to observe at gene body level [10,11]. This difference implies 3 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Network assessment of demethylation treatment effects in melanoma network scale, which remains difficult to detect and interpret, but preferably at a sub-network interactomic or modular scale, which can be algorithmically computed and evaluated in its sig- nificance [27–29]. In order to understand differential methylation patterns, also CpG island tracts are usefully studied, as from the exploration of coding and non-coding regions in cell lines, patient sam- ples, melanocytes of various types [30]. Here, early and late stage melanoma differentiated in retrotransposable elements and CpG island signatures, indicating methylation-driven progres- sion. Also, a co-expression network was built to establish the relationships between metastasis- linked genes subject to 5-aza treatment. An earlier study [31] pointed out gene expression pro- files differences between primary and metastatic melanoma in an attempt to identify a progres- sion-linked transition point. Specific gene sets appeared associated to the two conditions, and a transition period indicated a signature of the metastatic phenotype emergence (including oncogenes and suppressors). Asymmetry in expression was also found in another study through RNA-Seq of 3 distinctly pathogenic cell lines (normal, onset and metastasis) [32]. A special discriminatory role was here assigned to cytokine regulatory pathways, together with differential networks (cell death, cell cycle, cellular development). Rationale of the proposed study Our study aims to verify significant systemic effects of epigenetic treatment while informing on the influence exerted by melanoma metastatic states. All the significant evidences obtained from transcriptome and methylome profiles where mapped at the interactome scale, a natural ground for assessing intricate regulations and identifying possible targets. In such regards, net- works applied to large-scale ‘panomics’ have contributed to elucidate a series of hypotheses, such as identifying phenotypic drivers in targeted therapies (precision medicine approach) or the applicability of a reproducible cycle based on models, validations, and therapies acting inter-connectedly as a system (system medicine approach) [33]. Our use of the interactomes is aimed to the drawing of reference differential maps of coupled transcriptome-methylome DiP informative of the metastatic influences. Concerning such regulations, the final part of our work is dedicated to melanoma associ- ated antigens, and we use network inference in this context too. Several results have recently appeared showing T cell specific to neoantigens in cancer patients in view of developing cancer vaccines [34]. For instance, it is known that an immune response can be induced in melanoma patients, and thus melanoma cells express antigens as targets for immunotherapy [35,36]. These antigens are considered immune-activating and can provide diagnostic and prognostic utility. Melanoma has a quite hyper-mutated genome which increases the chances of neo-epi- tope formation [37]. This in turn explains why melanoma remains an excellent candidate for targeted immunotherapy. Several studies have considered somatic mutations for achieving predictive signatures associated with neo-antigen loads [38]. Antigens heterogeneity and pro- file stability are subject to variation depending on tumor, and especially cell proliferation stages [39]. It appears that T cell immunity acting against tumor-driven amino acid substitutions in melanoma patients presents a great potential outsourcing of antigens, which in turn requires systems analysis in order to identify targets of anticancer immunity. Melanoma cell state transitions These may not necessarily occur at a global PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 4 / 26 Cell cycle analysis After 72 hours of incubation with the culture medium containing (or not) 10 μM DAC, control and treated cells were harvested and their cell cycle phases were analysed by flow cytometry (FACS). For analysis, nuclei were stained with 10 μg/ml propidium iodide (PI) in hypotonic solution (1X PBS containing 0.1% sodium citrate and 0.1% Triton X-100) for 30 minutes at 4˚C in the dark. Cell cycle phases were analyzed by a FACS-Canto-II flow cytometer (BD Bio- sciences, San Jose, CA, USA) and data were analyzed with the FlowJo (Ashland, OR, USA) software. To evaluate the effect of DAC treatment on both melanoma cells, a one-way ANOVA was performed (cell cycle phases of treated vs control). Statistical significance thresh- old was set at a p-value < 0.05. The efficacy of different DAC concentrations administered at different time points to both melanoma cell lines was tested and following each treatment, and the effect on cell cycle was monitored by flow cytometric analysis. Fig 1 shows that treating both cell lines with 10μM DAC for 72h has induced a significant enrichment in sub-G1 peak (one-way ANOVA P < 0.05) in comparison to untreated cells. Since population in sub-G1 peak contains dying cells (apoptosis), it is an indicator of drug effect on cell viability. Network assessment of demethylation treatment effects in melanoma rate. In terms of cell culture and treatment, SKMEL-2 and HS294T were purchased by Ameri- can Type Culture Collection (ATCC) (Rockville, MD) and cultured under recommended con- ditions. Both human melanoma cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum, 2 mM L-glutamine and 1% penicillin-streptomycin, at split ratio of 1:3 twice a week and incubated at 37˚C in a 5% CO2 air atmosphere. For treatments, cells were seeded in 6-well plates at a density of 5 x 104 cells. After 24 hours from splitting, the culture medium was replaced with media containing no drug (control) or 10 μM 5-AZA-2’-DEOXYCYTIDINE (DAC) (treated cells) (Sigma- Aldrich) for 72 hours. Cell lines characteristics SKMEL-2 (ATCC HTB-68) is a cell line derived from patient skin malignant melanoma cells, and is known to express wild-type BRAF and mutant NRAS. HS294T (ATCC HTB-140) is a cell line derived from patient lymph node metastatic site, and has an enhanced proliferative 5 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 RNA-Seq The sequenced data were processed for DEG and ncRNA profiling using tools from the R envi- ronment. We used read counts to assess genomic coverage, and when less than 10 mapped reads were counted, the call was discarded. The read counts were then normalized by the edgeR package in Bioconductor [40]. The read counts were assessed for treatment and control samples under the negative binomial distributional assumptions. Differential expression in edgeR is assessed gene-wise by a Fisher’s exact test adapted to handle data over-dispersion. Variation estimates were performed by choosing a Generalized Linear Model (GLM) to account for gene-specific biological variation. Genes with \|logFC\|>1x and FDR<5% were established as DEGs. In choosing among various alternative models, the exact Test was found to generate less DEGs than GLM, actually providing subsets of those obtained with GLM. Hence, we selected GLM results (S1 Tables include DEGs). The differential values were detected in non-coding genomic regions too, and involved ncRNA categories further classified into major biotypes, such as lincRNA, pseudogenes, anti- sense, among all other biotypes that were found. This classification (S2 Tables) was obtained by using the Ensembl genome annotation (Homo_sapiens.GRCh38.85.gtf) as a guideline for annotation (S3 Tables). The parental genes associated with pseudogenes were determined by an in-house resource of pseudogenes-parental genes associations. The criteria for assessing transcribed evidence of significant associations are: 1) Reads mapped to the pseudogene sequence and not to the parental gene; 2) Reads mapped to both the pseudogene and to the parental gene, but with lower sequence similarity (<90%). Target parental genes were identi- fied by aligning the pseudogene sequences using BLAST against a database of the protein cod- ing CDNA sequences from Ensembl (v. 72). The best hit matches for a pseudogene sequence were selected based on e-value scores, and the best overall hit for a pseudogene was selected as its parental gene. The contextual analysis of lincRNAs with respect to the DEG targets was established on the basis of the physical proximal distance at chromosome level. The lncRNAs are generally classi- fied into cis- and trans-regulatory biotypes, influencing how they may target proteins and then determine RNA–protein interaction. One looks at whether the lncRNA regulates neighboring genes, i.e. genes on the same chromosomal regions where they are located, or instead distant genes, i.e. on other chromosomes. DNA and RNA extraction Genomic DNA was extracted from untreated SKMEL-2 and HS294T cells, using QIAamp DNA mini kit according to the manufacturer’s instructions. DNA concentration was deter- mined by NanoDrop spectrophotometer (Thermo Scientific). DNA integrity was checked by 1% agarose gel containing 1 μg/ml ethidium bromide. Fresh prepared DNA samples were sent Fig 1. Analysis of cell cycle phases of SK-MEL-2 (left) and HS294T (right) for both untreated (ctrl) and treated (DAC) cells with 10 μM DAC for 72h. The cells % in sub-G1 (apoptotic cells), G0-G1, S and G2-M cell cycle phases are reported in Y axis. Data are reported as mean ± sd. Statistical significance threshold set at p-value < 0.05. https://doi.org/10.1371/journal.pone.0206686.g001 Fig 1. Analysis of cell cycle phases of SK-MEL-2 (left) and HS294T (right) for both untreated (ctrl) and treated (DAC) cells with 10 μM DAC for 72h. The cells % in sub-G1 (apoptotic cells), G0-G1, S and G2-M cell cycle phases are reported in Y axis. Data are reported as mean ± sd. Statistical significance threshold set at p-value < 0.05. https://doi.org/10.1371/journal.pone.0206686.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 6 / 26 Network assessment of demethylation treatment effects in melanoma to BGI TECH SOLUTIONS (Hong Kong) for methtylation analysis by Reduced Representa- tion Bisulfite Sequencing (RRBS). Total RNAs were extracted from both treated and untreated melanoma cells using NucleoSpin RNA isolation kit (Macherey-Nagel) according to the man- ufacturer’s instructions. RNA concentration and purity was determined by NanoDrop spec- trophotometer (Thermo Scientific). Fresh prepared RNA samples were sent to BGI TECH SOLUTIONS (HongKong) for transcriptome sequencing by RNA-Seq. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Bioannotations Gene annotations (S6 Tables and S7 Tables) were retrieved from DAVID 6.8 (Database for annotation, Visualization and Integrated Discovery) (https://david.ncifcrf.gov/), an integrated database of data and tools like ENSEMBL, NCBI, UniProt, KEGG, BIOCARTA, PANTHER, BIND, GO and PUBMED etc. Following RNA-Seq, the identified ncRNAs were cross-checked with the lincRNome db (http://www.lncrnablog.com/tag/lincrnome/) and the lncrnadb (http://www.lncrnadb.org/) to understand the functionality details (S2 Text). From the DAnCER db (http://wodaklab.org/ dancer/) (Disease Annotated Chromatin Epigenetics Resource) [41] Recurring genes involved in chromatin modeling were also checked (S3 Text), and bio-annotations (molecular function and biological processes) were integrated also by BiNGO (http://apps.cytoscape.org/apps/ bingo) [42]. Networks tools Networks were generated from the STRING db (https://string-db.org/) by importing as sources the DEG and DMG (primarily, but not only, up-methylated) lists. The annotations reported on the maps have been obtained by the internal knowledgebase, and cross-checked within the GeneCards system (http://www.genecards.org/). The ncRNAs listed in the maps were man- ually curated and superimposed to the maps, after considering the previously computed associations. RNA-Seq Notably, lncRNAs interact directly or indirectly within genomic regions, sometimes through proteins performing specific biological functions, some- times through other neighbor lncRNAs in a coordinated way. In our application, locations of DE lincRNAs were obtained by simple steps: a) lincRNA at their genomic locations (start and end positions) and b) protein-coding genomic locations (start and end positions); c) lincRNA neighbors annotation. Genes at both left and right sides of starting and ending positions of lincRNAs, and within various intervals, say ±1, ±2, ±3 Mbps regions, were considered as puta- tive targets. Despite these intervals remain arbitrary, there is not a universally accepted defini- tion of an accurate range. In an attempt to explore neighbors of the lncRNA locus that confidently allow putative targets to be identified, we assigned priority to targets located at the closest possible distance from the locus of interest. confidently allow putative targets to be identified, we assigned priority to targets located at the closest possible distance from the locus of interest. 7 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Network assessment of demethylation treatment effects in melanoma As per the correlation between methylation on promoter region and gene expression, DEGs with \|logFC\| greater than 1.2x and methylation level on either promoter region or gene body greater than 0 were selected for performing correlation analysis. The methylation levels and expression values (FPKM) of treatment were normalized between 0 and 1. The linear model among expression of treated, methylation level on gene body and promoter region were accessed by lm (formula = treated ~ promoter + gene) and gls (model = treated ~ promoter + gene) functions in the R package. In both models, methylation level at promoter is signifi- cantly correlated with expression of treated (p<5%) while methylation level at gene body show no sign of correlation with expression (S1 Text). Extensive details on cross-profiles (transcrip- tome-methylome) are reported in S4 Tables and S5 Tables. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Detections Both protein coding genes and non-coding RNA expression values were considered compo- nents of interest for the DiP obtainable from each cell line by performing RNA-Seq. Table 1 summarizes these biological entities (details in S1 Tables). Fig 2 lists together with the detec- tions found in common between cell lines (Venn diagram), the genes considered putative tar- gets associated to a) lincRNAs, and retrieved within the flanking region of 1MB upstream to start position and downstream to end position; b) antisense; c) pseudogenes, considering their parental genes. Note that some of the antisense, pseudogenes and lincRNAs were also identi- fied as part of the DiP at the transcriptome level. Also note that metastatic values were half dis- tinctly detected and half shared with the DiP found in the non-metastatic cell line. Table 2 shows the various biotypes observed at transcript level and subsequently annotated. Table 3 shows annotations obtained by both DAVID and BiNGO. https://doi.org/10.1371/journal.pone.0206686.t001 Biological validations Drug administration for western blot analysis was performed in both HS294T and SKMel-2 by administering 10 μM 5-Aza-2’-Deoxycytidine (DAC) in culture medium for 72 hours. West- ern blot analysis was performed: cells were lysed in buffer containing 20 mM TRIS-HCl (pH = 7.5), 1% SDS, 1 mM Na3VO4, 1 mM PMSF, 5% beta-mercaptoethanol and protease inhibitors. After sonication, centrifugation and protein quantification by Bradford method, 15micrograms of proteins were subjected to SDS gradient gel (5–20%) electrophoresis and transferred into a nitrocellulose membrane. Incubation with primary antibodies: anti-PARP1 (Santa Cruz, sc-7150), anti-lamin A (Abcam ab26300) and anti-GAPDH (Millipore) was per- formed overnight at 4˚C. Secondary antibodies were applied for 20 minutes and immuno- blotted bands were revealed by Invitrogen ECL detection system. Immunoblotted bands were analysed by a BioRad Densitometer. Then, statistical analysis was performed from data over three independent experiments and using the Student’s t-test. Data were reported as mean val- ues with standard deviation (statistical significance values were associated to symbols as fol- lows: : p<0.05; : p<0.01; : p<0.001). 8 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Network assessment of demethylation treatment effects in melanoma Bio-annotations The biological annotations (see S6 Tables and S7 Tables) are summarized next. In SKMEL-2 the KEGG pathways point to the top-5 enriched terms that include: cell adhesion molecules (fdr = 6.59E-04; 35 genes); ECM-receptor integration (fdr = 0.002231; 25 genes); PI3K-Akt sig- naling (fdr = 0.006324; 61 genes); focal adhesion (fdr = 0.007964; 42 genes); leukocyte transen- dothelial migration (fdr = 0.024198; 28 genes). Other pathways have been enriched with reduced significance, as with proteoglycans, platelet activation, cytokine-cytokine receptor inter- action, Rap1 signaling, and calcium signaling. In terms of molecular functions, the most enriched terms are extracellular space and region (fdr = 6.52E-17, fdr = 2.88E-07, respectively) with 230 and 225 genes, respectively, and plasma membrane (fdr = 7.73E-09) with 498 genes. Cell adhesive properties refer to tumor plasticity and play a central role in the metastatic process. They involve morphological properties and integrin expression, but also migration and invasion potential signs. Metastasis drivers act in different directions. First, the ECM- receptor integration plays a key role during tumor progression through cross-talks between cancer spots and its surrounding regions. Second, joint with the local microenvironment (niche) that determines cancer development and in response to ECM signals, also PI3K-Akt regulates the cell cycle and deals with intracellular signaling, thus affecting cell metabolism, proliferation, growth, survival and angiogenesis. With HS294T the annotation evidenced the following terms: cytokine-cytokine receptor interaction (fdr = 0.01022), NF-kappa B signaling (fdr = 0.044757), leukocyte transendothelial migration (fdr = 0.048354), these all appearing with acceptable enrichment scores. In terms of molecular functions, the dominant one is extracellular region with 157 genes (fdr = 6.63E-18), Table 1. The number of differentially expressed biotypes in HS294T and SKMEL-2 cell lines. Cell lines Total n. of differentially expressed biotypes Cell-specific differentially expressed biotypes HS294T 1536 808 SKMEL-2 1993 1265 Number of shared differentially expressed biotypes 728 Note: The DE biotypes were selected based on criteria involving both expression and methylation levels in the treatment group, i.e. threshold of logFC > 50% quartile of logFC values, and hyper-methylation at the promoter of unregulated DE values. Table 1. The number of differentially expressed biotypes in HS294T and SKMEL-2 cell lines. Cell lines Total n. of differentially expressed biotypes Cell-specific differentially expressed biotypes HS294T 1536 808 SKMEL-2 1993 1265 Number of shared differentially expressed biotypes 728 Table 1. The number of differentially expressed biotypes in HS294T and SKMEL-2 cell lines. Bio-annotations Note: The DE biotypes were selected based on criteria involving both expression and methylation levels in the treatment group, i.e. threshold of logFC > 50% quartile of logFC values, and hyper-methylation at the promoter of unregulated DE values. Note: The DE biotypes were selected based on criteria involving both expression and methylation levels in the treatment group, i.e. threshold of logFC > 50% quartile of logFC values, and hyper-methylation at the promoter of unregulated DE values. Note: The DE biotypes were selected based on criteria involving both expression and methylation levels in the treatment group, i.e. threshold of logFC > 50% quartile of logFC values, and hyper-methylation at the promoter of unregulated DE values. https://doi.org/10.1371/journal.pone.0206686.t001 9 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Network assessment of demethylation treatment effects in melanoma Fig 2. Venn diagram of DE biotypes (top). Cell lines detections by biotype. https://doi.org/10.1371/journal.pone.0206686.g002 followed by cell differentiation with 201 genes (fdr = 3.19E-08), cell communication with 143 genes (fdr = 9.82E-06), cell motility with 132 genes (fdr = 5.05E-05), cytoskeletal proteins with 103 genes (fdr = 5.58E-05), cell cycle with 104 genes (fdr = 8.39E-05), and cell adhesion with 95 genes (fdr = 1.94E-04). A major role is played at the signaling level by immunological and inflammatory factors acting in response to disease and stressors through the binding to specific receptors of target cells. These factors typically involve cytokines, chemokines and adhesion molecules, and also leukocyte transmigration. followed by cell differentiation with 201 genes (fdr = 3.19E-08), cell communication with 143 genes (fdr = 9.82E-06), cell motility with 132 genes (fdr = 5.05E-05), cytoskeletal proteins with 103 genes (fdr = 5.58E-05), cell cycle with 104 genes (fdr = 8.39E-05), and cell adhesion with 95 genes (fdr = 1.94E-04). A major role is played at the signaling level by immunological and inflammatory factors acting in response to disease and stressors through the binding to specific receptors of target cells. These factors typically involve cytokines, chemokines and adhesion molecules, and also leukocyte transmigration. Note: Outcome of Generalized linear models (GLM) selected in the edgeR function. Chromatin remodeling By using DAnCER, forty DEGs were matched with the genes involved in chromatin in HS294T. Twentytwo of them were highly over-expressed, for instance H2AFB2, CTCFL, By using DAnCER, forty DEGs were matched with the genes involved in chromatin in HS294T. Twentytwo of them were highly over-expressed, for instance H2AFB2, CTCFL, Table 2. Decomposition of Differentially expressed biotypes in HS294T and SKMEL-2 cell lines. Hs294T cell line SKMEL-2 cell line Biotypes GLM edgeR Biotypes GLM edgeR NA 35 NA 71 antisense 17 TEC 1 lincRNA 22 antisense 30 pseudogene 15 lincRNA 38 processed_transcript 7 misc_RNA 1 protein_coding 1435 pseudogene 26 rRNA 1 processed_transcript 12 sense_overlapping 4 protein_coding 1810 scRNA 1 sense_intronic 1 sense_overlapping 1 snRNA 1 Note: Outcome of Generalized linear models (GLM) selected in the edgeR function. e 2. Decomposition of Differentially expressed biotypes in HS294T and SKMEL-2 cell lines. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 10 / 26 Network assessment of demethylation treatment effects in melanoma Table 3. Molecular functions annotated for both cell lines. source: DAVID source: BiNGO Cell line N. of detections Statistically significant (p-values <0.05) Statistically significant (exclusive) (p-values <0.05) Statistically significant (p-values <0.05) Statistically significant (exclusive) (p-values <0.05) HS294T 66 51 12 29 9 SKMEL-2 76 59 21 48 28 Common functions 51 38 20 Note: details provided in S6 Tables and S7 Tables. Table 3. Molecular functions annotated for both cell lines. SYCP3, AURKC, HIST1H2BG, HIST1H3D, HIST1H2BJ, SERPIND, and three were highly down-expressed, namely ZEB2, MGAM, LMNB1. Beyond being involved in DNA packaging and chromatin modelling, these genes play a role in cancer, for example the SERPIN family is involved in metastasis of cancer. Among the down-expressed genes, LMNB1 regulates PAK- 2p34 by protease mediated degradation, MGAM is involved in metabolism and immune system, ZEB2 is involved in the TGF-β signalling pathway. With SKMEL-2, thirtyfour DEGs were pres- ent also in DAnCER, of which nineteen genes overlapped with the HS294T cell-line. Instead, fif- teen DEGs were exclusively found in SKMEL-2, examples being SIRT7, PCSK4, H1F0, BCL6, SAP30, HIST1H3D, TLE4 etc. In particular, SIRT7 is involved in aging, cancer and circadian rhythm, while BCL6 is a transcriptional repressor involved in the immune system. Among the genes considered putatively involved in chromatin remodeling, two were pres- ent as down-expressed among our DEGs, POU3F2 (transcription factor, logFC = –1.549) classified as a melanoma gene, and IRF4 (interferon, logFC = -1.833) classified as a skin neo- plasma gene. Other two genes were matched, but with smaller negative expression, SOX10 (ERK signaling) and GLI2 (Wnt). With SKMEL-2 other matches were found, for instance NOS3 (logFC = 5.174), mediating VEGF-induced angiogenesis, ACTA2 (logFC = 4.862), an actin involved in cell motility. And GLI1 (logFC = 2.036), a member of the Kruppel family of zinc finger proteins encoding a transcription factor activated by the Hedgehog cascade and regulating stem cell proliferation and also p53 (negatively). PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Network assessment of demethylation treatment effects in melanoma Highly differential expressions of ncRNA targets appeared in relation with extracellular region functions. Considering HS294T, three identifications are worth mentioning: the over- expressed F5 and PRAP1, involved in cell adhesion, and TNC, which is down-expressed. With regards to SKMEL-2, of interest is the identification of CD14, involved in programmed cell death. Another group of functions is relevant, and involves cell junctions, i.e. multiprotein complexes that allow the contact between neighboring cells or between cells and the extracellu- lar matrix. Especially three identifications shared across the cell lines and highly over- expressed are especially relevant: CRB3 (cell polarity in epithelial cells, regulators of morpho- genesis), XIRP1 (involved with actin), and ARC (cell morphology, migration and cytoskeletal organization). Other DEGs were instead identified as uniquely over-expressed. For instance, in HS294T these are: ESAM (endothelial cell adhesion), involved in platelet activation and immune cell transmigration; GJB4 (gap junctions), providing intracellular communication; TNS4, involved in cell migration and possibly promoting apoptosis. With SKMEL-2 other highly over-expressed identifications were uniquely found, namely: CDHR2, a tumor suppres- sor; PECAM1, involved in leukocyte migration, angiogenesis and integrin activation; GPER1 (G-protein coupled receptor 1); S100A1, a tumor suppressor; SLC30A3, involved in trans- membrane transported activity. For the proteinaceous extracellular matrix, SKMEL-2 presents a few highly over-expressed DEGs, such as MMP9 (cell death regulator) and WNT10A (Wnt signaling), while HS294T presents SERPINA1 (hypoxia) as over-expressed and a series of down-expressed genes, such as MMP17 (metabolism), MGP (cell differentiation), CHL1 (cell adhesion and differentiation), MMP16 (proteolysis), COL11A1 (collagen). Of interest the sign concordance across cell lines of the over-expressed FBLN2 (cell adhesion) and PTPRZ1 (cell differentiation). Among the identifications uniquely found, a few appear in SKMEL-2 with the highly overexpressed WNT1 and WISP2 (Wnt), SOST (cell communication), ADAMTS14 (proteolysis), HAPLN4, ELN and COL8A2 (cell adhesion and proliferation), while then others identifications appear in HS294T as the under-expressed cell adhesion genes (among other processes), COL15A1, FREM1, GPC6 and FLRT1. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 ncRNAs, DEG targets, and a few specific annotations Considering the identified DE lincRNAs in SKMEL-2, the targets at 1-to-3MB distance from the genomic locus were identified. There are sixtynine targets, and some deserve attention. For instance, the over-expressed KLHDC8A, providing an alternative pathway for tumors for maintaining aggressiveness in the absence of epidermal GFR dependence. Then MZB1, which causes cell-specific regulation of apoptosis, among other functions, likewise TNFRSF25 and MX2 (interferon), both important for apoptosis. When looking at the matches of our detec- tions within the lncRNome db [43] (see S6 Tables), only LINC00337 was found, among those with target genes within the 1Mb of distance from locus. There are other seven target genes, namely KCNAB2, CHD5, GPR153, HES2, ESPN, TNFRSF25, PLEKHG5, and six are over- expressed and only KCNAB2 is down-expressed. Of these, CHD5 is a potential tumor suppres- sor regulating the expression of genes involved in cell proliferation and differentiation. Down- stream activated genes may include CDKN2A, which positively regulates the p53/TP53 pathway, which in turn, prevents cell proliferation. GPR153 and HES2 are involved in tran- scription activity, while TNRFSF25 induces apoptosis. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 11 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Differential network maps Cross-referencing of transcriptome and methylome DiP leads to network maps as a way to select regions in which the combined influence may exert effects visible through hubs, hierar- chies or modules. Since the majority of detected evidences turned into hyper-methylated over hypo-methylated genes, we focused on cancer genes undergoing inactivation most likely induced by hyper-methylation, in other words those subject to epigenetic silencing. Here, treatment via demethylation influences the re-activation of their expression levels. Being these genes involved in several pathways, an additional constraint was applied by mapping DEGs from both cell lines that were found upregulated. This implies re-activation of expression levels induced by treatment. Looking at Fig 3, the SKMEL-2 cell line network map, a number of nodes appear annotated in various ways. In some cases these are functionally relevant (gene/module annotations recalled by blue arrows) by either themselves, as with MAPK13 (light green envelope) or NOTCH3 (involved in several functions, i.e. differentiation, proliferation, apoptosis), or because of aggregates or modules. This is the case of WNT (WNT3 and WNT10A, yellow envelope), whose role in tumorigenesis is widely known in relation with proliferation and migration, or CDH3 and CDH15 (red envelope, bottom right), i.e. cadherin of P and M type, respectively. In particular, a paralog of CDH3 is CDH1, cadherin of the E type, a known growth and invasion suppressor whose loss of function contributes to cancer progression 12 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Network assessment of demethylation treatment effects in melanoma Fig 3. SKMEL-2 cell line map. Protein-protein Interaction map from STRINGdb obtained from DiP (DEGs and differentially methylated). The effects of treatment were analyzed in hyper-methylated DEGs to consider silencing effects on expression before treatment. The red names refer to ncRNAs, and lincRNAs depend on 1Mb distance between their locus and the associated target genes. Fig 3. SKMEL-2 cell line map. Protein-protein Interaction map from STRINGdb obtained from DiP (DEGs and differentially methylated). The effects of treatment were analyzed in hyper-methylated DEGs to consider silencing effects on expression before treatment. The red names refer to ncRNAs, and lincRNAs depend on 1Mb distance between their locus and the associated target genes. Fig 3. SKMEL-2 cell line map. Protein-protein Interaction map from STRINGdb obtained from DiP (DEGs and differentially methylated). The effects of treatment were analyzed in hyper-methylated DEGs to consider silencing effects on expression before treatment. Network assessment of demethylation treatment effects in melanoma Table 4. Cell adhesion in view of cadherins in SKMEL-2. SKMEL-2 Cell line TUMOR TREATED Log(FC) Log(CPM) Methylation Threshold Promoter Gene Body T > 2.24 M > 4.25 CDH1 0.64 5.14 2.86 3.76 3.44 21.02 T (yes) M (No) CDH3 0.28 2.09 2.72 2.35 25.99 9.28 T (yes) M (yes) CDH15 0.62 6.28 3.2 3.26 6.96 7.81 T (yes) M (yes) RET 0.01 0.75 7.88 0.91 4.9 3.22 T (yes) M (yes) Note: edgeR delivers logFC or log fold change and logCPM or log counts per million. Thresholds at transcriptome (T = 2.24) and methylome (M = 4.25) levels were computed according to quantiles (50%). Table 4. Cell adhesion in view of cadherins in SKMEL-2. d logCPM or log counts per million. Thresholds at transcriptome (T = 2.24) and methylome (M = 4.25) levels were Note: edgeR delivers logFC or log fold change and logCPM or log counts per million. Thresholds at transcriptome (T = 2.24) and methylome (M = 4.25) levels were computed according to quantiles (50%). significantly DEGs that were hyper-methylated in tumor data, at promoter and/or gene body, the effects of the epigenetic treatment over the inactivated cadherins is to induce a re-activation of their expression levels. This occurs in a non-metastatic context (SKMEL-2) and not in the metastatic one (HS294T). Considering this difference, and by seeing the epigenetic treatment under the lens of cadherin and its loss of function (cell adhesion), it appears that a functional inhibition by melanoma occurs at an early disease development stage, which is where the epi- genetic treatment exerts a clear impact. The same is not observed when the disease has already progressed to a metastatic stage. Back to RET, this gene is part of central modules (red envelope) including PDGFB and PDGFRA, which indicate platelet-derived growth factor involved in integrin signaling, cell migration and focal adhesion (FAK). GRB7is also part of this module, and is known to interact with integrin signaling, with EGFR and EFNB2 (Ephrin B2, a kinase being crucial for migration and adhesion), to bind with FAK, and also to communicate with the RET signaling network (see http://pathcards.genecards.org/card/ret_signaling). EFNB2 is annotated with three lincR- NAs. Two WNT components are also observed (yellow module) with WNT3 and WNT10A, both implicated in oncogenesis, DNA damage and PI3K Akt signaling. A list of lincRNAs is associated with PTK6 (green module), involved in tumor growth. Finally, MAPK13 is involved in proliferation and differentiation among other processes. Of particular interest is the associa- tion with the PANDAR ncRNA, which is thought to regulate the response to DNA damage, and whose deregulation induced cancer progression. We could not find it differentially expressed among our ncRNA detections. p g Looking at the metastatic HS294T cell line network map (Fig 4), fewer modules appeared relatively to before. MAPK13 (yellow envelope) is annotated here too, together with the associ- ated PANDAR and the negative regulator DUSP5. Among the different aggregates that were formed, one involves KISS1, relevant for cytoskeletal reorganization downstream cell matrix adhesion, a gene known to suppress metastases in melanoma and in some breast cancers too, by inhibiting invasion. Another gene, ESAM, i.e. endothelial cell adhesion molecule, is inter- acting with CLDN6 or claudin 6 (blue module), with possible sharing of tight junction func- tion to enable cell-to-cell adhesion. Claudins are examples of junctional proteins, i.e. transmembrane proteins that function to promote cell-cell adhesion, and are involved in the metastatic phenotype as both cancer promoters and tumor suppressors [45]. These proteins are natural candidates serving as therapeutic targets in cancer at metastatic stages. Associated to CLDN6 there is also a mini-list of ncRNAs, potential regulators whose functions remain largely unknown. Note that the network interactors were cross-referenced between STRING db knowledge base and GeneCards, while ncRNAs were inserted by inspecting the associations with ncRNA targets at given genomic distances from ncRNA loci. It is definitely less clear Looking at the metastatic HS294T cell line network map (Fig 4), fewer modules appeared relatively to before. MAPK13 (yellow envelope) is annotated here too, together with the associ- ated PANDAR and the negative regulator DUSP5. Among the different aggregates that were formed, one involves KISS1, relevant for cytoskeletal reorganization downstream cell matrix adhesion, a gene known to suppress metastases in melanoma and in some breast cancers too, by inhibiting invasion. Another gene, ESAM, i.e. endothelial cell adhesion molecule, is inter- acting with CLDN6 or claudin 6 (blue module), with possible sharing of tight junction func- tion to enable cell-to-cell adhesion. Claudins are examples of junctional proteins, i.e. transmembrane proteins that function to promote cell-cell adhesion, and are involved in the metastatic phenotype as both cancer promoters and tumor suppressors [45]. Differential network maps The red names refer to ncRNAs, and lincRNAs depend on 1Mb distance between their locus and the associated target genes. https://doi org/10 1371/journal pone 0206686 g003 https://doi.org/10.1371/journal.pone.0206686.g003 through augmented proliferation, invasion, and/or metastasis, and whose mutations have been correlated with various cancers. Table 4 is next shown to indicate the cadherin values computed in the SKMEL-2 cell line. In the HS294T cell line the significance of logFC was not high and thresholds were never met at both measurements together. The observed post-treat- ment effect inducing over-expressions in both CDH3 and CDH15 is therefore neutralizing the loss of cell-cell dependent adhesion and influencing melanoma development and progression. through augmented proliferation, invasion, and/or metastasis, and whose mutations have been correlated with various cancers. Table 4 is next shown to indicate the cadherin values computed in the SKMEL-2 cell line. In the HS294T cell line the significance of logFC was not high and thresholds were never met at both measurements together. The observed post-treat- ment effect inducing over-expressions in both CDH3 and CDH15 is therefore neutralizing the loss of cell-cell dependent adhesion and influencing melanoma development and progression. Interestingly, note that also RET (red central envelope) is marked with reference to cad- herin, and indeed it is considered an atypical cadherin belonging to a group of cadherins endowed with a diversity of unique structures and functions but still playing a role in cell adhe- sion and beyond [44]. A relatively long list of lincRNAs is associated with RET. Considering Table 4 and the interaction network maps, it appears that the role of cadherin genes is more substantial in SKMEL-2 than in HS294T. Because the maps satisfy two constraints, namely 13 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 https://doi.org/10.1371/journal.pone.0206686.t004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 These proteins are natural candidates serving as therapeutic targets in cancer at metastatic stages. Associated to CLDN6 there is also a mini-list of ncRNAs, potential regulators whose functions remain largely unknown. Note that the network interactors were cross-referenced between STRING db knowledge base and GeneCards, while ncRNAs were inserted by inspecting the associations with ncRNA targets at given genomic distances from ncRNA loci. It is definitely less clear 14 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Network assessment of demethylation treatment effects in melanoma Fig 4. HS294T cell line map. Protein-protein Interaction map from STRINGdb obtained from DiP (DEGs and differentially methylated). The effects of treatment were analyzed in hyper-methylated DEGs to consider silencing effects on expression before treatment. The red names refer to ncRNAs, and lincRNAs depend on 1Mb distance between their locus and the associated target genes. expression before treatment. Fig 4. HS294T cell line map. Protein-protein Interaction map from STRINGdb obtained from DiP (DEGs and differentially methylated). The effects of treatment were analyzed in hyper-methylated DEGs to consider silencing effects on expression before treatment. The red names refer to ncRNAs, and lincRNAs depend on 1Mb distance between their locus and the associated target genes. expression before treatment. https://doi.org/10.1371/journal.pone.0206686.g004 https://doi.org/10.1371/journal.pone.0206686.g004 and surely less pervasive the effects induced by the demethylant with HS294T, which suggests a limited impact of the treatment in the reprogramming action against the metastatic process. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Network assessment of demethylation treatment effects in melanoma Table 5. HS294 antigens. Table 5. HS294 antigens. Gene name Protein ID Length Antigenic peptide name Antigenic peptide seq Loc LogFC Methylation level promoter Methylation level gene body BIRC3 XP_016873132.1 604 T000939 RLQEERTCKV 550 1.808209 BCL2 XP_011524437.1 224 T000947 NIALWMTEYL 172 -2.10161 1.083 9.978 DCT XP_011519351.1 456 T000114 LLGPGRPYR 134 -2.79781 EPHA3 XP_005264772.1 982 T000393 DVTFNIICKKCG 356 -2.39375 FLT1 XP_016875974.1 1300 T000789 GVLLWEIFSL 1048 -1.46441 7.443 1.712 GFl1 XP_005270806.1 422 T001014 QPR(S)PGPDYSL [QPRSPGPDYSL] 17 3.3185 18.065 12.974 LCK XP_011539755.1 516 T000743 DYLRSVLEDF 495 7.500057 MAGEB2 XP_011543814.1 319 T000252 FLWGPRAYA 273 1.8108824 MDM2 XP_005268929.1 491 T000674 YTMKEVLFYL 48 1.815603 0.397 17.749 NFATC2 XP_011527126.1 940 T000980 KPY(S)PLASL[KPYSPLASL] 70 -1.81647 18.28 22.764 CLCA2 XP_011540750.1 616 T000385 LLGNCLPTV 425 2.206476 MSLN XP_005255091.1 621 T000796 FLLFSLGWV 23 1.457531 3.75 4.516 TRPM8 XP_011510112.1 1115 T000883 GLMKYIGEV 187 -1.61673 ARHGAP30 XP_005245127.1 1044 T001046 RPAK(S)MDSL [RPAKSMDSL] 323 4.162052 MAGEA2B XP_016884895.1 314 T000162 TTINYTLWR 73 -8.76344 MAGEA9B XP_005278249.1 315 T000196 VALELVHFLL 112 2.124184 N l i i d h l h h ld Note: values passing transcriptome and methylome thresholds. https://doi.org/10.1371/journal.pone.0206686.t005 logFC<-1.5) and methylome (2.9 at promoter level, 8.39 at gene body level, as a result of 50% quintiles) values. The top unconstrained map shows the presence of various singletons, such as FMNL1 (formin-like protein 1) involved in morphogenesis, cytokinesis and cell polarity, plus logFC<-1.5) and methylome (2.9 at promoter level, 8.39 at gene body level, as a result of 50% quintiles) values. The top unconstrained map shows the presence of various singletons, such as FMNL1 (formin-like protein 1) involved in morphogenesis, cytokinesis and cell polarity, plus logFC<-1.5) and methylome (2.9 at promoter level, 8.39 at gene body level, as a result of 50% quintiles) values. The top unconstrained map shows the presence of various singletons, such as FMNL1 (formin-like protein 1) involved in morphogenesis, cytokinesis and cell polarity, plus Table 6. SKMEL-2 antigens. Systems analysis of T-Antigens Table 5 and Table 6 refer to the list of antigens searched via the TANTIGEN db (Tumor T-cell Antigen Database) [46]. This is a resource on human tumor antigens based on HLA ligands, predicted binding peptides and T cell epitopes, and including reference to gene expression, isoforms, mutations. Two types of results were obtained from our profiles cross-referencing, namely the ‘best match’ and the ‘one-mismatch’ (tabulated evidences are reported in S8 Tables). Looking at the results, a few considerations follow. First, the networks establish co- expressions between nodes and/or interactions. Fig 5 for HS294T shows two panels: at the top level, one with all the gene selections with best-matched antigens; at the bottom level, one with the gene list restricted by the application of thresholds in the transcriptome (logFC>1.5, PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 15 / 26 Note: values passing transcriptome and methylome thresholds. Note: values passing transcriptome and methylome thresholds. https://doi.org/10.1371/journal.pone.0206686.t006 Note: values passing transcriptome and methylome thresholds. https://doi.org/10.1371/journal.pone.0206686.t006 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 16 / 2 Network assessment of demethylation treatment effects in melanoma members of the MAGE family of antigens (especially tumor specific proteins belonging to CTA group), whose exact functions is not completely known but appear to regulate cell cycle progression and apoptosis. Of interest are the connectivity paths, and these are visible in the unconstrained map, less so in the constrained maps. In the former context, the following chain of nodes is noted: TTK, a mitotic kinase associated with cell proliferation through mitosis because critical for the regu- lation of cell division; TOP2A, a gene known as target for anticancer agents whose mutations are linked to drug resistance, and which encodes an enzyme altering DNA during transcrip- tion and replication; BCL2, encoding an anti-apoptotic protein (affecting lymphocytes, for instance), and the associated BCL2L1, also inhibiting cell death, and BIRC3, an inhibitor of apoptosis; MSLN, or megakaryocyte potentiating factor, involved in mesothelin generation, which is a sort of cell adhesion protein appearing overexpressed in some cancers; MDM2, which can promote tumor formation by targeting tumor suppressor proteins such as p53 and whose overexpression has been seen in various cancers; FLT1, or Fms-related Tyrosine Kinase 1, encoding a member of VEGFR of relevance to angiogenesis and vasculogenesis; LCK, of the Src family of protein tyrosin kinases and key signaling molecule involved with T-cells by bind- ing to various cell surface receptors, also playing a key role in the T-cell antigen receptor- linked signaling transduction patwhays; EPHA3, of the ephrin receptor (part of the protein tyrosine kinases), involved in cell-cell adhesion, cytoskeletal organization and cell migration processes. When the same analysis is performed over the SKMEL-2 map in Fig 6, much smaller con- nectivity is observed and the presence of singletons is dominant under constraints. The appli- cation of such constraints dissolves the connectivity observed with the entire list of genes for which the antigens were matched, and in which two paths had appeared: a) OCA2, a trans- membrane protein involved in the transport of tyrosine, linked to TYR (tyrosinase) and GPR143 (tyrosine binding); b) BCL2, LCK, BCL2L1 and BIRC3. MAGE and XAGE (member of GAGE family, useful markers in melanoma and associated with poor prognosis) genes are present too. Biological validation Features of programmed cell death consist of a very particular nuclear behavior involving DNA cleavage, destruction of the structural chromatin organization and proteolysis of nuclear membrane and nuclear lamina components [47]. Poly(ADP-ribose) polymerase 1 (PARP1) and Lamin A/C are cleaved in apoptotic nuclei by proteolitic enzymes, called caspases, at the late stage of apoptotic process [48, 49]. Therefore, we used these markers and tested their apo- ptotic cleavage by western blot analysis. Both HS294T and SKMEL-2 melanoma cells under- went apoptosis after 72 hours of treatment with 5-Aza-2’-Deoxycytidine (DAC) as indicated by cleavage of PARP1 and lamin A/C (S1 Fig). The densometric analysis indicates that PARP1 cleavage (85 kDa) was detectable in both DAC-treated cell types while significantly increasing in DAC-treated SKMEL-2, and cleaved Lamin A (45 kDa) significantly increased in both DAC- treated cell lines. Gene name Protein ID Length Antigenic peptide name Antigenic peptide seq Loc LogFC Methylation level promoter Methylation level gene body PLIN2 XP_016869748.1 445 T000366 SVASTITGV 129 5.632593 13.151 25.123 BIRC3 XP_016873132.1 604 T000939 RLQEERTCKV 550 2.404825 FMNL1 XP_006722125.1 1164 T000850 RLPERMTTL 799 2.028542 7.675 8.02 GFI1 XP_005270806.1 422 T001014 QPR(S)PGPDYSL [QPRSPGPDYSL] 17 3.026243 11.808 11.394 LCK XP_011539755.1 516 T000743 DYLRSVLEDF 495 7.499478 OCA2 XP_011519942.1 852 T000618 IMLCLIAAV 427 -2.63585 2.826 13.916 PTHLH XP_011519076.1 177 T000876 FLHHLIAEI 59 1.809974 7.344 1.106 ABCC3 XP_005257820.1 1463 T000825 LYAWEOSFL 439 4.109376 0.99 5.013 IGF2BP1 XP_005257012.2 565 T000906 KTVNELQNL 496 2.309944 27.031 3.641 SYNPO XP_005268427.1 903 T000979 RPSRS(S)PGL[RPSRSSPGL] 615 1.504201 RAB38 XP_016872944.1 177 T000663 VLHWDPETV 50 -1.22612 0.428 0.892 ARHGAP30 XP_005245127.1 1044 T001046 RPAK(S)MDSL[RPAKSMDSL] 323 1.867114 MAGEA2B XP_016884895.1 314 T000162 TTINYTLWR 73 8.94263 CCDC88B XP_006718582.1 1560 T001017 SPEKAGRR(S)SL [SPEKAGRRSSL] 588 2.423258 XAGE1B XP_016885238.1 146 T000840 CATWKVICKSCISQTPG 98 1.782175 MAGEA9B XP_005278249.1 315 T000196 VALELVHFLL 112 3.747172 Table 6. SKMEL-2 antigens. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 16 / 26 Discussion Gene expression profiles from melanoma cell lines may display different transcriptome states, such as of proliferative and invasive ones, which tend to switch when temporally observed and the initially observed proliferative-to-invasive state transition seems induced by chromatin- dependent transcriptional changes, among other regulatory network dynamics [25]. These states prefigure two transcriptional signatures beyond the influence of genetic mutations, as PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 17 / 26 Network assessment of demethylation treatment effects in melanoma these alone are not sufficient to explain the reprogramming and also reversible dynamics, and most likely influenced by the microenvironment [50]. Mutations involved in the growth of tumor are rare but may generate aberrations (i.e. amino acid substitutions) in protein sequences, making thus the latter potential targets in view of tumor-specific immune response Fig 5. HS294T Antigen-driven map. Protein-protein interaction map from STRING db obtained from all best matched antigens (top panel)—see Table 5 and S8 Tables. https://doi.org/10.1371/journal.pone.0206686.g005 Network assessment of demethylation treatment effects in melanoma Fig 5. HS294T Antigen-driven map. Protein-protein interaction map from STRING db obtained from all best matched antigens (top panel)—see Table 5 and S8 Tables Fig 5. HS294T Antigen-driven map. Protein-protein interaction map from STRING db obtained from all best matched antigens (top panel)—see Table 5 and S8 Tables. Fig 5. HS294T Antigen-driven map. Protein-protein interaction map from STRING db obtained from all best matched antigens (top panel)—see Table 5 and S8 T bl Protein-protein interaction map from STRING db obtained from all best matched antigens (top panel)—see Table 5 and S8 https://doi.org/10.1371/journal.pone.0206686.g005 https://doi.org/10.1371/journal.pone.0206686.g005 these alone are not sufficient to explain the reprogramming and also reversible dynamics, and most likely influenced by the microenvironment [50]. Mutations involved in the growth of tumor are rare but may generate aberrations (i.e. amino acid substitutions) in protein sequences, making thus the latter potential targets in view of tumor-specific immune response [51]. 18 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Network assessment of demethylation treatment effects in melanoma Our melanoma data were obtained from two cell lines, only one being metastatic. Coupled profiling was performed, and methylomes are expected to reflect characteristics that are unique to each cancer type, thus justifying differences in patterns or signatures. We Fig 6. SKMEL-2 Antigen-driven map. Protein-protein interaction map from STRING db obtained from all best matched antigens (top panel)—see Table 6 and S8 Tables. Discussion In our study we noticed negatively cor- related behavior between transcriptome and methylome levels at promoters, but could not observe this pattern at gene-body levels. We supported these evidences with a model employ- ing simple statistical regressions and testing the significance for the coefficients in relation with the two profiles at both promoter and gene body level. When considering profile coverage of coding and non-coding genomic regions, correlations were assessed only at the coding regions, which is where methylome measures were obtained by reduced sequencing. Aberrant DNA methylation affects many cancer genes, suggesting a potential use as bio- markers for early diagnosis, prognosis and also prediction of therapy response [55]. Due to the reversibility of such aberrations, there is potential for therapeutic targeting combining DNA demethylation with candidate target selection. Since hyper-methylated genes can be reacti- vated after treatment with methylation inhibitors, mapping these genes onto networks may elucidate possible but hard to measure correlation with differential expression. The advantage of using networks is that connected paths identified among DEGs represent a robust measure of association at a biological level. Functional epigenetic modules have been recently indicated as good instruments for integrated use with scaffold networks, especially but not only with protein networks. They are aimed at identifying hotspots, i.e. significant epigenetic dysregula- tion associated to key phenotypes. Examples of integrative tools are provided by FEM [56], BioNet [57] and SMITE [58]. Other tools have been provided without explicit use of correlated profiles (see EMDN [12]). In the context of our cell lines, a feature shared by the two cell lines is MAP-kinase signaling associated with regulation of cell proliferation, something already noticed with different can- cers and analyzed through network oncomarkers [12]. Several studies have pointed out the re- activation of the MAPK pathway as an effector of mechanisms of acquired resistance, and holding especially in view of MITF or SOX10 activity [59]. In our study, the modular protein interaction networks evidenced MAPK13, a target associated with the PANDAR ncRNA of relevance to therapy because aberrantly expressed across various cancers. However, since it is the diversity from metastatic levels that we are interest in, we observed the effects of demethyl- ation under such cell-specific metastatic potential. Under non-metastatic conditions various re-activated pathways appear deeply involved in cancer hallmarks. Discussion https://doi.org/10.1371/journal.pone.0206686.g006 Network assessment of demethylation treatment effects in melanoma Fig 6. SKMEL-2 Antigen-driven map. Protein-protein interaction map from STRING db obtained from all best matched antigens (top panel)—see Table 6 and S8 Tables. Fig 6. SKMEL-2 Antigen-driven map. Protein-protein interaction map from STRING db obtained from all best matched a T bl Fig 6. SKMEL-2 Antigen-driven map. Protein-protein interaction map from STRING db obtained from all best matched antigens (top panel)—see Table 6 and S8 Tables Protein-protein interaction map from STRING db obtained from all best matched antigens (top panel)—see Table 6 and S8 Antigen-driven map. Protein-protein interaction map from STRING db obtained from all best matched antigens (top panel)— https://doi.org/10.1371/journal.pone.0206686.g006 Our melanoma data were obtained from two cell lines, only one being metastatic. Coupled profiling was performed, and methylomes are expected to reflect characteristics that are unique to each cancer type, thus justifying differences in patterns or signatures. We PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 19 / 26 Network assessment of demethylation treatment effects in melanoma hypothesize that by these signatures we might better decipher complex regulation mechanisms underlying the observed data. Transcriptomes and methylomes have been combined in many studies, but there is still not a clear understanding of how this merge can improve early diagno- sis, prognosis and prediction of therapeutic response. The advantage of investigating DNA hyper-methylation versus standard tumor biomarkers is that the former can be highly relevant in predictive terms, such as monitoring the stages of cancer progression, and including early and premalignant conditions. The best example provided by our study concerns cadherins, in particular CDH3 and CDH15, for which a clear mark of post-treatment appears through the over-expressions of both genes. Treatment therefore contrasts the loss of cell-cell dependent adhesion underpinning melanoma development and progression, and indicating the opportu- nity for targeted clinical intervention similarly to what has been observed in the so-called ‘actin-diseases’, i.e. those with a disruption of the E-cadherin and actin connection [52]. Gene expression is known to be affected by DNA methylation. In particular, a repressive epigenetic mark is usually investigated at both promoter and gene-body located sequences. In general, methylated promoters are negatively correlated with gene expression because associ- ated with gene silencing, while non-methylated promoters may associate independently on transcription states [53,54]. In fact, it is still to be clearly assessed whether gene body methyla- tion levels are more or less predictive than TSS regions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Network assessment of demethylation treatment effects in melanoma of ncRNAs detected as differentially expressed and contextualized in network maps with their putative targets. Both the map configuration and the presence of ncRNAs change substantially under metastatic conditions, as the more advanced stage of melanoma progression implies a different degree of involvement of pathways. Interestingly, this emerging differentiality indi- cates a demethylation impact stronger at the early disease stage, most likely the best time for therapeutic intervention. The other instruments explored in this study are the differential antigen maps, and here are a few remarks. Tumor antigens refer to tumor molecules interactive with the immune system, being some specific (not present in normal tissue) and some associated (overexpressed in tumor compared to normal) [46]. These types can be recognized by T cells and present on the cell surface by human by so-called HLA or human leukocyte antigen molecules. T cells can reject tumor due to such molecules thus eliciting immune response. Our evidences point to a few directions, all deserving a few final remarks that we summa- rize in five points. First, when antigen maps are created to capture differential configurations in the two cell line scenarios, a superior connectivity appears in the HS294-T cell line vs the SKMEL-2 cell line. While some commonalities persist when considering the unconstrained gene lists, the established thresholds enable constraints that lead only for the HS294-T map to trackable and interpretable paths. These differential network signatures seem to reflect the metastatic potential of the cell lines. Therefore, by exploiting the identified antigens the con- nected target genes suggest that targets are better actionable in the presence of an increased metastatic potential. Second, the expression thresholds that we adopted exert substantial effects, and expectedly constrain the systems under study by reducing the potential of exploitable target connectivity observed under unconstrained scenarios. This effect shows up by complete link depletion in the non-metastatic case. The thresholds are surely affected by the re-activation of expression levels induced by the demethylation treatment. However, an increased number of re-activated genes doesn’t necessarily means that more melanoma targets become available. Indeed these targets appear as associated to the metastatic power. In other terms, while the protein-protein interaction network maps revealed increased treatment effects in the context of non-metastatic cell line, the antigen network maps showed more actionable targets with the metastatic cell line. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Discussion Due to hyper-methylation induced by tumor, these pathways were altered, and the epigenetic treatment is reconfiguring them in an intricate map of network relationships. Such complex regulations include the roles PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 20 / 26 Supporting information Supporting information S1 Tables. DEG profiles and ncRNA biotypes. (XLSX) S2 Tables. Biotype classification. (XLSX) S3 Tables. Biotype annotation. (XLSX) S4 Tables. Cross-profiles (links between differential expression and differential methyla- tion values). (XLSX) S5 Tables. Measurements of methylation levels. (XLSX) S6 Tables. DiP bio-annotations SKMEL-2. (XLSX) S7 Tables. DiP bio-annotations HS294T. (XLSX) S8 Tables. TANTIGEN results. (XLSX) S1 Text. Implementation of gls function in R. (DOCX) S2 Text. lncRNA (lincRNome) cross-referencing. (PDF) S3 Text. Chromatin (DAnCER) cross-referencing. (PDF) S1 Fig. Biological validation. (RTF) Acknowledgments The authors want to thank all colleagues involved in the project. Author Contributions Conceptualization: Caterina Cinti, Enrico Capobianco. Data curation: Zhijie Jiang, Sakshi Singh, Rimpi Khurana. Formal analysis: Zhijie Jiang, Caterina Cinti, Monia Taranta, Nicholas F. Tsinoremas, Enric Capobianco. S1 Tables. DEG profiles and ncRNA biotypes. (XLSX) S1 Tables. DEG profiles and ncRNA biotypes. (XLSX) S2 Tables. Biotype classification. (XLSX) S3 Tables. Biotype annotation. (XLSX) S5 Tables. Measurements of methylation levels. (XLSX) S6 Tables. DiP bio-annotations SKMEL-2. (XLSX) S7 Tables. DiP bio-annotations HS294T. (XLSX) S8 Tables. TANTIGEN results. (XLSX) S1 Text. Implementation of gls function in R. (DOCX) S2 Text. lncRNA (lincRNome) cross-referencing. (PDF) S3 Text. Chromatin (DAnCER) cross-referencing. (PDF) Target actionability in a network context adheres strictly to the property of network con- nectivity, and the topological measures such as degree and centrality that can be derived. Intui- tively, the presence of connected target paths translates into increased chances to be able to use if not directly these targets, at least their close interactors. Third, any current evidence must be seen in light of technological limitations, one being that the database resources here used are specific. In general, a systematic and/or comparative evaluation of putative antigens as targets of antitumor immunity is not yet available [60]. Con- versely, the impact of cancer immunotherapy is constantly growing, particularly in light of the fact that the mutational load is a limited marker by itself. Whether T cell activity is the ultimate effector mechanism is something deserving further study, in association with other aspects making more complete the present cancer-immune interactome, and thus refining the “cancer immunogram” towards personalized treatments [61]. Fourth, while significant methylation appears at both promoter and gene body levels, it is quite hard to measure the influence of these values over the potential of connected targets at varying metastatic stages in view of tumor T-cell antigens. It is a topic that deserves further investigation, and here probably suffers from the incompleteness of the available data for which a clear coupled-profiles correlation is observed only at promoter level. Nonetheless, the complex regulations in the presence of differential metastatic potential have pinpointed inter- esting identifications that may guide the choice of candidate genes for further validation stages. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 21 / 26 Network assessment of demethylation treatment effects in melanoma Fifth and last point is a general limitation in our study. The observed patterns may provide insights into biological processes driving metastasis transition, but the limitations imposed by the scale of the experiments must be considered too. Therefore, further confirmation of the findings relative to the observed phenotypes require additional verification that can ultimately be achieved through scaled up validations and applications of biological models. Acknowledgments The authors want to thank all colleagues involved in the project. References 1. Knijnenburg TA, Ramsey SA, Berman BP, Kennedy KA, Smit AF, Wessels LF, et al. Multiscale repre- sentation of genomic signals. Nat Methods. 2014; 11(6):689–94. https://doi.org/10.1038/nmeth.2924 PMID: 24727652 2. Nguyen M, Dobosz P. 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Author Contributions Conceptualization: Caterina Cinti, Enrico Capobianco. Conceptualization: Caterina Cinti, Enrico Capobianco. Conceptualization: Caterina Cinti, Enrico Capobianco. Data curation: Zhijie Jiang, Sakshi Singh, Rimpi Khurana. Formal analysis: Zhijie Jiang, Caterina Cinti, Monia Taranta, Nicholas F. Tsinoremas, Enrico Capobianco. 22 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206686 November 28, 2018 Network assessment of demethylation treatment effects in melanoma Funding acquisition: Caterina Cinti, Enrico Capobianco. Investigation: Zhijie Jiang, Sakshi Singh, Enrico Capobianco. Methodology: Zhijie Jiang, Enrico Capobianco. Resources: Nicholas F. Tsinoremas, Enrico Capobianco. Supervision: Caterina Cinti, Enrico Capobianco. Validation: Caterina Cinti, Monia Taranta, Elisabetta Mattioli, Elisa Schena, Giovanna Lattanzi. Visualization: Rimpi Khurana. Funding acquisition: Caterina Cinti, Enrico Capobianco. 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https://openalex.org/W3198817659	https://www.revistas.usp.br/rlae/article/download/191885/176832	English	null	Content analysis of the Global Alliance for Nursing and Midwifery discussion forum: an online community of practice	Revista latino-americana de enfermagem	2,021	cc-by	4,828	Rev. Latino-Am. Enfermagem 2021;29:e3431 DOI: 10.1590/1518-8345.4878.3431 www.eerp.usp.br/rlae Original Article * This article refers to the call “Human Resources in Health and Nursing: Training and Practice in the Americas”. 1 Johns Hopkins University, School of Nursing, Baltimore, MD, United States of America. Content analysis of the Global Alliance for Nursing and Midwifery discussion forum: an online community of practice* Objective: to examine the usage and content of the Global Alliance for Nursing and Midwifery (GANM) discussion forum in relation to nursing and midwifery education and practice. Method: a qualitative conventional content analysis was performed. Subject lines from 1689 discussion board threads were extracted and used as the unit of analysis. A-priori codes were developed based on topical relevance (e.g. maternal health) and typical discussion board usage (e.g. announcing educational opportunities). Emerging codes were further identified while coding the data (e.g. infectious diseases). Results: the GANM discussion forum was used most frequently for information exchange (43.8%), such as dissemination of new information on evidence-based practice, and to announce educational opportunities (24.8%). The most frequently discussed topics were nursing (14.2%; e.g. the role of nurses in primary care, nursing education, etc.) and maternal health (13.8%; e.g. postpartum care, maternal mortality, etc.). Infectious diseases were discussed in 9% of threads, 40% of which concerned the current coronavirus pandemic. Conclusion: findings reinforce the utility of the GANM as a platform for professional development and continuing education. As a platform for disseminating empirical research, the GANM can be leveraged to have an influence on real-world, evidence- based practice. Hillary Chu1 https://orcid.org/0000-0001-8933-8734 Ashley Gresh1 https://orcid.org/0000-0002-7181-8219 Valentina Bolanos1 https://orcid.org/0000-0002-2899-4583 Nancy Reynolds1 https://orcid.org/0000-0002-5023-1953 Conclusion: findings reinforce the utility of the GANM as a platform for professional development and continuing education. Descriptors: Collaboration; Content Analysis; Continuing Education; Knowledge Exchange; Professional Development; Online Community of Practice. and Nursing: Training and Practice in the Americas”. 1 Johns Hopkins University, School of Nursing, Baltimore, MD, United States of America. 1 Johns Hopkins University, School of Nursing, Baltimore, MD, United States of America. Method A qualitative content analysis was performed. This method was selected because it is commonly used in nursing studies and is a practical method for analyzing communication messages(7). A qualitative approach was used to explore content areas discussed on the GANM discussion board. The content analysis is an appropriate methodology because it provides a “systematic and objective means of describing and quantifying phenomena”(8). Using this approach, a priori codes were developed by the research team covering a range of maternal and child health topics and typical discussion board posts as identified by GANM moderators. New codes were added as new themes emerged. These codes were then grouped into categories derived from the data. One research team member coded all of the data and regular meetings were held with the research team to establish intercoder reliability. These strategies for maximizing education in nursing and midwifery are essential for building competency and preparedness, especially in times of outbreaks such as the COVID-19 pandemic. According to Brand(2), nurses are key players in a country’s response to a national or international crisis. This means that they are not only providing care at the individual or population level, they are also improving health systems(2). Thus, a commitment to continuous education is necessary to maximize the response of nurses in the face of a pandemic. To achieve such goals, an educational approach for a new infectious disease must address clinical knowledge, buildup of competencies, and accessibility to high quality, evidence- based material(2-3). Online communities of practice can serve as an essential tool to provide access to up to date resources to respond to healthcare needs for nurses and midwives globally(4-5). Discussion board posts from the GANM Knowledge Gateway were first extracted for analysis from its inception in 2006 to October 2019. However, in early 2020, with the onset of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, members began sharing new information about the virus and the disease it causes, COVID-19. At the time, scientific knowledge about the virus and disease was still emerging, and concern about the spread of misinformation was growing. Because we were interested in the utility of the GANM forum in sharing evidence-based information, a second round of data extraction was performed in early May 2020. How to cite this article Chu H, Gresh A, Bolanos V, Reynolds N. Content analysis of the Global Alliance for Nursing and Midwifery discussion forum: an online community of practice. Rev. Latino-Am. Enfermagem. 2021;29:e3431. [Access day month year ]; Available in: URL . DOI: http://dx.doi.org/10.1590/1518-8345.4878.3431 2 Rev. Latino-Am. Enfermagem 2021;29:e3431. Introduction The objective of this study was to examine the usage and content of the Global Alliance for Nursing and Midwifery (GANM) discussion forum in relation to nursing and midwifery education and practice. Given that 2020 is the year of the nurse and midwife, it is worth highlighting the importance of having a competent and educated workforce across different system levels and settings(1). This entails providing the most up to date information pertaining to evidence-based practice and opportunities for continuous professional growth as means to build upon the competence of nurses and midwives globally(1). Educational information exchange requires collaboration at the intra and interprofessional level to foster professional development and collaboration(1). www.eerp.usp.br/rlae Method In 2006, the Johns Hopkins School of Nursing PAHO/ WHO Collaborating Center established the Global Alliance for Nursing and Midwifery (GANM), an online community of practice that provides an innovative forum for knowledge exchange. Currently the GANM has over 4,000 members from 163 countries comprised of practitioners, researchers, and others. The GANM’s mission is to provide a platform for knowledge exchange globally, using low band-width technology. As of May 2020, the GANM consisted of 4,036 members in 162 countries. A survey of GANM members conducted in August and September 2019 indicated that most respondents were licensed or registered nurses (36%), midwives (33%), or licensed advanced practice nurses [15% Certified Nurse-Midwife (CNM), 6% Nurse Practitioner (NP), 5% Doctorate of Nursing Practice (DNP), 2% Clinical Nurse Specialist (CNS), and 1% nurse anesthetist]. Twelve percent were public health specialists, including Master’s in Public Health and PhD. Sixty-two percent of respondents identified English as their primary language, 29% identified Spanish, and 24% identified another language, with Portuguese, French, Nepali, Vietnamese and Arabic being the most common other languages spoken(9). The GANM Knowledge Gateway supports a discussion board and an online library to provide a mechanism for knowledge dissemination globally. The discussion board serves as an online forum for sharing information and ideas, particularly related to nursing and midwifery. What makes GANM’s discussion forum unique is that it is moderated by graduate students at Johns Hopkins School of Nursing. Moderators perform supportive tasks such as providing help to members, as well as administrative tasks such as deleting spam(6). Of relevance to the current study, all messages posted to the GANM discussion board must first be approved by a moderator. Nearly all messages were approved, with the exception of posts that were selling a product or contained inappropriate content. All threads from the inception of the discussion board in August 2006 until May 2020 were included. A www.eerp.usp.br/rlae 3 3 Chu H, Gresh A, Bolanos V, Reynolds N. new thread (or topic) begins when a member shares information or asks a question by posting a message with a subject line on the discussion board. Other members participate in the discussion by responding to the original post. Often, there are multiple conversations about similar topics, but each conversation has its own thread. Additionally, the same topic may have more than one thread. Method For example, a member may wish to share information about an upcoming conference by posting a message on the discussion board. They may send reminders to the community about the conference in the form of a new message, which would start a new, separate thread. for the differentiation between conversations initiated by moderators and discussion board usage driven by members at large. Messages were also cross-coded to identify the language in which they were posted. Emerging codes were further identified while coding the data. For example, many posts discussed infectious diseases, including HIV, Ebola, and COVID-19 (Topics that comprised less than 3% of the total discussion threads were coded under “other health topics.”). Each subject line could have more than one code; for example, a subject line announcing a GANM blog post about nursing during COVID-19 written in Spanish was coded under “GANM,” “nursing,” “infectious diseases,” and “Spanish.” The text of the discussion board was imported into an Excel file, where the subject lines from each thread were extracted and used as the unit of analysis. The text of the thread, names of GANM members who participated in the discussion, and dates on which messages were posted were omitted, so any personal identifying information was removed for the analysis. The subject lines only were then copied into a separate Excel file, which was then imported into the qualitative data analysis software F4 Analyze. F4 Analyze was used to facilitate data management and analysis. It was assumed that each subject line reflected the content of its corresponding thread, and was written in the same language. Additionally, subject lines that discussed health in a specific country or geographical region were coded under the corresponding WHO region: African Region (AFRO), Region for the Americas (AMRO), Eastern Mediterranean Region (EMRO), European Region (EURO), South-East Asia Region (SEARO), and Western Pacific Region (WPRO). One research team member coded all of the data and conferred with the team where there was ambiguity to establish intercoder reliability. For example, threads about engaging men and boys in family planning were not initially coded under “maternal health” because they address male participation. However, after conferral, the authors decided that because these threads centered around family planning, “maternal health” was an appropriate code. Additionally, threads with 20 or more posts were reviewed to identify topics that generated the most participation. Method When GANM members participate in a conversation thread, each response is recorded as a post within the thread. This analysis was done because in the method described above, each thread is counted exactly once. By examining threads with greater than 20 posts we were able to identify specific topics of interest, creating a fuller picture of what GANM members choose to discuss and engage with on the discussion board. The study was exempted from IRB review; discussion posts were de-identified and analyzed anonymously. www.eerp.usp.br/rlae Results Between August 2006 and May 2020, the GANM discussion forum had 1689 unique discussion board threads with approximately 20 new posts each month. Results are presented based on usage (e.g. how members used the discussion forum) and content (e.g. what was discussed) in order to provide a comprehensive exploration of the discussion board. A priori codes were developed based on maternal and child health content areas and typical discussion board usage. For example, because the GANM is an online community of practice for nurses and midwives, maternal health was often discussed. Typical discussion board usage consists of such activities as announcing upcoming events, including educational opportunities (i.e., conferences, online courses, webinars, etc.); posting job vacancies; and sharing information in the form of practice guidelines, press releases, and newsletters. Threads related to GANM activities (i.e., new blog posts, additions to the online community library, and GANM surveys) were written by a moderator and coded as “GANM.” It should be noted that GANM threads represent instances of information exchange categorized under their own code because they were generated by the moderators. Coding moderator-generated threads separately allowed Usage GANM members primarily use the discussion board to share opportunities for professional development, which was the purpose of 85.5% of all threads. Professional development was subdivided into five categories: information exchange, continuing education, GANM posts, job announcements, and other. As shown in Table 1, the most frequent use of the discussion board was for information exchange (43.8% of all threads), such as dissemination of new information on evidence- based practice and sharing press releases, policy briefs, and newsletters. The second most frequent use was 4 Rev. Latino-Am. Enfermagem 2021;29:e3431. Rev. Latino-Am. Enfermagem 2021;29:e3431. for announcing opportunities for continuing education (24.8%), including conferences, online courses, and webinars, which accounted for nearly half (46.5%) of all educational opportunities. GANM threads, which represent instances of moderator-generated information exchange, comprised 12.3% of all posts. These threads primarily disseminated new ideas and evidence-based information in the forms of expert blog posts and article summaries of empirical research published in open access peer-reviewed journals, respectively. Infectious diseases were discussed in 9% of threads. Guidance around disease outbreaks were frequently shared, including guidelines for the Ebola outbreaks that began in 2013 and 2018, in West Africa and the Democratic Republic of Congo, respectively; the Zika outbreak that began in South America in 2015; and the current COVID-19 pandemic that began in 2019 in China. COVID-19 was specified in 40% of all threads discussing infectious diseases. Midwifery comprised 10% of all threads, and included midwifery education, midwifery competencies from the International Council of Midwives, and information about the Virtual International Day of the Midwife. Threads about midwifery also tended to receive many messages, indicating that there was interest among members in discussing this topic. Table 1 - Usage of the GANM discussion board Professional Development N % Information exchange 740 43.8% Continuing education 419 24.8% GANM* (moderator-generated) 208 12.3% Job announcements 77 4.5% Other 58 3.4% GANM = Global Alliance for Nursing and Midwifery Table 1 - Usage of the GANM discussion board Professional Development N % Information exchange 740 43.8% Continuing education 419 24.8% GANM (moderator-generated) 208 12.3% Job announcements 77 4.5% Other 58 3.4% GANM = Global Alliance for Nursing and Midwifery The threads that generated the most discussion among members (greater than 20 posts) were entitled, “Doulas” (53 messages) and “Looking for collaborators” (52 messages), followed by, “Traditional Midwives (Untrained)” (50 messages) and “Direct entry midwifery programme” (44 messages), as show in Table 3. Usage A thread entitled, “2020 Year of the Nurse, celebrating nursing and midwifery!” generated the fifth most number of responses (43 total messages), many of which advocated for the profession of midwifery to be recognized separately from nursing. For example, one contributor to the thread wrote:“Fantastic we are celebrating nursing and midwifery but...midwifery should not be under the umbrella name of nursing. Globally midwifery should be recognised as a separate profession”. In contrast, another member shared a different perspective, writing: “I think historically the two professions have so much commonalities, deeply intertwined and complements each other. My inclinations is that we should focus on celebrating and rejoicing 2020 as the year of the nurses and midwives together given their historical linkage in many parts of the world, especially in developing countries!”. GANM = Global Alliance for Nursing and Midwifery Content 27 Institutional Violence against women in childbirth globally 26 HOPE Nurses Conference \| HIV in Women - Announcements 25 New York Times Article: American Way of Birth Costliest in the World 24 Human Resources for Health and Nursing 23 “Essential medicines out of reach for most people” 21 International Day of the Midwife ideas 21 Invitation to participate in 3rd Trimester Screening Ultrasound project to prevent maternal and neonatal mortality 21 The World needs Midwives with Straight Hair More than Ever 21 Birth Centers and home births 20 News: Royal College of Midwives ends 12-year campaign against caesarians, epidurals, inductions and use of instruments 20 MNH = Maternal and Neonatal Health News: Royal College of Midwives ends 12-year campaign against caesarians, epidurals, inductions and use of instruments practitioners, policymakers, researchers, and students the GANM forum is both an impactful and efficient vehicle for disseminating important information, including practice guidelines from the WHO and other international and professional organizations. One hundred and fifty-six threads (9.2%) discussed health in a specific country or region. The most frequently discussed region was the Americas (AMRO; 37.8%). The African region (AFRO) was also frequently discussed (28.8%), as was South-East Asia (SEARO; 10.9%). As a platform for disseminating empirical research, the GANM can be leveraged to have an influence on real- world, evidence-based practice. In particular, the GANM is used to share practice guidelines and professional competencies from international organizations, an important function of online communities of practice(4). The ability to learn from other health professionals’ experiences is also a valuable purpose of online communities as networking, knowledge sharing, and participation and interaction are initiated among participants(5,10-11). A minority of threads were written in Spanish (13.5%). While a handful of subject lines were written in languages other than English or Spanish (notably, Portuguese and French), these comprised a negligible percentage of threads. www.eerp.usp.br/rlae MNH = Maternal and Neonatal Health Content In terms of content, the most frequently discussed topics were nursing (14.2%) and maternal health (13.8%), as shown in Table 2. Discussions about nursing included conversations about the role of nurses in primary health care, workplace safety for nurses, disaster nursing, and advancing nursing in global settings. Announcements about such campaigns as the Year of the Nurse and Midwife and Nursing Now, and the State of the World’s Nursing Report were also included. Maternal health topics included family planning, postpartum care, maternal mortality reports, and more recently, COVID-19 and pregnancy and breastfeeding. Table 2 - Discussion board content areas Topic N % Nursing 239 14.2% Maternal health 233 13.8% Other health topics 178 10.5% Midwifery 169 10.0% Infectious diseases 152 9.0% Women’s health 106 6.3% Technology 78 4.6% Child health 75 4.4% www.eerp.usp.br/rlae 5 5 Chu H, Gresh A, Bolanos V, Reynolds N. Table 3 - Discussion board threads that generated the most discussion (N represents the number of messages within the thread) Discussion Board Thread Title N Doulas 53 Looking for collaborators 52 Traditional Midwives (Untrained) 50 Direct entry midwifery programme 44 2020 Year of the Nurse, celebrating nursing and midwifery! 43 Information 33 Partograph: Does it Improve MNH* Outcomes? 31 HELP TO MOTHERS: fundal pressure 29 Medical Aid Films New Films on Identifying a Sick Child in the Community 29 Simulation Education and Research Dialogue 29 Controversy Surrounding Bed-Sharing 28 Digest of Greetings for 2014 28 Worsening Situation for Nurses and Midwives - Numbers Dropping 28 Alternativas no farmacológicas para el alivio del dolor en el trabajo de parto 27 RE: Request for primary studies on respectful maternity care 27 What is your position on Workforce Migration? Discussion The findings of this qualitative content analysis reinforce the utility of the GANM as a platform for professional development. Information exchange was shown to be a primary function of the discussion forum. This is consistent with previous research findings that online discussion groups for health professionals are used more frequently for information sharing, communication, and networking(5). With a global membership of over 4,000 Opportunities for continuing education were plentiful and, due to the value that the nursing profession places on lifelong learning, significant. In addition to sharing real-world continuing education activities (conferences, online courses, workshops, etc.), the GANM facilitates several aspects of lifelong learning, including knowledge 6 Rev. Latino-Am. Enfermagem 2021;29:e3431. as a dynamic process, collaborative learning, appropriate learning environment, and the act of seeking opportunities for learning(12-13). The discussion forum offers a means to implement lifelong learning by serving as an interactive space for members to access evidence-based information, ask questions, and share experiences, creating a dynamic learning environment and opportunities for collaboration. By sharing article summaries of open access peer-reviewed empirical research hosted on the online community library, the GANM also offers opportunities for practitioners to actively seek out new knowledge. information known about members (for example, their country) is self-reported. A limitation with the research design is that only discussion board thread subject lines were used for analysis without delving into the content of each post. Future studies should examine the content of each post to further explore the information delivered to the members of the online community of practice. Understanding how this online community of practice is being used for knowledge dissemination is important to impact the advancement of scientific knowledge for nursing and midwifery. The GANM team can use this information to continue to provide a platform for knowledge exchange and to disseminate information that we now know is most discussed and utilized among members. In addition, this study provides insight for researchers and policymakers to leverage online communities of practice to ensure that the latest evidence-based research and practice is being disseminated to wide audiences. With contributing members from over 60 countries, the GANM is an innovative platform for connecting practitioners worldwide for potential collaboration. This is most singularly exemplified by a post entitled, “Looking for collaborators”, which generated over 50 responses. Conclusion This conventional qualitative content analysis sought to understand how members of the GANM online community of practice use the GANM discussion forum for communication and knowledge dissemination. The findings of this study show that the utility of the GANM centers on opportunities for professional development, continuing education, and worldwide collaboration. Exchange of empirical research and high-quality, evidence-based information is also a key function of the discussion forum, as it helps practitioners build professional competency and combats the spread of misinformation. This study sheds light on the role of online communities of practice in sharing evidence-based information about emerging health issues. The COVID-19 pandemic is a global crisis that has ushered in an era of information sharing spurred by a collective sense of urgency. During this time, the GANM discussion board has been used to share evidence-based, peer-reviewed research articles regarding COVID-19 and such topics as pregnancy, maternal and neonatal health, and the health of vulnerable populations. Webinars from such authoritative organizations as the Consortium of Universities for Global Health, the IBP Network, and Johns Hopkins University have also been announced. Of relevance to nursing and midwifery, guidance from the WHO has also been shared, including protocols for maintaining maternal, newborn, child and adolescent health and resources for addressing violence against women during social distancing. Such online training materials help nurses and midwives build competencies, particularly in treating patients with COVID-19. In light of the present COVID-19 pandemic, the ability to access reliable clinical knowledge is especially critical for nurses and midwives to maximize their impact of care. Because of social distancing, it is even more critical to be able to access practice guidelines, training webinars, and evidence-based research articles from reputable sources online. Doing so allows nurses and midwives to build their competencies from the safety of their own homes while also slowing the spread of SARS-CoV-2. The GANM discussion forum represents a transformative approach for knowledge acquisition during a global public health emergency with implications for nursing and midwifery practice. As a platform for disseminating empirical research, this low band-width technology offers nurses and midwives opportunities for professional growth that can have an impact on their real- world practice when caring for patients with COVID-19. Limitations of this study include the lack of detailed demographic information about the GANM’s membership and possible lack of representation from other health professions. Discussion The fact that a call for partnership garnered interest from members across the globe suggests that there is enthusiasm among members in using the GANM discussion forum for networking purposes. Indeed, previous research has shown that networking is a primary reason that health professionals use online discussion groups(5). Additionally, the level of engagement within the debate surrounding the topic, “2020 Year of the Nurse, celebrating nursing and midwifery!” demonstrates that the GANM discussion board offers a platform for real dialogue. www.eerp.usp.br/rlae References 9. Chu H. GANM survey results: informing and connecting our community. [Internet]. 2019 Oct 7 [cited Jul 31, 2020]. Available from: https://ganm.nursing.jhu.edu/ ganm-survey-results-informing-and-connecting-our- community/ 1. World Health Organization. Global strategic directions for strengthening nursing and midwifery 2016-2020. [Internet]. Geneva: WHO; 2016 [cited Jul 31, 2020]. Available from: https://www.who.int/hrh/nursing_ midwifery/global-strategic-midwifery2016-2020.pdf?ua=1 2. Brand R. When disaster strikes: nurse leadership, nursing care, and teamwork saves lives. [Internet]. 2016 May 1 [cited Jul 31, 2020]. Available from: https://www. rwjf.org/en/library/research/2016/05/when-disaster- strikes.html 1. World Health Organization. Global strategic directions for strengthening nursing and midwifery 2016-2020. [Internet]. Geneva: WHO; 2016 [cited Jul 31, 2020]. Available from: https://www.who.int/hrh/nursing_ midwifery/global-strategic-midwifery2016-2020.pdf?ua=1 2. Brand R. When disaster strikes: nurse leadership, nursing care, and teamwork saves lives. [Internet]. 2016 May 1 [cited Jul 31, 2020]. Available from: https://www. rwjf.org/en/library/research/2016/05/when-disaster- strikes.html 10. Abidi SSR. Healthcare knowledge management. New York: Springer; 2007. Cap. 6, Healthcare knowledge sharing: purpose, practices, and prospects, p. 67-86. 11. Ferrante JM, Friedman A, Shaw EK, Howard J, Cohen DJ, Shahidi L. Lessons learned designing and using an online discussion forum for care coordinators in primary care. Qual Health Res. 2016 Nov;26(13):1851-61. doi: 10.1177/1049732315609567 3. Veenema TG, Friese CR, Meyer D. The increasing demand for critical care beds- recommendations for bridging the RN staffing gap. [Internet]. Clinicians’ Biosecurity News. 2020 March 30 [cited Jul 31, 2020]. Available from: https://www.centerforhealthsecurity.org/ cbn/2020/cbnreport-03302020.html 12. Davis L, Taylor H, Reyes H. Lifelong learning in nursing: a Delphi study. Nurse Educ Today. 2014 Mar 1;34(3):441-5. doi: 10.1016/j.nedt.2013.04.014 13. Qalehsari MQ, Khaghanizadeh M, Ebadi A. Lifelong learning strategies in nursing: a systematic review. Electron Physician. 2017 Oct;9(10):5541. doi: 10.19082/5541 4. Billings DM. Online communities of professional practice. J Nurs Educ. 2003 Aug 1;42(8):335-6. doi: 10.3928/0148-4834-20030801-03 4. Billings DM. Online communities of professional practice. J Nurs Educ. 2003 Aug 1;42(8):335-6. doi: 10.3928/0148-4834-20030801-03 5. Dieleman C, Duncan EA. Investigating the purpose of an online discussion group for health professionals: a case example from forensic occupational therapy. BMC Health Services Research. 2013 Dec;13(1):1-8. doi: 10.1186/1472-6963-13-253 5. Dieleman C, Duncan EA. Investigating the purpose of an online discussion group for health professionals: a case example from forensic occupational therapy. BMC Health Services Research. 2013 Dec;13(1):1-8. doi: 10.1186/1472-6963-13-253 Conclusion For example, doulas and untrained midwives were popular topics of conversation, but it is unknown how many doulas and untrained midwives participated in these discussion threads. Furthermore, the demographic 7 Chu H, Gresh A, Bolanos V, Reynolds N. Copyright © 2021 Revista Latino-Americana de Enfermagem This is an Open Access article distributed under the terms of the Creative Commons (CC BY). This license lets others distribute, remix, tweak, and build upon your work, even commercially, as long as they credit you for the original creation. This is the most accommodating of licenses offered. Recommended for maximum dissemination and use of licensed materials. Corresponding author: Hillary Chu E-mail: hillary@mail.harvard.edu https://orcid.org/0000-0001-8933-8734 www.eerp.usp.br/rlae Corresponding author: Hillary Chu E-mail: hillary@mail.harvard.edu https://orcid.org/0000-0001-8933-8734 Authors’ contribution: Study concept and design: Hillary Chu, Ashley Gresh, Valentina Bolanos, Nancy Reynolds. Obtaining data: Hillary Chu, Ashley Gresh, Valentina Bolanos. Data analysis and interpretation: Hillary Chu, Ashley Gresh, Valentina Bolanos. Drafting the manuscript: Hillary Chu, Ashley Gresh, Valentina Bolanos. Critical review of the manuscript as to its relevant intellectual content: Nancy Reynolds. 6. Smedley RM, Coulson NS. A thematic analysis of messages posted by moderators within health-related 6. Smedley RM, Coulson NS. A thematic analysis of messages posted by moderators within health-related asynchronous online support forums. Patient Educ Couns. 2017 Sep 1;100(9):1688-93. doi: 10.1016/j. pec.2017.04.008 6. Smedley RM, Coulson NS. A thematic analysis of messages posted by moderators within health-related asynchronous online support forums. Patient Educ Couns. 2017 Sep 1;100(9):1688-93. doi: 10.1016/j. pec.2017.04.008 asynchronous online support forums. Patient Educ Couns. 2017 Sep 1;100(9):1688-93. doi: 10.1016/j. pec.2017.04.008 7. Elo S, Kyngäs H. The qualitative content analysis process. J Adv Nurs. 2008 Apr;62(1):107-15. doi: 10.1111/j.1365-2648.2007.04569.x 8. Elo S, Kääriäinen M, Kanste O, Pölkki T, Utriainen K, Kyngäs H. Qualitative content analysis: a focus on trustworthiness. SAGE Open. 2014 Feb 5;4(1):2158244014522633. doi: 10.1177/2158244014522633 7. Elo S, Kyngäs H. The qualitative content analysis process. J Adv Nurs. 2008 Apr;62(1):107-15. doi: 10.1111/j.1365-2648.2007.04569.x 7. Elo S, Kyngäs H. The qualitative content analysis process. J Adv Nurs. 2008 Apr;62(1):107-15. doi: 10.1111/j.1365-2648.2007.04569.x All authors approved the final version of the text. All authors approved the final version of the text. Conflict of interest: the authors have declared that there is no conflict of interest. Conflict of interest: the authors have declared that there is no conflict of interest. 8. Elo S, Kääriäinen M, Kanste O, Pölkki T, Utriainen K, Kyngäs H. Qualitative content analysis: a focus on trustworthiness. SAGE Open. 2014 Feb 5;4(1):2158244014522633. doi: 10.1177/2158244014522633 8. Elo S, Kääriäinen M, Kanste O, Pölkki T, Utriainen K, Kyngäs H. Qualitative content analysis: a focus on trustworthiness. SAGE Open. 2014 Feb 5;4(1):2158244014522633. doi: 10.1177/2158244014522633 Received: Aug 19th 2020 Accepted: Sep 27th 2020 Received: Aug 19th 2020 Accepted: Sep 27th 2020 Received: Aug 19th 2020 Accepted: Sep 27th 2020 Associate Editor: Associate Editor: Evelin Capellari Cárnio Copyright © 2021 Revista Latino-Americana de Enfermagem This is an Open Access article distributed under the terms of the Creative Commons (CC BY). This license lets others distribute, remix, tweak, and build upon your work, even commercially, as long as they credit you for the original creation. This is the most accommodating of licenses offered. Recommended for maximum dissemination and use of licensed materials. ( ) This license lets others distribute, remix, tweak, and build upon your work, even commercially, as long as they credit you for the original creation. This is the most accommodating of licenses offered. Recommended for maximum dissemination and use of licensed materials. www.eerp.usp.br/rlae
https://openalex.org/W2766465006	https://scindeks-clanci.ceon.rs/data/pdf/1821-4487/2017/1821-44871703174V.pdf	English	null	Effects of seed coat colour on the seed quality and initial seedling growth of red clover cultivars (Trifolium pratense)	Journal on processing and energy in agriculture	2,017	cc-by	3,544	Biblid: 1821-4487 (2017) 21; 3; p 174-177 UDK: 581.48 Biblid: 1821-4487 (2017) 21; 3; p 174-177 UDK: 581.48 Original Scientific Paper Originalni naučni rad REZIME Cilj istraživanja bio je da se ispita uticaj boje semena četiri sorte crvene deteline proizvedene u Srbiji tokom dve godine na njihov kvalitet (klijanje, dormantnost (tvrdo seme), mrtvo seme i početni porast klijanaca (vigor)). U ovom istraživanju su prvo vizuelno razdvojena semena po boji, a zatim su razdvojena digitalnim kolor separatorom na svetla, tamna i mešovita. Rezultati su pokazali da boja semena crvene deteline može biti dobar pokazatelj kvaliteta semena i početnog porasta klijanaca. Rezultati su pokazali da su svetlo obojena semena sorte crvene deteline imala snažniji vigor i bolji kvalitet od drugih boja. Nije bilo jasnog zaključka o uticaju regiona i godine na praćene parametre kvaliteta semena. Zabeležena je visoka varijabilnost za tvrda (CV = 22,22%) i mrtva semena (CV = 40,18%) sorti crvene deteline. Za klijanje je zabeležena manja varijabilnost svetlih i mešovitih semena (CV = 4,53% i CV = 8,53%). Boja semena može biti značajan faktor kvaliteta semena crvene deteline i zbog toga je moguće povećati klijavost uklanjanjem tamno obojenih semena. Ključne reči: vigor, crvena detelina, boja semenjače, kvalitet semena. Many studies have demonstrated that seed colour influences water uptake (Powell et al., 1986), gas diffusion, seed dormancy (Baskin et al., 2000), seed quality, germination and seedling emergence (Mavi, 2010) in some crop plants, owing to colour pigments located in the seed coat (Powell et al., 1986; Abdullah et al., 1991). The seed coat allows the passage of water and gasses to the tissues, and it features a varying colour. Differences in the seed colour within a species are associated with harvesting seeds in different developmental stages of fruit and some genetic differences. In red clovers, the seed colour is determined by two loci. If two loci are homozygous and recessive, the colour will be yellow. If two loci are heterozygote and dominant, the seed colour will be light purple. If two loci are homozygous and dominant, the seed colour will be purple (Bortnem and Boe, 2003). The formation of brown and red colour in red clover seed lots is a consequence of seed aging. ABSTRACT The objective of this study was to investigate the influence of seed coat colour on the seed quality of four red clover cultivars (germination, dormancy (hard seeds), dead seeds and initial seedling growth (vigour)) produced in Serbia over a period of two years. The seeds analyzed were first separated visually into bright, dark and mixed seed groups, followed by subsequent digital colour measurements. The results obtained showed that the seed coat colour of red clover could be considered a good indicator of seed quality and seedling growth ability. The results furthermore showed that bright-coloured seeds of red clover cultivars indicated increased vigour and seed quality compared to other colours. The impact of the production area and year on the seed quality parameters was inconclusive. A high variability in hard (CV = 22.22 %) and dead seeds (CV = 40.18 %) was recorded between different seed lots of red clover. A lower variability was found in the germination parameter of bright- and mixed-coloured seeds (CV = 4.53 % and CV = 8.53 %). The seed coat colour could be an important factor in determining the quality of red clover seeds, potentially increasing germination simply by removing dark-coloured seeds. Keywords: vigour, red clover, seed coat colour, seed quality. Keywords: vigour, red clover, seed coat colour, seed quality. Journal on Processing and Energy in Agriculture 21 (2017) 3 EFFECTS OF SEED COAT COLOUR ON THE SEED QUALITY AND INITIAL SEEDLING GROWTH OF RED CLOVER CULTIVARS (Trifolium pratense) UTICAJ BOJE SEMENJAČE NA KVALITET SEMENA I POČETNI PORAST KLIJANACA SORATA CRVENE DETELINE (Trifolium pratense) Nataša VELIJEVIĆ, Ratibor ŠTRBANOVIĆ, Dobrivoj POŠTIĆ, Rade STANISAVLJEVIĆ, Lana ĐUKANOVIĆ Institute for Plant Protection and Environment, 11040 Belgrade, Teodora Drajzera 9, Serbia e-mail: natasvelijeviocc@gmail.com MATERIALS AND METHODS seeds were between the dark and bright-coloured lots. In like fashion, the values recorded for hard seeds were the highest (28– 44 %) in the dark-coloured seed lots, whereas the lowest values (18 %) were in bright seeds. The highest CV (56.77 %) was calculated for dead seeds in the cultivar K–39. The initial seedling growth of the tested red clover cultivars was significantly different according to each coat colour: the root was 1.8 cm in bright-coloured seeds compared to 1.4 cm in dark seeds, whereas the shoot was 4.3 cm in bright-coloured seeds compared to 3.1 cm in dark seeds. This was confirmed by another study which showed that brown (dark) and mixed- coloured seed lots indicated lower vigour than yellow (bright)- coloured seed lots (Atis et al., 2010). Over a period of two years (2015 and 2016), the experiment was conducted under laboratory conditions, including seeds of four diploid red clover cultivars (K-39, Sana, K-17, and Una), i.e. the second cut of the cultivars grown in different locations in Serbia. After harvesting, the seeds were dried to a moisture content of 12 % and separated into three colour groupings: dark, mixed and bright. The relationship (%) between the colours of the seed coat of each cultivar (Table 1) was established thereafter on the basis of the following parameters: germination, hard or dormant seeds, dead seeds and initial seedling growth. The analysis of seed germination was performed two months after the harvest, which corresponds to the fall planting period (September to October). The ANOVA showed that the seedling weights significantly changed relative to the change in the colour of red clover seed lots. The lowest seedlings weight of red clovers was recorded in dark-coloured seed lots (1.176 g), whereas the highest seedling weight of red clovers was recorded in bright-coloured seed lots (1.605 g) (Table 2). A germination test with 4x100 seeds was carried out in two plastic pots, using filter paper at a temperature of 20 °C (in the dark). The germination was recorded on the 10th day in accordance with the ISTA rules (ISTA, 2016). The Tetrazolium Chloride (TZ) test was applied on hard seeds in order to separate dead seeds from the hard ones (ISTA 2008). The initial growth of seedlings was determined in germinating seeds by measuring the following parameters: shoot length (cm), root length (cm) and seedling weight (g). Table 2. INTRODUCTION The red clover (Trifolium pratense L.) is one of the main forage species found natively in the temperate regions of Southern Europe and Southern Eurasia (Taylor and Quesenberry, 1996; Algan and Buyukkartal, 2000; Herrmann et al., 2006). Although Mediterranean in origin, it is widely adapted to many climatic conditions around the world (Taylor and Smith, 1979). The red clover has a high nutritive value, and it is of immense importance to the environment and soil due to its ability to fix atmospheric nitrogen. Furthermore, it is an important component of grass-legume mixtures used to achieve high-quality forage production, particularly high-quality silage (Dias et al., 2008; Knežević, 2013; Knežević et al., 2014). From a botanical perspective, the red clover is a perennial species which generally persists in pastures two to three years under normal agricultural conditions (Ulloa et al., 2003). This annual legume can be used in various manners such as renovation, pasture improvement, erosion control or soil restoration programs. Harvesting and seed processing of forage legumes is highly important for producing high-quality seeds (Đokić et al., 2012; 2015). Although such variations in the colour of clover seeds have been acknowledged by researchers for more than 100 years (Brown and Hillman, 1906), the specialist literature dealing with the effect of red clover seed colour on the seed quality is fairly scarce. The objective of this study was to determine a relationship between the seed coat colour, seed quality and initial seedling growth of the selected red clover cultivars. Journal on Processing and Energy in Agriculture 21 (2017) 3 174 Velijević, Nataša et al./ Effec. of Seed Coat Col. on The Seed Qual. and Initial Seedling Growth of Red Clover Culti. (Trifolium P.) MATERIALS AND METHODS Effects of different seed coat colour of red clover cultivars on the seed quality and initial seedling growth (Note: Lower case letters in the bars indicate the statistical significance between the seed colour means, whereas different numbers indicate the statistical significance (p < 0.05) between different seed colour means of the tested red clover cultivars) The data collated were analyzed by the analysis of variance (ANOVA – F test) adapted to a randomized block design, whereas the evaluation of difference significance was performed by the mean Tukey test method. Tukey's multiple range tests were applied to establish differences between the treatments. The coefficient of variation (CV, %) was calculated and the relationship between the traits was established by the Pearson's correlation test (r). The program Minitab 16.1.0 (statistics software package) was used for statistical analysis. The results obtained are presented in Table 1, 2 and 3. f Cultivars of red clovers K-39 Sana K-17 Una CV% a) Germination Dark 61c 51c 56c 57c 7.3 Bright 80a 75a 81a 78a 3.4 Mixed 70b 67b 71b 70b 2.5 CV% 13.5 19.0 18.2 15.5 b) Hard seed Dark 33a 45a 39a 38a 12.7 Bright 18c 18c 14c 18c 11.8 Mixed 26b 27b 23b 25b 6.8 CV% 29.2 45.8 49.9 37.6 c) Dead seed Dark 6a 4b 5a 5a 16.3 Bright 2b 7a 5a 4b 46.3 Mixed 4b 6ab 6b 5a 18.2 CV% 50.0 26.9 10.8 12.4 d) Root Dark 1.4a 1.3b 1.2b 1.7a 15.4 Bright 1.6a 2.1a 1.6a 1.8a 13.3 Mixed 1.3a 1.4b 1.2b 1.5a 9.6 CV% 10.7 27.2 17.3 9.2 e) Shoot Dark 3.5a 2.8b 3.6ab 2.6c 15.9 Bright 4.5a 4.2a 4.5a 4.0a 5.7 Mixed 4.1a 3.6ab 4.2a 3.4b 10.1 CV% 12.5 19.9 11.2 21.1 f) Seedling weight Dark 1.158b 1.035b 1.282c 1.229c 9.1 Bright 1.395a 1.417a 1.932a 1.677a 15.7 Mixed 1.317ab 1.432a 1.427b 1.411b 3.8 CV% 9.4 17.4 22.1 15.6 Journal on Processing and Energy in Agriculture 21 (2017) 3 REFERENCES determined (P≤0.01) in mixed-coloured seeds, whereas the correlation value was not significant in bright-coloured seeds (in contrast with dark- and mixed-coloured seeds which indicated a more significant correlation) (Table 3). Abdullah, W.D., A.A. Powell and S. Matthews, (1991). Association of differences in seed vigour in long bean with testa colour and imbibition damage. Journal of Agriculture Science, 116: 259–264 d Abdullah, W.D., A.A. Powell and S. Matthews, (1991). Association of differences in seed vigour in long bean with testa colour and imbibition damage. Journal of Agriculture Science, 116: 259–264 d Table 3. The correlation coefficient (r) of the tested red clover parameters and seed coat colours Table 3. The correlation coefficient (r) of the tested red clover parameters and seed coat colours Seed coat colour Parameter Germ. Hard seed Dead seed Root Shoot Seedling weight Dark Germination % - -0.999* 0.993* 0.119 ns 0.483 ns 0.507 ns Mixed Germination % - -0.845** -0.302 ns 0.333 ns 0.523 ns -0.271 ns Bright Germination % - -0.629 ns -0.666 ns 0.195 ns 0.667 ns 0.562 ns Statistical significance level: P≤ 0.05;P≤0.01; P≤0.001; NS= not significant Table 3. The correlation coefficient (r) of the tested red clover parameters and d l Atis, I., M. Atak, E. Can and Mavi K., (2011). Seed coat color effects on seed quality and salt tolerance of red clover (Trifolium pratense). International Journal of Agriculture and Biology, 13: 363–368 g gy Algan, G. and H.N.B. Buyukkartal, (2000). Ultrastructure of seed coat development in the natural tetraploid Trifolium pratense L. Journal of Agronomy and Crop Science, 182: 205–213 Differences in the seed coat colour within red clover lots can be visually identified and easily discerned using a digital colour meter (Table 1). Therefore, the colour evaluation in this study was performed using a digital colour meter. The seed lots were classified according to the seed coat colour using the image analysis method in flax (Dana and Ivo, 2008) and Ambrosia trifida (Sako et al., 2001) species. Our research showed that this method can be used to classify red clover seed lots according to the seed coat colour. Seed colour is an important distinguishing feature between hard-seeded and soft-seeded lines (Juan et al., 1994; Brochmann, 1992). The hard seeds of Vicia sativa are smaller and lighter than the soft seeds. REFERENCES The soft seeds are light brown in colour, whereas the hard seeds are black, indicating certain degree of chemical or mechanical differences (Büyükkartal et al., 2013). Differences in seed colour also refer to differences in the amount of colour pigments in the seed coat. The water uptake and tolerance of seeds to excessive water were closely associated with the seed colour in some rape species, and coloured seeds showed a slow water uptake, low electrical conductivity and high tolerance to excessive water (Zhang et al., 2008). The seed coat protects seeds from water penetration in the initial germination stage. Additionally, the seed coat does not allow the passage of water and/or oxygen, thus hindering the overall germination (Mohamed-Yassen et al., 1994). Seed coat colour affects the water uptake in many legume species, especially in bright-coloured varieties and cultivars which absorb water faster than those of darker colour and have a lower germination percentage (Powell et al., 1986). The red clover seed lots ought to be harvested in a timely manner in order minimise the adverse effects of seed coat on the quality of seeds. Baskin, J.M., C.C. Baskin and Li X., (2000).Taxonomy, anatomy and evolution of physical dormancy in seeds. Plant Species Biology, 15: 139–152 Bortnem, R. and A. Boe, (2003). Color index for red clover seed. Crop Sci., 43: 2279–2283 Brochmann, C, (1992). Pollen and seed morphology of Nordic Draba (Brassicaceae), phylogenetic and ecological implications. Nordic Journal of Botany. 12: 657–673 Brown, E. and F.H. Hillman, (1906). Seed of Red Clover and its Impurities. Farmers' Bulletin, No: 260 Büyükkartal, H.N., Çölgeçen, H., Pinar, N.M., Erdoğan N., (2013). Seed coat ultrastructure of hard-seeded and soft-seeded varieties of Vicia sativa. Turkish Journal of Botany, 37: 270- 275 Dana, W. and W. Ivo, (2008). Computer image analysis of seed shape and seed color for flax cultivar description. Computers and Electronics in Agriculture, 61: 126–135 Demir, I., S. Ermis, K. Mavi, Matthews S., (2008). Mean germination time of pepper seed lots (Capsicum annuum L.) predicts size and uniformity of seedlings in germination tests and transplant modules. Seed Science and Technology, 36: 21– 30 Dias, P.M.B., B. Julier, J.P. Sampoux, P. Barre, Dall’Agnol M., (2008). Genetic diversity in red clover (Trifolium pratense L.) revealed by morphological and microsatellite (SSR) markers. Euphytica, 160: 189–205 Đokić, D., Stanisavljević, R., Terzić, D., Milenković, J., Radivojević, G., Koprivica, R., Štrbanović R., (2015). REFERENCES Efficiency of alfalfa seed processing with different seed purity. Journal of Processing and Energy in Agriculture, 19: 166 – 168. RESULTS AND DISCUSSION The cultivars analyzed showed a significant variability in the seed colour (dark CV = 15.89 %, mixed CV = 15.89 %, bright CV = 12.76 %) (Table 1). Table 1. Relation (%) between the seed colour of the tested red clover cultivars Table 1. Relation (%) between the seed colour of the tested red clover cultivars Cultivar Colour K-39 Sana K-17 Una CV (%) Dark (1) 23 % 25 % 21 % 17 % 15.89 Bright (2) 55 % 46 % 54 % 63 % 12.76 Mixed (3) 22 % 31 % 25 % 20 % 15.89 Using the F-test, it was determined that the year of the experiment had no significant effects on the tested parameters (results are shown in average values), whereas the colour and the cultivar were greatly affected (P≤ 0.05 or P≤0.01). Visually inspected and separated seeds were subjected to digital colour measurements in order to perform colour classification. The statistical analysis showed that the seed coat colour was significantly different. As expected, the germination values were the highest for bright seeds and the lowest for dark seeds, i.e. 57 % for black-coloured and 78 % for bright-coloured seeds (Table 2). The findings are in accordance with those of Atis et al. (2010) who confirmed that the brown-coloured seed lots had the lowest total germination percentage (58 %), whereas the yellow-coloured seed lots had the highest total germination (99 %). The total germination percentages for mixed-coloured Using the Pearson's correlation test (r), the most significant correlation between germination and dead seeds (P≤0.001) was recorded in dark-coloured seeds, whereas a negative correlation was determined between germination and hard seeds. A significant relationship between germination and hard seeds was Journal on Processing and Energy in Agriculture 21 (2017) 3 175 Velijević, Nataša et al./ Effec. of Seed Coat Col. on The Seed Qual. and Initial Seedling Growth of Red Clover Culti. (Trifolium P.) Velijević, Nataša et al./ Effec. of Seed Coat Col. on The Seed Qual. and Initial Seedling Growth of Red Clov Velijević, Nataša et al./ Effec. of Seed Coat Col. on The Seed Qual. and Initial Seedling Growth of Red Clover Culti. (Trifolium P.) determined (P≤0.01) in mixed-coloured seeds, whereas the correlation value was not significant in bright-coloured seeds (in contrast with dark- and mixed-coloured seeds which indicated a more significant correlation) (Table 3). RESULTS AND DISCUSSION Differences in the seed coat colour within red clover lots can REFERENCES Abdullah, W.D., A.A. Powell and S. Matthews, (1991). Association of differences in seed vigour in long bean with testa colour and imbibition damage. Journal of Agriculture Science, 116: 259–264 Atis, I., M. Atak, E. Can and Mavi K., (2011). Seed coat color effects on seed quality and salt tolerance of red clover (Trifolium pratense). International Journal of Agriculture and Biology, 13: 363–368 Algan, G. and H.N.B. Buyukkartal, (2000). Ultrastructure of seed coat development in the natural tetraploid Trifolium pratense L. Journal of Agronomy and Crop Science, 182: 205–213 Table 3. The correlation coefficient (r) of the tested red clover parameters and seed coat colours Seed coat colour Parameter Germ. Hard seed Dead seed Root Shoot Seedling weight Dark Germination % - -0.999* 0.993* 0.119 ns 0.483 ns 0.507 ns Mixed Germination % - -0.845 -0.302 ns 0.333 ns 0.523 ns -0.271 ns Bright Germination % - -0.629 ns -0.666 ns 0.195 ns 0.667 ns 0.562 ns Statistical significance level: P≤ 0.05;P≤0.01; **P≤0.001; NS= not significant CONCLUSION Đokić, D., Stanisavljević, R., Marković, J., Terzić, D., Anđelković B., 2012. Impurities in alfalfa seed and their impact on processing technology. Journal of Processing and Energy in Agriculture, 16: 75 – 78. In the present study, seeds of four red clover cultivars were examined. The results obtained showed that the colour of red clover seeds can be a good indicator of the seed quality and initial seedling growth. Light-coloured cultivars of red clover seeds indicated stronger vigour and were of better quality than those of other colours. The seed coat colour could be an important factor in determining the quality of red clover seeds, potentially increasing germination simply by removing dark- coloured seeds. Differences in the seed colour of red clovers can be used as seed quality markers. Moreover, dark-coloured seeds are readily separated in seed lots using digital colour measuring equipment. Herrmann, D., Boller B., Studer B., Widmer F., Kölliker R., (2006). QTL analysis of seed yield components in red clover (Trifolium pratense L.). Theor. Appl. Genet., 112: 536–545 ISTA. (2016). International Rules for Seed Testing: Edition 2003 ISTA, Bassersdorf. Juan, R., Pastor, J.,Fernandez I., (1994). Seed morphology in Veronica L. (Scrophulariaceae) from south-west Sapin. Botanical Journal of the Linnean Society, 115: 133–143 Ć Knežević, J., Aksić, M., Ćirić, S., Stevović, V., Tomić, D., Stanisavljević R., 2014. Application of cornmeal at ensiling of alfalfa cocksfoot and their mixture. Acta Agriculturae Serbica, 19: 143-150. ACKNOWLEDGMENTS: This research was financed by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Project TR-31057 and TR-31018 (2011-2017). Journal on Processing and Energy in Agriculture 21 (2017) 3 176 Velijević, Nataša et al./ Effec. of Seed Coat Col. on The Seed Qual. and Initial Seedling Growth of Red Clover Culti. (Trifolium P.) Mavi, K., (2010). The relationship between seed coat color and seed quality in watermelon Crimson sweet. Horticiculture Science, 37: 62–69 Ulloa, O., F. Ortega, H. Campos, (2003). Analysis of genetic diversity in red clover (Trifolium pratense L.) breeding populations as revealed by RAPD genetic markers. Genome, 46: 529–535 Mohamed-Yasseen, Y., S.A. Barringer, W.E. Splittstoesser, S. Costanza, (1994). The role of seed coats in seed viability. Botanical Review, 60: 426–439 Zhang, X.K., J. Chen, L. Chen, H.Z. Wang, J.N. Li, (2008). Imbibition behavior and flooding tolerance of rapeseed seed (Brassica napus L.) with different testa color. Genetic Resources and Crop Evolution, 55: 1175–1184 Powell, A.A., M.A. Journal on Processing and Energy in Agriculture 21 (2017) 3 CONCLUSION Oliveira, Matthews S., (1986). The role of imbibition damage in determining the vigour of white and coloured seed lots of dwarf french beans (Phaseolus vulgaris). Journal of Experimental Botany, 37: 716–722 Knežević, J., (2013). Posebno ratarstvo, industrijsko i krmno bilje. Univerzitet u Prištini, Poljoprivredni fakultet, Kosovska Mitrovica – Lešak. Sako, Y., E.E. Regnier, T. Daoust, K. Fujimura, S.K. Harrison, M.B. McDonald, (2001). Computer image analysis and classification of giant ragweed seeds. Weed Science, 49: 738– 745 Taylor, N.L. and K.H. Quesenberry, (1996). Red Clover Science, p: 226. Kluwer Academic Publishers Taylor, N.L. and R.R. Smith, (1979). Breeding and genetics of red clover. Advances in Agronomy, 31: 125–154 Accepted: 12. 04. 2017. Received: 21. 02. 2017. 177
https://openalex.org/W3206139477	https://www.frontiersin.org/articles/10.3389/fnins.2021.721773/pdf	English	null	Increased Signal Delays and Unaltered Synaptic Input Pattern Recognition in Layer III Neocortical Pyramidal Neurons of the rTg4510 Mouse Model of Tauopathy: A Computer Simulation Study With Passive Membrane	Frontiers in neuroscience	2,021	cc-by	20,344	ORIGINAL RESEARCH published: 18 October 2021 doi: 10.3389/fnins.2021.721773 Increased Signal Delays and Unaltered Synaptic Input Pattern Recognition in Layer III Neocortical Pyramidal Neurons of the rTg4510 Mouse Model of Tauopathy: A Computer Simulation Study With Passive Membrane Attila Somogyi1,2 and Ervin Wolf1* 1 Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 2 Department of Emergency Medicine, University of Debrecen, Debrecen, Hungary 1 Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 2 Department of Emergency Medicine, University of Debrecen, Debrecen, Hungary Abnormal tau proteins are involved in pathology of many neurodegenerative disorders. Transgenic rTg4510 mice express high levels of human tau protein with P301L mutation linked to chromosome 17 that has been associated with frontotemporal dementia with parkinsonism. By 9 months of age, these mice recapitulate key features of human tauopathies, including presence of hyperphosphorylated tau and neuroﬁbrillary tangles (NFTs) in brain tissue, atrophy and loss of neurons and synapses, and hyperexcitability of neurons, as well as cognitive deﬁciencies. We investigated effects of such human mutant tau protein on neuronal membrane, subthreshold dendritic signaling, and synaptic input pattern recognition/discrimination in layer III frontal transgenic (TG) pyramidal neurons of 9-month-old rTg4510 mice and compared these characteristics to those of wild- type (WT) pyramidal neurons from age-matched control mice. Passive segmental cable models of WT and TG neurons were set up in the NEURON simulator by using three- dimensionally reconstructed morphology and electrophysiological data of these cells. Our computer simulations predict leakage resistance and capacitance of neuronal membrane to be unaffected by the mutant tau protein. Computer models of TG neurons showed only modest alterations in distance dependence of somatopetal voltage and current transfers along dendrites and in rise times and half-widths of somatic Excitatory Postsynaptic Potential (EPSPs) relative to WT control. In contrast, a consistent and statistically signiﬁcant slowdown was detected in the speed of simulated subthreshold dendritic signal propagation in all regions of the dendritic surface of mutant neurons. Predictors of synaptic input pattern recognition/discrimination remained unaltered in model TG neurons. This suggests that tau pathology is primarily associated with failures/loss in synaptic connections rather than with altered intraneuronal synaptic integration in neurons of affected networks. Keywords: tauopathy, mouse frontal cortex, morphology and dendritic signaling, conservation of synaptic input pattern recognition, computer simulations Edited by: Athanasios Metaxas, European University Cyprus, Cyprus Reviewed by: Hugo Geerts, Certara UK Limited, United Kingdom KongFatt Wong-Lin, Ulster University, United Kingdom Correspondence: Ervin Wolf wolf.ervin@anat.med.unideb.hu Edited by: Athanasios Metaxas, European University Cyprus, Cyprus Reviewed by: Hugo Geerts, Certara UK Limited, United Kingdom KongFatt Wong-Lin, Ulster University, United Kingdom Correspondence: Ervin Wolf wolf.ervin@anat.med.unideb.hu Specialty section: This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience Received: 07 June 2021 Accepted: 22 September 2021 Published: 18 October 2021 Citation: Somogyi A and Wolf E (2021) Increased Signal Delays and Unaltered Synaptic Input Pattern Recognition in Layer III Neocortical Pyramidal Neurons of the rTg4510 Mouse Model of Tauopathy: A Computer Simulation Study With Passive Membrane. Front. Neurosci. 15:721773. doi: 10.3389/fnins.2021.721773 Specialty section: This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience Specialty section: This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience Specialty section: This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience Received: 07 June 2021 Accepted: 22 September 2021 Published: 18 October 2021 Received: 07 June 2021 Accepted: 22 September 2021 Published: 18 October 2021 INTRODUCTION and in humans (Callahan et al., 1999; Ginsberg et al., 2000) with tauopathies have been observed and investigated, studying possible eﬀects of tau on individual neurons’ electrophysiological properties gained less attention. However, electrophysiological alterations in layer III neocortical pyramidal neurons have been investigated during advanced tauopathy in rTg4510 mice (Rocher et al., 2010; Crimins et al., 2012). These authors recorded electrophysiological data from individual neurons of rTg4510 and age-matched wild-type (WT) mice and found that cortical neurons in rTg4510 mice have some altered active and passive properties. On average, transgenic (TG) neurons showed depolarized resting membrane potential, increased depolarizing sag potential and increased action potential ﬁring rate in response to current steps, all of which indicate hyperexcitability. On the other hand, these TG neurons had unaltered input resistances and membrane time constants. Following whole-cell patch-clamp recordings, all neurons were ﬁlled with biocytin and dendritic arborizations, and somata of neurons were 3- dimensionally reconstructed and subjected to morphometric analysis. Morphological analysis revealed a reduction in spine density both in apical and basal dendrites and a decrease in size of apical tuft in TG neurons, but no statistically signiﬁcant diﬀerence was found in soma and total neuron surface areas, dendritic diameters, or in horizontal and vertical extents of dendrites. Two key proteins involved in pathophysiology of Alzheimer disease (AD), the most common form of dementia, are amyloid β (Aβ) and tau proteins. These proteins are present in extracellular senile plaques and intracellular neuroﬁbrillary tangles (NFTs), respectively (Kosik et al., 1986; Dickson et al., 1988), which are well-known pathological features of AD. Although these two culprits of AD have been identiﬁed in the 1980s (Glenner et al., 1984; Brion et al., 1986), their entire role in the etiology of this disease is still to be elucidated. Mutant tau alone was demonstrated to be involved in neuroﬁbrillary pathology, synaptic loss, and neurodegeneration (Dickstein et al., 2010; Kopeikina et al., 2013a), but abnormal tau protein is also linked to eﬀects of Aβ as tau protein was shown to be essential to Aβ-induced neuronal degeneration in AD (Rapoport et al., 2002; Bennett et al., 2004; Revett et al., 2013). Mutant tau, if present in addition to Aβ, increases severity of memory deﬁcit in mice (Rhein et al., 2009). INTRODUCTION Besides AD, many other neurodegenerative disorders are also associated with tau inclusions, and these diseases are collectively called tauopathies, including frontotemporal dementia with parkinsonism linked to chromosome 17 (Hutton et al., 1998; Poorkaj et al., 1998; Spillantini et al., 1998b), Pick disease, progressive supranuclear palsy, argyrophilic grain disease, certain prion diseases, and several genetic forms of Parkinson disease (Lee et al., 2001; Ludolph et al., 2001; Tolnay and Probst, 2003; Williams, 2006). Transgenic mouse models have signiﬁcantly increased our understanding on role of toxic tau protein in the development of these diseases. The rTg(tau301L 4R0N) 4510 mouse model (SantaCruz et al., 2005) expresses high levels of human tau (up to 13-fold of its normal murine level) with a mutation that has been linked to familial frontotemporal dementia, the second most prevalent neurodegenerative disease (Clark et al., 1998; Dumanchin et al., 1998; Hutton et al., 1998; Spillantini et al., 1998a). By 9 months of age, these mice recapitulate many pathological changes seen in tauopathies: tangle-like tau inclusions in their brain, neuronal and synaptic loss, atrophy of dendrites, changes in electrophysiological properties of pyramidal neurons, and signs of cognitive and motoric impairments (Ramsden et al., 2005; SantaCruz et al., 2005; Rocher et al., 2010; Crimins et al., 2012; Kopeikina et al., 2013a,b; Scott et al., 2016; Holton et al., 2020; Kubota and Kirino, 2021). In vitro, P301L mutation was shown directly to enhance formation of paired helical ﬁlaments and promote β-sheet structure during aggregation (Barghorn et al., 2000; von Bergen et al., 2001; Fischer et al., 2007). These pathological alterations are well documented, but the precise causal link between the tau protein and the alterations of neural activities is still not well understood. To comprehend mutant tau-related alterations of neural activities, we need to study the changes in functional synaptic connections within such vulnerable networks, and we also need These combined electrophysiological and morphological studies (Rocher et al., 2010; Crimins et al., 2012) demonstrated that in the rTg4510 mouse model, cortical TG neurons suﬀer from variable degree of morphological regression, and parallel with this, some electrophysiological properties of these neurons get altered in advanced tauopathy. MATERIALS AND METHODS To comprehend mutant tau-related alterations of neural activities, we need to study the changes in functional synaptic connections within such vulnerable networks, and we also need to determine the eﬀects of mutant tau protein on the neurons, the building blocks of neural assemblies that conduct and integrate Postsynaptic Potentials (PSPs). While loss of synaptic contacts and shifting of diﬀerent neurotransmitter systems in transgenic animals (Katsuse et al., 2004, 2006; Kopeikina et al., 2013a) INTRODUCTION However, regarding intrinsic neuronal properties, it remained unclear (1) if membrane properties of WT and TG neurons diﬀer from each other, (2) whether dendrites of WT and TG neurons diﬀer in their dendritic signaling properties, and (3) whether WT and TG neurons are diﬀerent in their synaptic input pattern recognition/discrimination abilities that are important in network functions and memory? Study of these possible tau- mediated alterations in intraneuronal properties is mandatory to understand how dysfunctions in individual neurons may contribute to pathological activities of neural networks in diﬀerent forms of tauopathies. Therefore, for the ﬁrst time, we studied questions 1–3 by utilizing multicompartmental computational models of these TG and WT neurons based on spatial neuron reconstructions and electrophysiological measurements. 1Neuromorpho.org Citation: Somogyi A and Wolf E (2021) Increased Signal Delays and Unaltered Synaptic Input Pattern Recognition in Layer III Neocortical Pyramidal Neurons of the rTg4510 Mouse Model of Tauopathy: A Computer Simulation Study With Passive Membrane. Front. Neurosci. 15:721773. doi: 10.3389/fnins.2021.721773 October 2021 \| Volume 15 \| Article 721773 1 Frontiers in Neuroscience \| www.frontiersin.org Dendritic Signaling in Tauopathy Somogyi and Wolf Frontiers in Neuroscience \| www.frontiersin.org Neuron Samples Files containing the detailed three-dimensional (3D) morphology of layer III frontal pyramidal neurons of WT and littermate age- matched rTg4510 tau mutant (P301L) TG 9-month-old mice were downloaded from the NeuroMorpho database1 (Rocher October 2021 \| Volume 15 \| Article 721773 2 Dendritic Signaling in Tauopathy Somogyi and Wolf by a Zeiss LSM-510 confocal laser-scanning microscope. Spatial reconstructions of neurons were based on integrated volumetric datasets obtained by a Volume Integration and Alignments System (VIAS) (Rodriguez et al., 2003). Z-stack stitching of 40 × confocal images was then exported to Neurolucida neuron reconstruction system with AutoNeuron and NeuroExplorer (MBF Bioscience, Williston, VT) software for automatic tracing, which were then manually corrected, and used for morphometric analysis. Spine detection and analysis were performed by the NeuronStudio on full resolution stacks by VIAS, which was followed by manual checking and corrections, if needed. et al., 2010). Altogether 28 WT and 23 TG neurons were suitable for computer modeling and used for this study (Figure 1). TG neurons were analyzed whether they contain NFTs, and it was found that NFT−and NFT+ neurons have identical somatic neuron resistance and membrane time constants and show the same general morphological characteristics (Rocher et al., 2010). Therefore, we did not distinguish NFT−and NFT+ neurons in our study. Sex diﬀerences were not taken into account in the earlier study, where morphology and electrophysiology of these neurons were investigated; thus, we could not account for possible sex diﬀerences. p Details on tissue preparations, labeling procedures, and 3D reconstruction of neurons have been described in the earlier paper (Rocher et al., 2010). Brieﬂy, mice were sacriﬁced by decapitation, and brains were placed in oxygenated (95% O2, 5% CO2) ice-cold Ringer solution. Frontal 300-µm-thick cortical slices (8–10/hemispheres) were made by a vibrating microtome and then left in oxygenated Ringer’s solution at room temperature for at least 1 h before whole-cell patch-clamp recordings. During ∼15 min long recordings slices were still superfused (2.5 ml/min) with Ringer solution. Layer III frontal (dorsal premotor) cortical pyramidal neurons were identiﬁed by infrared-diﬀerential interference contrast microscope. Passive properties were determined electrophysiologically by analyzing voltage responses to 200-ms current steps. Calculation of neuron resistance was based on slope of a regression line ﬁtted to linear portion of the voltage–current plot. Membrane time constant was measured by ﬁtting a single exponential function to membrane voltage in response to 10-pA hyperpolarizing current step. Compartmentalization of Neurons Morphologically faithful compartmental models of pyramidal neurons were created in the NEURON (version 7.3–7.5) simulation environment (Hines and Carnevale, 1997, 2001) based on the neurons’ 3D data ﬁles containing length, diameter, and branching topology of dendrites (Figure 2). NEURON software numerically calculates solution of the spatially and temporally discrete approximation of linear cable equation, which has the form: Cm ∂V ∂t + V Rm = r 2Ra ∂2V ∂x2 where V, x, and t are membrane potential, location, and time; Cm and Rm are speciﬁc membrane capacitance and resistance, respectively, Ra is the axial resistivity, and r is radius. Model neurons had single soma compartments with individually assigned soma surface areas according to reconstructions of respective cells. These mean soma surface areas were 146.2 ± 26.3 and 205.2 ± 41.4 µm2 for WT and TG neurons, respectively. The numbers of dendritic compartments in model neurons were between 17 and 79 (25–66) in WT and 15 and 81 (25–60) in TG neurons in their apical and (basal) dendritic arbors, depending on the complexity of arborization pattern and size of dendrites of individual neurons. FIGURE 1 \| Morphology of layer III wild-type (WT) and transgenic (TG) neurons of Tg4510 mice. Representative neurons with illustrations of dendritic diameters. Neurons are from the NeuroMorpho database (neuromorpho.org) and were 3D reconstructed and analyzed morphologically and electrophysiologically by Rocher et al. (2010) and Crimins et al. (2012). Scale bars are 100 µm. Neuron Samples Following recordings, all neurons were ﬁlled with 1% biocytin; slices were ﬁxed in 4% paraformaldehyde in 0.1 M phosphate-buﬀered saline for 4 days at 4◦C. To allow visualization, slices were incubated in streptavidin–Alexa 546 for 2 days. Confocal images for 3D reconstructions were obtained Only those neurons were included in the ﬁnal dataset, which had membrane potentials ≤−55 mV, were able to ﬁre trains of action potentials in response to sustained depolarizing current, had well-labeled dendrites with no distal cuts, and showed intact soma. Modeling Spines PSPs were simulated by injecting constant or 50-Hz sinusoid current or by conductance changes at various locations of the modeled dendrites (D). Transfers, delays of simulated PSPs to the soma, and rise times/half-widths of somatic EPSPs were measured by simultaneous recordings from dendritic and somatic compartments. 228 ± 23 M for TG cells) was matched (Rocher et al., 2010). During these simulations, somatic neuron resistance (Rin) was computed by measuring voltage changes at the soma in response to constant current injection (Rin = 1V/I, where 1V is amplitude of somatic depolarization, and I is injected current), just like in real electrophysiological experiments. of cylindrical compartments in our computational models (Shelton, 1985; Holmes, 1989; Larkman, 1991; Jaslove, 1992; Stuart and Spruston, 1998; Schmidt-Hieber et al., 2007; Somogyi et al., 2016). In the ﬁrst step of this procedure, relative increase in total surface area (q) caused by spines was calculated for each dendritic compartment according to the following formula: q = (As + Ad)/Ad, where As is total surface area of spines received by the dendritic compartment, and Ad is surface area of the “smooth” cylindrical compartment without spines. Then, the adjusted speciﬁc membrane capacitance (Cm∗) and conductance (Gm∗) of the compartment were assigned individually for each compartment as Cm∗= Cm . q and Gm∗= Gm . q, where Cm and Gm are measurable capacitance and conductance for unit membrane area of the spiny neuron. y g In the second step of our manual ﬁtting procedure, speciﬁc membrane capacitance (Cm) was set by varying Cm (with the Rm found in the previous step of ﬁtting) until simulated membrane time constants (τ) matched those measured electrophysiologically in WT (32.5 ± 4.1 ms) and rTg4510 (35.2 ± 3.4 ms) mice (Rocher et al., 2010). Simulated membrane time constants were calculated from voltage responses of model neurons to depolarizing current steps by measuring time needed for the soma of model neurons to reach 63% of peak depolarization. Axial resistance (Ra) of cytosol was 150 cm (Trevelyan and Jack, 2002; Kabaso et al., 2009) in all model neurons. Integration time was 0.025 ms in all simulations (Hines and Carnevale, 1997, 2001). A summary of model parameters is presented in Table 1. Membrane Models of Neurons Initiation of Postsynaptic Potentials and Measures of Dendritic Signal Transfer Postsynaptic potentials were simulated by steady-state or sinusoidal (50 Hz) current injections to dendritic sites or by local dendritic conductance changes according to an alpha function. The alpha function has the following form: Modeling Spines As loss of dendritic spines is one element of dendritic atrophy in the rTg4510 mouse model (Rocher et al., 2010; Crimins et al., 2012) and also in human tauopathies (Ferrer and Gullotta, 1990; Ferrer et al., 1990), we considered the eﬀects of spines on dendritic impulse propagation in our computational models. Linear spine densities of model neurons were 1.25 and 1.00 spines/µm for the WT and TG neurons, respectively (Crimins et al., 2012) with common 1.5-µm2 spine surface areas (Larkman et al., 1992; Middei et al., 2008). Spines were not modeled as individual structures but by proportionally increasing speciﬁc membrane capacitance and conductance of dendritic compartments. This way, we accounted for electrical eﬀects caused by increase in dendritic surface area due to dendritic spines without actually modifying length and diameter FIGURE 1 \| Morphology of layer III wild-type (WT) and transgenic (TG) neurons of Tg4510 mice. Representative neurons with illustrations of dendritic diameters. Neurons are from the NeuroMorpho database (neuromorpho.org) and were 3D reconstructed and analyzed morphologically and electrophysiologically by Rocher et al. (2010) and Crimins et al. (2012). Scale bars are 100 µm. October 2021 \| Volume 15 \| Article 721773 Frontiers in Neuroscience \| www.frontiersin.org 3 Dendritic Signaling in Tauopathy Somogyi and Wolf FIGURE 2 \| Basic concept of compartmental models used in the study. Based on 3D reconstructed morphology of neurons (A), dendrites were divided into cylindrical compartments, while the soma was represented by a single spherical compartment (B). Compartments were replaced and coupled together by electrical equivalent circuits (C) with resistors and capacitors representing speciﬁc membrane resistance (Rm) and capacitance (Cm), as well as axial resistance of the dendroplasm (Ra). PSPs were simulated by injecting constant or 50-Hz sinusoid current or by conductance changes at various locations of the modeled dendrites (D). Transfers, delays of simulated PSPs to the soma, and rise times/half-widths of somatic EPSPs were measured by simultaneous recordings from dendritic and somatic compartments. FIGURE 2 \| Basic concept of compartmental models used in the study. Based on 3D reconstructed morphology of neurons (A), dendrites were divided into cylindrical compartments, while the soma was represented by a single spherical compartment (B). Compartments were replaced and coupled together by electrical equivalent circuits (C) with resistors and capacitors representing speciﬁc membrane resistance (Rm) and capacitance (Cm), as well as axial resistance of the dendroplasm (Ra). Modeling Spines Regarding the many thousands of spines per neuron, such modeling is feasible as we were not interested in signal propagation within spines but wanted to explore possible changes in dendritic impulse propagation along dendrites by taking eﬀects of spine loss into consideration. Frontiers in Neuroscience \| www.frontiersin.org Initiation of Postsynaptic Potentials and Measures of Dendritic Signal Transfer Details of distribution and kinetics of various ion channels over somadendritic surface area of layer III pyramidal neurons of WT and Tg4510 mice are not available. Therefore, we restricted our investigations to passive membrane, and in our canonical membrane model, speciﬁc resistance and capacitance of the plasma membrane (transmembrane resistance and capacitance for the unit membrane area) were considered to be uniformly distributed over soma and dendritic compartments. To set speciﬁc membrane resistances (Rm) of neurons in the computer model, ﬁrst speciﬁc resistance was varied by hand in each WT and TG neuron model until the electrophysiologically determined mean somatic neuron resistance (197 ± 23 M for WT and Postsynaptic potentials were simulated by steady-state or sinusoidal (50 Hz) current injections to dendritic sites or by local dendritic conductance changes according to an alpha function. The alpha function has the following form: gsyn(t) = gmax · t/tpeak · exp(1 −t/tpeak) where gsyn and gmax are actual and maximum synaptic conductance and tpeak is the time when the conductance has its maximum value (gmax), t is time. In our simulations to mimic October 2021 \| Volume 15 \| Article 721773 Frontiers in Neuroscience \| www.frontiersin.org 4 Dendritic Signaling in Tauopathy Somogyi and Wolf TABLE 1 \| Summary of ﬁxed morphological and physiological data used as input parameters of the model and free parameters computed by ﬁtting procedures, as well as a set of simulation output data. current injections. In addition, 10–90% rise times and half-widths of somatic Postsynaptic Potentials (EPSPs) were also studied, whereas dendritic PSPs were simulated by local dendritic conductance changes. Voltage transfer was deﬁned as the ratio of amplitudes of somatic and dendritic PSPs measured during dendritic current injections. Current transfer was deﬁned as the fraction of electrical charge that reached the soma relative to the total charge injected at the dendritic site. Total delays associated with propagation of PSPs between dendritic points and the soma were measured as sum of time needed for injected current to develop a local dendritic PSP (local delay) and the time needed for this locally developing potential change to reach the soma (propagation delay). Local delay was quantiﬁed as the time delay between centroids of voltage–time and current–time curves at the site of current injection. Frontiers in Neuroscience \| www.frontiersin.org Electrotonic distances Initiation of Postsynaptic Potentials and Measures of Dendritic Signal Transfer Propagation delay was computed as the time diﬀerence between centroids of the voltage–time curves at the soma and at the dendritic injection site (Agmon-Snir and Segev, 1993; Zador et al., 1995). This way, total delay (delay from this point on), which is the sum of local and propagation delays, measures total time that elapses between synaptic current ﬂow at a working dendritic synapse and the development of somatic voltage response. Fixed parameters Free parameters Simulation output Name Value Source 3D dendritic trajectory and soma From individual data ﬁles Neuromor pho.org Speciﬁc membrane capacitance (µF/cm2) Transfers Spine density (1/µm) 1.25 (WT); 1.0 (TG) Crimins et al., 2012 Speciﬁc membrane resistance (·cm2) Rise times Spine surface area (µm2) 1.5 Larkman et al., 1992; Middei et al., 2008 Half-widths Time constant (ms) 32.5 ± 4.1 (WT); 35.2 ± 3.4 (TG) Rocher et al., 2010 Delays Input resistance (M) 197 ± 23 (WT); 228 ± 23 (TG) Rocher et al., 2010 Electrotonic distances Axial resistance (·cm) 150 Trevelyan and Jack, 2002; Kabaso et al., 2009 Resting membrane potential (mV) −75 (WT); −65 (TG) Rocher et al., 2010 Alpha synapse max conductance (nS) 0.25 Sarid et al., 2007 Peak time of alpha synapse conductance (ms) 0.5 Sarid et al., 2007 Integration time step (ms) 0.025 Hines and Carnevale, 1997, 2001 In order to compare dendritic impulse propagation in WT and TG neurons, two types of comparison graphs were created. (1) To assess transfers/delays of propagating PSPs and rise times/half- widths of somatic EPSPs in the function of site of PSP generation in WT and TG neurons, these descriptors of dendritic impulse propagation were graphed as a function of path distance of the site of PSP generation (as a function of synaptic loci) measured from the soma. (2) In addition, to assess relative weight of any given descriptor among the many location-dependent values, distributions of dendritic surface areas (a good approximation of distributions of synapses received by dendrites; see later) were graphed in the function of these descriptors of dendritic impulse propagation. To account for the diﬀerent degrees of morphological alterations seen in apical and basal arbors of TG neurons (Rocher et al., 2010; Crimins et al., 2012), the two types of comparison graphs were computed for apical and basal dendritic arbors separately in both WT and TG neurons. AMPAR-mediated single-synapse conductance changes gmax was 0.25 nS and tpeak was 0.5 ms (Sarid et al., 2007). Distribution of Dendritic Surface Area as a Function of Path Distance From the Soma Approximates Distribution of Excitatory Synapses Received by Dendrites of Wild-Type and Transgenic Pyramidal Neurons Based on observations that mutant human tau protein alters morphology of pyramidal neurons, we aimed to explore some of the possible diverse functional consequences of tau burden on pyramidal neurons. More speciﬁcally, we wanted to characterize features of subthreshold alterations of intradendritic signaling in pyramidal neurons of rTg4510 mice. Quantiﬁcation and comparison of dendritic impulse propagation were achieved by using comparison graphs. In these graphs, diﬀerent descriptors (transfers, delays of propagating dendritic PSPs and rise times, and half-widths of somatic EPSPs) of somatopetal dendritic signaling were depicted in function of path distance of the simulated synaptic site from the soma. To account for the many diﬀerent values of location-dependent descriptors, we also wanted to give weights to them, representing the fraction of dendritic synapses with similar descriptors. One way to estimate fraction of synapses with similar descriptors is to use the fraction of dendritic surface area from where PSPs have similar descriptor values as they travel toward the soma. To justify the use of this fraction of dendritic surface area for the approximation of dendritic synapses with similar descriptors, we addressed the question of how good this approximation can be. Based on this thorough analysis, we computed these two metrics in each WT and TG neuron. Then, we used these values as predictors to test if synaptic input pattern recognition/discrimination gets altered in TG neurons aﬀected by mutant tau. Mean and variance of electrotonic distances of synapses were estimated from electrotonic distances of dendritic injections sites (loci of simulated synapses) from the soma in each model neuron. To estimate mean electrotonic distance of synapses, electrotonic distances of midpoints of dendritic segments from the soma were averaged in each WT and TG neuron individually according to the following formula: 1/n 6 Li, where n is number of dendritic segments in the neuron, and Li is the electrotonic distance of the midpoint of the ith dendritic segments from the soma. Li = 6 (lk / λk), where lk is geometrical length of the kth segment on route between the soma and the midpoint of the ith segment, and λk is the space constant of the kth segment. Initiation of Postsynaptic Potentials and Measures of Dendritic Signal Transfer Thus, value of synaptic current (Isyn) is AMPAR-mediated single-synapse conductance changes gmax was 0.25 nS and tpeak was 0.5 ms (Sarid et al., 2007). Thus, value of synaptic current (Isyn) is To quantify the size overall alteration in each descriptor of dendritic impulse propagation in TG neurons, area weighed arithmetic means of the descriptors were also computed for each WT and TG neuron. The formula used in these calculations is 6Ai · Di / 6Ai, where Ai is the surface area of the ith dendritic segment and Di is the value of descriptor describing somatopetal propagation of the PSP when the PSP was initiated at midpoint of the ith segment, and 6Ai is the total surface area of dendrites in the subject neuron. Isyn(V, t) = gsyn(t) · (V(t) −Esyn) where Esyn and V are reversal potential, taken as 0 mV, and the membrane potential, respectively. Resting membrane potentials in simulations were −75 mV for WT, and −65 mV for TG neurons (Table 1; Rocher et al., 2010). where Esyn and V are reversal potential, taken as 0 mV, and the membrane potential, respectively. Resting membrane potentials in simulations were −75 mV for WT, and −65 mV for TG neurons (Table 1; Rocher et al., 2010). Finally, to test whether our major ﬁndings are independent of the natural within-group variations, both types of comparison graphs were created and analyzed on normalized scales as well. In these normalized graphs, distance dependence and weight of descriptors were analyzed over a normalized path distance scale and over normalized scale of descriptors, respectively. Normalized path distances and descriptors were measured as percentage of maximum path distances and percentage of maximum values of descriptors that occur in diﬀerent neurons. Distance between neighboring injection sites (modeled synaptic loci) was never farther than 37 µm or 0.2 space constant resulting in 83–270 injection sites per neuron, depending on size and arborization pattern of dendrites. Subthreshold somatopetal dendritic impulse propagation was studied and compared in models of TG and WT neurons by analyzing current transfers, steady-state and sinusoidal voltage transfers, and delays of dendritic PSPs, generated by local October 2021 \| Volume 15 \| Article 721773 Frontiers in Neuroscience \| www.frontiersin.org 5 Dendritic Signaling in Tauopathy Somogyi and Wolf Distribution of Dendritic Surface Area as a Function of Path Distance From the Soma Approximates Distribution of Excitatory Synapses Received by Dendrites of Wild-Type and Transgenic Pyramidal Neurons The space constant of the kth segment is λk = (dk · Rmk/4 · Ra)1/2, where dk and Rmk are the diameter and speciﬁc dendritic membrane resistance of the kth segment after correction for the spine surface area of that segment, and Ra is axial resistance. In neocortical pyramidal neurons, more than 90% of excitatory synapses is received by dendritic spines, and in the most cases, a single spine receives one synapse only (Nimchinsky et al., 2002). It was also shown that only 3.6% of spines do not receive synapses in mouse neocortex (Arellano et al., 2007). These observations suggest that distribution of spines could be a good estimate for distribution of excitatory synapses over the dendrites. As linear spine density (number of spines along the unit length of dendrite) is nearly constant in layer III pyramidal neurons of WT and rTg4510 mice (Rocher et al., 2010; Measures of Synaptic Input Pattern Recognition Capabilities in Wild-Type and Transgenic Neurons of electrotonic distances and their variances, speciﬁc membrane resistances, and capacitances of WT and TG neurons, Mann– Whitney test was used. Comparison graphs showing distance dependence of descriptors of propagating PSPs and dendritic surface areas with identical descriptors were compared by two-way analysis of variance (ANOVA) tests to reveal overall alterations in dendritic signaling in TG neurons. Bonferroni post hoc tests were used to identify path distance regions or intervals of descriptors where WT and TG neurons diﬀer. These post hoc tests were performed only in intervals with at least three data from both populations of neurons. Level of statistical signiﬁcance was 0.05 in all cases. Percentage of distance regions and descriptor intervals with statistically signiﬁcant diﬀerences between WT and TG neurons was used to quantify the degree of alterations in dendritic signaling. Synaptic input pattern recognition/diﬀerentiation capabilities in WT and TG neurons were compared by predictors of these capabilities based on a detailed study (de Sousa et al., 2015) involving hundreds of thousands of model neurons with diﬀerent morphologies and with passive and active membrane conductances. These authors used a Hebbian learning rule (use- dependent synaptic facilitation) in their computational model. Diﬀerent, randomly chosen sets of synapses were activated over dendrites of a postsynaptic neuron, and synapses that were activated multiple times during this “training phase” became stronger (their synaptic conductance was gradually increased). Following this “training phase,” a new “novel” pattern of synapses was activated, and the diﬀerence between recognition of “learnt” and “novel” patterns (synaptic input pattern discrimination) was quantiﬁed. This quantiﬁcation was based on the ratio of somatic EPSPs or, in case of active membranes, by the ratio of the number of spikes produced by the postsynaptic neuron in response to activation of the respective synaptic patterns. It was found (de Sousa et al., 2015) that synaptic input pattern recognition/discrimination was inversely proportional to two simple metrics, mean electrotonic distance of synapses, and within-cell variance of these electrotonic distances, in neurons with both passive and active dendritic membranes. Frontiers in Neuroscience \| www.frontiersin.org Signiﬁcance of Different Measures of Dendritic Impulse Propagation Steady-state voltage transfer measures the extent to which a dendritic synapse with relatively slow time course [e.g., those mediated by N-methyl-D-aspartate (NMDA) receptors] can inﬂuence membrane potential dynamics at the soma (at the nearby axon hillock). Synapses at loci with higher steady-state voltage transfers may have bigger inﬂuence in shaping somatic potential and therefore ﬁring activity of the postsynaptic neuron than synapses at loci with smaller transfer values (assuming other factors are equal). Based on these arguments, percentages of total dendritic surface area of neurons were used to estimate fractions of total number of synapses generating PSPs with similar descriptors. Passive Membrane Properties Remain Unaltered in Transgenic Neurons To set up segmental cable models of WT and TG neurons, we had to estimate resistance and capacitance of a unit area of neuronal membrane, which are currently not directly available from electrophysiological experiments. These estimates were carried out in anatomically faithful compartmental models of the neurons and were based on ﬁtting somatic DC neuron resistances and membrane time constants measured electrophysiologically (see section “Materials and Methods”). Estimated speciﬁc resistances and capacitances in TG neurons were proven to be identical to those in WT neurons (Mann–Whitney test, p = 0.267 and p = 0.083), which were not aﬀected by the mutant tau protein and used as control (Figure 4). Voltage transfers depend on the frequency of signal because of non-zero membrane capacitance. As a consequence of this, PSPs with short time constants (e.g., those mediated by AMPA receptors) attenuate diﬀerently than PSPs with slower kinetics. Therefore, we extended our steady-state voltage transfer analysis with computation of the 50-Hz sinusoid voltage transfer proﬁles. In some cases, predominantly when voltage perturbations are small, the amount of electrical charge reaching the soma predicts chances for ﬁring an action potential better than amplitude of somatic membrane potential (Jack et al., 1975). To account for this notion, current transfer was measured as fraction of electric charge reaching the soma relative to the total charge injected locally at the locus of modeled dendritic synapse. Other interpretation of this measure is based on equality between the somatopetal charge transfer from a dendritic point to the soma and somatofugal voltage transfer from the soma to the same dendritic point (Zador et al., 1995). This equality is held in any dendritic tree and is valid for any dendritic point. Therefore, somatopetal current transfer may be interpreted as a measure of eﬃciency of passive back-propagation of action potentials along the dendrites. Statistical Analysis Statistical tests were performed, and ﬁgures were plotted by using the Microsoft Oﬃce (Microsoft Corp.), PAST (Hammer et al., 2001), and SigmaPlot for Windows version 14 (Systat Software, Inc., San Jose, CA, United States) software. Identity of distributions of dendritic lengths and dendritic surface areas was tested by a Kolmogorov–Smirnov test. For pairwise comparisons October 2021 \| Volume 15 \| Article 721773 Frontiers in Neuroscience \| www.frontiersin.org 6 Dendritic Signaling in Tauopathy Somogyi and Wolf of synapses with bigger or smaller transfers (bigger or smaller ability to aﬀect the soma potential) gets signiﬁcantly altered in TG neurons, then this alteration is considered as tau-induced modiﬁcation in dendritic signaling relative to control, WT neurons. Idea behind a similar analysis of delays and rise times and half-widths of somatic EPSPs is the same. Timing and shape of somatic EPSPs can also aﬀect action potential generation. Thus, if either location dependence of these descriptors or fraction of synapses with bigger or smaller descriptors changes signiﬁcantly in TG neurons, then such a change is treated as an indication of an altered intrinsic signaling mechanism in the aﬀected neuron in response to toxic eﬀect of mutant tau protein. Both types of comparison graphs were created for apical and basal dendritic arbors separately and were graphed over normalized and absolute scales as well. Crimins et al., 2012), distribution of dendritic length provides a good estimate of distribution of spines and excitatory synapses as well. We compared the fraction of dendritic length and dendritic surface area as a function of path distance from the soma, and these distributions were statistically identical in WT and TG neurons (Figure 3, Kolmogorov–Smirnov test, p > 0.999). This means that the percentage of total dendritic surface area within a path distance range could be used for estimating percentage of total number of excitatory synapses received by the respective dendritic surface. Distribution of inhibitory synapses cannot be related directly to distribution of spines as they are usually received by dendritic shaft. However, the ratio of symmetrical to asymmetrical synapses was found to be nearly constant on dendritic shafts of diﬀerent regions of reconstructed neocortical dendrites in mouse, and the ratio of the total number of symmetrical and asymmetrical synapses was also constant (Hersch and White, 1982). Statistical Analysis Many other studies have also shown evidence for close interrelationship between the size of dendritic receptive surface, number of synapses, and number of spines (Colonnier, 1968; Feldman, 1975; Feldman and Dowd, 1975; Muller et al., 1984). Frontiers in Neuroscience \| www.frontiersin.org Rationale of Analysis of Dendritic Signaling of PSPs of certain local synapses get too long relative to delays of PSPs generated by other synapses, then these modiﬁcations may prevent delayed PSPs from summating eﬀectively with other simultaneously generated PSPs that arrive at the soma much earlier. If half-width of somatic PSPs gets increased, time window for eﬀective summation of multiple PSPs arriving at the soma will widen, and coincidence detection capability will worsen. Finally, longer rise times may postpone action potential FIGURE 3 \| Distributions of dendritic length (solid lines) and surface area (dotted lines) as a function of path distance measured from the soma. Distributions are statistically identical (Kolmogorov–Smirnov test, p > 0.999 both in apical (upper panels) and basal (lower panels) dendritic arbors of WT (left panels) and TG (right panels) neurons. FIGURE 3 \| Distributions of dendritic length (solid lines) and surface area (dotted lines) as a function of path distance measured from the soma. Distributions are statistically identical (Kolmogorov–Smirnov test, p > 0.999 both in apical (upper panels) and basal (lower panels) dendritic arbors of WT (left panels) and TG (right panels) neurons FIGURE 4 \| Estimated speciﬁc membrane resistances and capacitances of WT and TG neurons. Dot and box plots comparing speciﬁc membrane resistances (left panel) and capacitances (right panel) of WT (blue) and TG (red) neurons. None of these membrane parameters get altered signiﬁcantly by the mutant tau protein (Mann–Whitney test, p = 0.267 and p = 0.083 for resistances and capacitances, respectively). Lower and upper boundaries of the boxes indicate the 25th and 75th percentiles; a line within each box marks the median value; whiskers below and above the boxes show the 10th and 90th percentiles; lower and upper closed circles represent the 5th and 95th percentiles. FIGURE 4 \| Estimated speciﬁc membrane resistances and capacitances of WT and TG neurons. Dot and box plots comparing speciﬁc membrane resistances (left panel) and capacitances (right panel) of WT (blue) and TG (red) neurons. None of these membrane parameters get altered signiﬁcantly by the mutant tau protein (Mann–Whitney test, p = 0.267 and p = 0.083 for resistances and capacitances, respectively). Lower and upper boundaries of the boxes indicate the 25th and 75th percentiles; a line within each box marks the median value; whiskers below and above the boxes show the 10th and 90th percentiles; lower and upper closed circles represent the 5th and 95th percentiles. Rationale of Analysis of Dendritic Signaling g g Signiﬁcantly diverse tau-induced morphological alterations combined with the unaltered passive membrane properties in TG neurons led us to analyze possible functional consequences of observed morphological alterations on intraneuronal dendritic signaling. In this analysis, we utilized two types of comparison graphs: one type shows location dependence of propagation- related descriptors of locally generated dendritic PSPs, and the other type shows proportion of dendritic synapses (estimated as the proportion of total dendritic surface area) with similar values of descriptors of dendritic signaling. This latter type of comparison graph was meant to estimate the weight of any given value of a signaling property among the many diﬀerent location- dependent values. Rationale of this is that if either the location dependence of signal transfers along the dendrites or proportion Possible tau-induced alterations in delays of PSPs or in shape of somatic PSPs can also aﬀect summation of dendritic signals arriving at the cell body. Summation of PSPs arriving at the soma in response to a given pattern of simultaneous synaptic inputs to a neuron may be diﬀerent if delays of PSPs or the shape of summating somatic PSPs gets altered. In case delays October 2021 \| Volume 15 \| Article 721773 7 Dendritic Signaling in Tauopathy Somogyi and Wolf FIGURE 3 \| Distributions of dendritic length (solid lines) and surface area (dotted lines) as a function of path distance measured from the soma. Distributions are statistically identical (Kolmogorov–Smirnov test, p > 0.999 both in apical (upper panels) and basal (lower panels) dendritic arbors of WT (left panels) and TG (right panels) neurons. FIGURE 4 \| Estimated speciﬁc membrane resistances and capacitances of WT and TG neurons. Dot and box plots comparing speciﬁc membrane resistances (left panel) and capacitances (right panel) of WT (blue) and TG (red) neurons. None of these membrane parameters get altered signiﬁcantly by the mutant tau protein (Mann–Whitney test, p = 0.267 and p = 0.083 for resistances and capacitances, respectively). Lower and upper boundaries of the boxes indicate the 25th and 75th percentiles; a line within each box marks the median value; whiskers below and above the boxes show the 10th and 90th percentiles; lower and upper closed circles represent the 5th and 95th percentiles. Delays of Postsynaptic Potentials Both distance dependence of delays and distributions of dendritic surface areas showed statistically signiﬁcant overall alterations in TG neurons (Figure 8, two-way ANOVA, p < 0.001 and p = 0.004) except for the distribution of dendritic surface area along normalized scale. We detected a general and statistically signiﬁcant slowdown in dendritic signaling (increase in delays) both in apical and in basal dendritic arbors of TG neurons along normalized and absolute path distance scales too (two- way ANOVA, p < 0.001). A rearrangement of the distribution of dendritic surface area according to delays was also revealed along absolute scale of delays and this alteration was present in the apical and basal arbors as well (two-way ANOVA, p = 0.004 and p < 0.001). Such a rearrangement increased the fraction of dendritic surface area in TG neurons where locally generated PSPs are associated with longer delays during their propagation toward the cell body (Bonferroni test, p < 0.05). In other words, alteration in distribution of dendritic surface area in TG neurons showed a shift toward bigger delays, indicating presence of a bigger percentage of synapses at loci associated with longer delays of PSPs (Figures 8C,G). This led to a statistically signiﬁcant increase in area weighted average delays of PSPs both in apical and basal dendrites of TG neurons relative to control (40.6 ± 1.90 ms vs. 37.1 ± 0.45 ms in apical and 35.9 ± 0.75 ms vs. 33.8 ± 0.13 ms in basal dendrites, Mann–Whitney test, p = 0.003 and p < 0.001). Dendritic signaling was studied by analysis of eight comparison graphs for each transfer property (current transfer and steady- state and sinusoid voltage transfers) and for delays of PSPs. In case of current transfer, two of eight comparison graphs showed statistically signiﬁcant overall tau-induced alteration (Figure 5, two-way ANOVA, p = 0.012 and p < 0.001, location dependence in apical dendrites over normalized and absolute scales, respectively). For voltage transfers, there was only one comparison graph with statistically signiﬁcant overall alteration for steady-state signals (Figure 6) and one for sinusoidal signals (Figure 7, two-way ANOVA, p < 0.001 and p = 0.002 for location dependence of steady-state and sinusoid voltage transfers in basal dendrites). Delays of Postsynaptic Potentials All these comparison graphs on current and voltage transfers with statistically signiﬁcant overall diﬀerences between WT and TG neurons showed alterations in distance dependence of transfers, and no comparison graph showed statistically signiﬁcant overall alteration in the distribution of dendritic surface area (distribution of synapses) as a function of current or voltage transfer neither in apical nor in basal dendritic arbors (two-way ANOVA, p > 0.05). During analysis of comparison graphs with distance dependence of voltage and current transfers, Bonferroni post hoc tests detected only one or two distance regions per graph with signiﬁcantly altered transfers in TG neurons, indicating spatially restricted alterations in transfers. At distances where transfers in WT and TG neurons were found to be signiﬁcantly diﬀerent, the steady-state and sinusoid voltage transfers were always bigger, and current transfers were always smaller in TG neurons relative to control, regardless that absolute or normalized path distance scales were considered. Rationale of Analysis of Dendritic Signaling of PSPs of certain local synapses get too long relative to delays of PSPs generated by other synapses, then these modiﬁcations may prevent delayed PSPs from summating eﬀectively with other simultaneously generated PSPs that arrive at the soma much earlier. If half-width of somatic PSPs gets increased, time window for eﬀective summation of multiple PSPs arriving at the soma will widen, and coincidence detection capability will worsen. Finally, longer rise times may postpone action potential October 2021 \| Volume 15 \| Article 721773 Frontiers in Neuroscience \| www.frontiersin.org 8 Dendritic Signaling in Tauopathy Somogyi and Wolf WT neurons, p = 0.016, Mann–Whitney test). This better voltage transfer is in line with increased general excitability of neurons seen in mutant tau-TG animals (Rocher et al., 2010; Crimins et al., 2012) and during seizures in humans (Sanchez et al., 2018) with tauopathies. generation by delaying somatic membrane potential to reach the voltage threshold. generation by delaying somatic membrane potential to reach the voltage threshold. Rise Times and Half-Widths of Somatic Excitatory Postsynaptic Potential Rise times of simulated somatic EPSPs as a function of path distance showed statistically signiﬁcant overall alterations when PSPs started from apical but not from basal dendrites of TG neurons. This was the case both on relative and absolute scales of distances (Figures 9B,D, two-way ANOVA, p = 0.002 and p < 0.001). Rise times of somatic EPSPs were invariably longer in TG neurons regardless the distance region where dendritic PSP was initiated, but these changes reached statistically signiﬁcant levels (Bonferroni test, p < 0.024) in the 450- to 550-µm distance region only (Figure 9D). Distributions of dendritic surface area as a function of rise time did not get altered in TG neurons (two-way ANOVA, p > 0.05). For distributions of dendritic surface area as a function of transfers, ANOVA tests did not show any statistically signiﬁcant overall alteration in TG neurons (p > 0.05). However, Bonferroni post hoc tests revealed a reorganization of dendritic surface. In TG neurons, smaller fraction of dendritic surface area (smaller fraction of synapses) is associated with lower transfers, and bigger fraction is associated with higher transfers. Such a reorganization means that the average transfer value of multiple PSPs generated by localized synaptic activities may get bigger under the eﬀect of mutant human tau protein, potentially increasing the general excitability of TG neurons. This modiﬁcation in distribution of dendritic surface area (distribution of synapses) was consistently present both in apical and basal dendritic arbors and over normalized and absolute scales as well. Alteration was the most obvious in case of steady-state voltage transfers of basal dendritic arbors (Figures 6E,G), where such a rearrangement in distribution of dendritic receptive ﬁeld led to statistically signiﬁcantly bigger area weighted average steady-state voltage transfer in TG neurons (0.68 ± 0.01 vs. 0.65 ± 0.01 in TG vs. Analysis of half-widths of somatic EPSPs showed statistically signiﬁcant overall alterations only in the function of path distances of the site of PSP initiation (Figures 10B,F,H, two- way ANOVA, p = 0.014, p < 0.001, and p = 0.001, respectively), but distributions of dendritic surface areas as a function of half-widths exhibited no overall alteration (two-way ANOVA, p > 0.05). Frontiers in Neuroscience \| www.frontiersin.org Rise Times and Half-Widths of Somatic Excitatory Postsynaptic Potential Asterisks mark path distance regions and transfer intervals with statistically signiﬁcant differences between WT and TG neurons (Bonferroni post hoc test, p < 0.05). Distance regions and transfer intervals with less than 3–3 data points in TG-WT comparisons were omitted from statistical tests. dendritic arbors of TG neurons (Figures 10F,H). In addition, post hoc tests revealed a shift in distribution of dendritic surface areas toward longer half-widths in basal arbors of mutant tau-aﬀected neurons (Bonferroni test, p = 0.035 and p = 0.046; Figure 10G). to percentages taking the total number of intervals where statistical tests could be performed (there were at least 3–3 data points for WT and TG neurons) as 100%. These calculations were performed separately for comparison graphs with absolute and normalized scales and by combining results along both scales (Figure 11). Area weighed mean rise times and half-widths of somatic EPSPs increased for PSPs arriving from apical and basal arbors of TG neurons relative to WT control, but these increases never represented statistically signiﬁcant diﬀerence between WT and TG neurons (Mann–Whitney test, p > 0.05). A general observation shown by Figure 11 is diﬀerent vulnerability of apical and basal dendritic arbors to tau- induced alterations in dendritic signaling. The biggest contrast in vulnerabilities of apical and basal arbors was detected when mutant-tau–induced alterations of half-widths of somatic EPSPs were investigated. In this case, half-widths were signiﬁcantly altered in TG neurons relative to WT control only if PSPs started from the basal arbor, whereas no alteration was found when PSPs started from any region of apical arbors. Generally, basal arbors suﬀer from a higher degree of alterations in other descriptors of dendritic impulse propagation too (the only exception is current transfer and rise time of somatic EPSPs, whose alterations were the smallest among the descriptors). This is in line with morphological observation that basal dendritic arbors of pyramidal neurons get altered earlier during the course of tau-induced neurotoxicity than the apical arbors in rTg4510 mice (Crimins et al., 2012) suggesting diﬀerent degrees of vulnerabilities of the two dendritic arbors. Rise Times and Half-Widths of Somatic Excitatory Postsynaptic Potential Wherever Bonferroni post hoc test detected signiﬁcant alteration in lengths of half-widths of somatic EPSPs starting from a given distance region, this alteration was always a lengthening of the half-width when PSPs started from the basal October 2021 \| Volume 15 \| Article 721773 Frontiers in Neuroscience \| www.frontiersin.org 9 Dendritic Signaling in Tauopathy Somogyi and Wolf FIGURE 5 \| Alterations in current transfers of locally generated PSPs in advanced tauopathy. Comparison graphs showing alterations in dendritic impulse propagation of TG (red lines) neurons relative to control, WT neurons (blue lines). Two types of comparison graphs are presented: one of them shows percentages of dendritic surface area (an approximation of the fraction of dendritic synapses) with similar transfers of locally generated PSPs (A,C,E,G); the other one displays location dependence of the current transfer (B,D,F,H). Comparison graphs were computed both along absolute (right half) and normalized scales (left half), where path distances and transfers were measured as percentages of their maximum values. Apical (upper panels) and basal (lower panels) dendritic arbors were analyzed separately. Overall statistical evaluations of comparison graphs were made by two-way ANOVA tests. Signiﬁcance levels of these ANOVA tests were indicated where statistically signiﬁcant overall alteration in comparison graphs was found (elsewhere p > 0.05). Asterisks mark path distance regions and transfer intervals with statistically signiﬁcant differences between WT and TG neurons (Bonferroni post hoc test, p < 0.05). Distance regions and transfer intervals with less than 3–3 data points in TG-WT comparisons were omitted from statistical tests. FIGURE 5 \| Alterations in current transfers of locally generated PSPs in advanced tauopathy. Comparison graphs showing alterations in dendritic impulse propagation of TG (red lines) neurons relative to control, WT neurons (blue lines). Two types of comparison graphs are presented: one of them shows percentages of dendritic surface area (an approximation of the fraction of dendritic synapses) with similar transfers of locally generated PSPs (A,C,E,G); the other one displays location dependence of the current transfer (B,D,F,H). Comparison graphs were computed both along absolute (right half) and normalized scales (left half), where path distances and transfers were measured as percentages of their maximum values. Apical (upper panels) and basal (lower panels) dendritic arbors were analyzed separately. Overall statistical evaluations of comparison graphs were made by two-way ANOVA tests. Signiﬁcance levels of these ANOVA tests were indicated where statistically signiﬁcant overall alteration in comparison graphs was found (elsewhere p > 0.05). Frontiers in Neuroscience \| www.frontiersin.org Differential Mutant Human Tau-Induced Alterations in Dendritic Signaling of Neurons Vulnerabilities of layer III pyramidal neurons to diﬀerent, human mutant tau-induced alterations of dendritic impulse propagation were quantiﬁed and compared. This analysis was based on comparison graphs (Figures 5–10), where intervals with statistically signiﬁcant diﬀerences (Bonferroni test, p < 0.05) between descriptors of dendritic signaling of WT and TG neurons were marked by asterisks. We counted the number of intervals, where statistically signiﬁcant diﬀerence was detected between WT and TG cells in location dependence of a given descriptor plus in distribution of dendritic surface area as a function of that descriptor. These counts were then converted Regarding variable degrees of alterations in diﬀerent descriptors of dendritic signaling, the most widespread alteration was found October 2021 \| Volume 15 \| Article 721773 Frontiers in Neuroscience \| www.frontiersin.org Frontiers in Neuroscience \| www.frontiersin.org 10 Dendritic Signaling in Tauopathy Somogyi and Wolf d Wolf Dendritic Signaling in Tauopathy 6 \| Alterations in steady-state voltage transfers in advanced tauopathy. Comparison graphs showing alterations in steady-state voltage transfers of TG (red rons relative to control, WT neurons (blue lines). Overall statistical evaluations of comparison graphs were made by two-way ANOVA tests. Signiﬁcance hese ANOVA tests were indicated where statistically signiﬁcant overall alteration in comparison graphs was found (elsewhere p > 0.05). Asterisks mark nce regions and transfer intervals with statistically signiﬁcant differences between WT and TG neurons (Bonferroni post hoc test, p < 0.05). Distance nd transfer intervals with less than 3–3 data points in TG-WT comparisons were omitted from statistical tests. 7 \| Alterations in sinusoid voltage transfers in advanced tauopathy. Comparison graphs showing alterations in sinusoid voltage transfers of TG (red lines) elative to control, WT neurons (blue lines). Overall statistical evaluations of comparison graphs were made by two-way ANOVA tests. Signiﬁcance levels of OVA tests were indicated where statistically signiﬁcant overall alteration in comparison graphs was found (elsewhere p > 0.05). Asterisks mark path egions and transfer intervals with statistically signiﬁcant differences between WT and TG neurons (Bonferroni post hoc test, p < 0.05). Distance regions fer intervals with less than 3–3 data points in TG-WT comparisons were omitted from statistical tests. FIGURE 6 \| Alterations in steady-state voltage transfers in advanced tauopathy. Comparison graphs showing alterations in steady-state voltage transfers of TG (red lines) neurons relative to control, WT neurons (blue lines). Overall statistical evaluations of comparison graphs were made by two-way ANOVA tests. Differential Mutant Human Tau-Induced Alterations in Dendritic Signaling of Neurons Signiﬁcance levels of these ANOVA tests were indicated where statistically signiﬁcant overall alteration in comparison graphs was found (elsewhere p > 0.05). Asterisks mark path distance regions and transfer intervals with statistically signiﬁcant differences between WT and TG neurons (Bonferroni post hoc test, p < 0.05). Distance regions and transfer intervals with less than 3–3 data points in TG-WT comparisons were omitted from statistical tests. FIGURE 6 \| Alterations in steady-state voltage transfers in advanced tauopathy. Comparison graphs showing alterations in steady-state voltage transfers of TG (red lines) neurons relative to control, WT neurons (blue lines). Overall statistical evaluations of comparison graphs were made by two-way ANOVA tests. Signiﬁcance levels of these ANOVA tests were indicated where statistically signiﬁcant overall alteration in comparison graphs was found (elsewhere p > 0.05). Asterisks mark path distance regions and transfer intervals with statistically signiﬁcant differences between WT and TG neurons (Bonferroni post hoc test, p < 0.05). Distance regions and transfer intervals with less than 3–3 data points in TG-WT comparisons were omitted from statistical tests. FIGURE 7 \| Alterations in sinusoid voltage transfers in advanced tauopathy. Comparison graphs showing alterations in sinusoid voltage transfers of TG (red lines) neurons relative to control, WT neurons (blue lines). Overall statistical evaluations of comparison graphs were made by two-way ANOVA tests. Signiﬁcance levels of these ANOVA tests were indicated where statistically signiﬁcant overall alteration in comparison graphs was found (elsewhere p > 0.05). Asterisks mark path distance regions and transfer intervals with statistically signiﬁcant differences between WT and TG neurons (Bonferroni post hoc test, p < 0.05). Distance regions and transfer intervals with less than 3–3 data points in TG-WT comparisons were omitted from statistical tests. FIGURE 7 \| Alterations in sinusoid voltage transfers in advanced tauopathy. Comparison graphs showing alterations in sinusoid voltage transfers of TG (red lines) neurons relative to control, WT neurons (blue lines). Overall statistical evaluations of comparison graphs were made by two-way ANOVA tests. Signiﬁcance levels of these ANOVA tests were indicated where statistically signiﬁcant overall alteration in comparison graphs was found (elsewhere p > 0.05). Asterisks mark path distance regions and transfer intervals with statistically signiﬁcant differences between WT and TG neurons (Bonferroni post hoc test, p < 0.05). Distance regions and transfer intervals with less than 3–3 data points in TG-WT comparisons were omitted from statistical tests. Differential Mutant Human Tau-Induced Alterations in Dendritic Signaling of Neurons Outside this delay range, fractions of dendritic surface area quickly converge to zero in both TG and WT neurons. Overall statistical evaluations of comparison graphs were made by two-way ANOVA tests. Signiﬁcance levels of these ANOVA tests were indicated where statistically signiﬁcant overall alteration in comparison graphs was found (elsewhere p > 0.05). Asterisks mark path distance regions and transfer intervals with statistically signiﬁcant differences between WT and TG neurons (Bonferroni post hoc test, p < 0.05). Distance regions and transfer intervals with less than 3–3 data points in TG-WT comparisons were omitted from statistical tests. TG and WT neurons are red and blue lines, respectively. in delays of PSPs in TG neurons, whereas the degree of alterations was the most limited in current transfers and rise times. Steady state and sinusoidal voltage transfers were also diﬀerentially altered. Alterations were present in 2.5 times higher percentage of intervals in relation to steady-state than sinusoidal voltage transfers in basal dendritic arbors (33.3% vs. 13.3%). Diﬀerential alterations in steady-state and sinusoidal voltage transfers may reﬂect a relative shift or modulation in spread of NMDA- and non–NMDA receptor–mediated PSPs with slower and faster kinetics. Such a diﬀerential alteration is supported by direct experimental ﬁndings on diﬀerential modulation of glutamatergic signaling in neurodegenerative diseases (Di Lazzaro et al., 2003; Revett et al., 2013) and by the fact that memantine, approved to treat moderate and severe forms of AD, modulates disturbed glutamatergic system (Frost et al., 2015). systematically study trends in alterations of diﬀerent aspects of dendritic signaling mediated by the presence of mutant tau protein. However, neurons usually receive not a single, but multiple synaptic potentials, which overlap in time and summate on axon hillock near the cell body. The result of this summation determines ﬁring activity of postsynaptic neurons. Recognition and diﬀerentiation between activation patterns of dendritic synapses are a vital process in neuronal information processing, and it is also involved in learning and memory, which are known to be aﬀected in tauopathies. Therefore, we investigated possible alterations in levels of synaptic input pattern recognition capability in TG neurons related to simultaneous activation of multiple synapses. These estimates for levels of synaptic input recognition/discrimination were based on a thorough study by de Sousa et al. (2015). Differential Mutant Human Tau-Induced Alterations in Dendritic Signaling of Neurons This study, by using an extremely large number of model neurons with diﬀerent morphologies and with active and passive membranes, concluded that means and variances of electrotonic distances of synapses correlate strongly with level of synaptic input pattern recognition (see section Materials and Methods for a brief summary). Thus, in the next step, we investigated if these simple metrics undergo any changes in neurons of tau-TG mice to assess whether presence of mutant human tau protein leads to pathological alterations in synaptic input pattern recognition. All the aforementioned features of degrees of alterations showed qualitative and quantitative similarities along absolute and normalized scales, suggesting that our observations on alterations in subthreshold dendritic signaling are independent of the choice of scale in comparison graphs. Differential Mutant Human Tau-Induced Alterations in Dendritic Signaling of Neurons October 2021 \| Volume 15 \| Article 721773 11 Frontiers in Neuroscience \| www.frontiersin.org Dendritic Signaling in Tauopathy Somogyi and Wolf FIGURE 8 \| Alterations in delays of PSPs in advanced tauopathy. See legend for analog Figure 5 with the difference that instead of transfers; here time delays of individually generated PSPs were graphed. For better visibility, distribution of dendritic surface area over absolute scale of delays is shown for the 30–50-ms interval only, where statistically signiﬁcant alterations in TG neurons were detected. Outside this delay range, fractions of dendritic surface area quickly converge to zero in both TG and WT neurons. Overall statistical evaluations of comparison graphs were made by two-way ANOVA tests. Signiﬁcance levels of these ANOVA tests were indicated where statistically signiﬁcant overall alteration in comparison graphs was found (elsewhere p > 0.05). Asterisks mark path distance regions and transfer intervals with statistically signiﬁcant differences between WT and TG neurons (Bonferroni post hoc test, p < 0.05). Distance regions and transfer intervals with less than 3–3 data points in TG-WT comparisons were omitted from statistical tests. TG and WT neurons are red and blue lines, respectively. FIGURE 8 \| Alterations in delays of PSPs in advanced tauopathy. See legend for analog Figure 5 with the difference that instead of transfers; here time delays of individually generated PSPs were graphed. For better visibility, distribution of dendritic surface area over absolute scale of delays is shown for the 30–50-ms interval only, where statistically signiﬁcant alterations in TG neurons were detected. Outside this delay range, fractions of dendritic surface area quickly converge to zero in both TG and WT neurons. Overall statistical evaluations of comparison graphs were made by two-way ANOVA tests. Signiﬁcance levels of these ANOVA tests were indicated where statistically signiﬁcant overall alteration in comparison graphs was found (elsewhere p > 0.05). Asterisks mark path distance regions and transfer intervals with statistically signiﬁcant differences between WT and TG neurons (Bonferroni post hoc test, p < 0.05). Distance regions and transfer intervals with less G G FIGURE 8 \| Alterations in delays of PSPs in advanced tauopathy. See legend for analog Figure 5 with the difference that instead of transfers; here time delays of individually generated PSPs were graphed. For better visibility, distribution of dendritic surface area over absolute scale of delays is shown for the 30–50-ms interval only, where statistically signiﬁcant alterations in TG neurons were detected. Synaptic Input Pattern Recognition Is Preserved in Tauopathy In previous phases of our analysis, we explored various tau- related pathological alterations in dendritic propagation of locally generated single PSPs. This was useful because we could Analysis of means and variances of electrotonic distances between modeled synaptic sites and the cell body in WT and TG groups of neurons revealed no statistically signiﬁcant October 2021 \| Volume 15 \| Article 721773 Frontiers in Neuroscience \| www.frontiersin.org 12 Dendritic Signaling in Tauopathy Somogyi and Wolf FIGURE 9 \| Alterations in rise times of somatic EPSPs in advanced tauopathy. Dendritic PSPs were generated by local dendritic synaptic conductance changes at different distances from the soma, according to an alpha function with kinetic parameters corresponding to AMPAR-mediated single-synapse activities. For better visibility, distribution of dendritic surface area over absolute scale of rise times is shown up to rise times where at least 3–3 data points for WT (blue lines) and TG neurons (red lines) were present and statistical tests could be performed. Outside this range, percentages of dendritic surface areas converged to zero. Asterisks mark path distance regions where simulated PSPs, after traveling to the soma, led to statistically signiﬁcant rise times of somatic EPSPs in WT and TG neurons (Bonferroni post hoc test, p < 0.05). FIGURE 9 \| Alterations in rise times of somatic EPSPs in advanced tauopathy. Dendritic PSPs were generated by local dendritic synaptic conductance changes at different distances from the soma, according to an alpha function with kinetic parameters corresponding to AMPAR-mediated single-synapse activities. For better visibility, distribution of dendritic surface area over absolute scale of rise times is shown up to rise times where at least 3–3 data points for WT (blue lines) and TG neurons (red lines) were present and statistical tests could be performed. Outside this range, percentages of dendritic surface areas converged to zero. Asterisks mark path distance regions where simulated PSPs, after traveling to the soma, led to statistically signiﬁcant rise times of somatic EPSPs in WT and TG neurons Bonferroni post hoc test, p < 0.05). FIGURE 10 \| Alterations in half-widths of somatic EPSPs in advanced tauopathy. Half-widths of somatic EPSPs, in response to locally generated dendritic synaptic conductance changes at different distances from the soma, were compared in WT and TG neurons (blue and red lines, respectively). These conductance changes mimicked AMPAR-mediated single synaptic activities. Synaptic Input Pattern Recognition Is Preserved in Tauopathy Asterisks mark path distance and half-width intervals where simulated PSPs, after traveling to the soma, led to statistically signiﬁcant half-widths of somatic EPSPs in WT and TG neurons (Bonferroni post hoc test, p < 0.05). FIGURE 9 \| Alterations in rise times of somatic EPSPs in advanced tauopathy. Dendritic PSPs were generated by local dendritic synaptic conductance changes at different distances from the soma, according to an alpha function with kinetic parameters corresponding to AMPAR-mediated single-synapse activities. For better visibility, distribution of dendritic surface area over absolute scale of rise times is shown up to rise times where at least 3–3 data points for WT (blue lines) and TG neurons (red lines) were present and statistical tests could be performed. Outside this range, percentages of dendritic surface areas converged to zero. Asterisks mark path distance regions where simulated PSPs, after traveling to the soma, led to statistically signiﬁcant rise times of somatic EPSPs in WT and TG neurons (Bonferroni post hoc test, p < 0.05). FIGURE 10 \| Alterations in half-widths of somatic EPSPs in advanced tauopathy. Half-widths of somatic EPSPs, in response to locally generated dendritic synaptic conductance changes at different distances from the soma, were compared in WT and TG neurons (blue and red lines, respectively). These conductance changes mimicked AMPAR-mediated single synaptic activities. Asterisks mark path distance and half-width intervals where simulated PSPs, after traveling to the soma, led to statistically signiﬁcant half-widths of somatic EPSPs in WT and TG neurons (Bonferroni post hoc test, p < 0.05). FIGURE 10 \| Alterations in half-widths of somatic EPSPs in advanced tauopathy. Half-widths of somatic EPSPs, in response to locally generated dendritic synaptic conductance changes at different distances from the soma, were compared in WT and TG neurons (blue and red lines, respectively). These conductance changes mimicked AMPAR-mediated single synaptic activities. Asterisks mark path distance and half-width intervals where simulated PSPs, after traveling to the soma, led to statistically signiﬁcant half-widths of somatic EPSPs in WT and TG neurons (Bonferroni post hoc test, p < 0.05). October 2021 \| Volume 15 \| Article 721773 13 Frontiers in Neuroscience \| www.frontiersin.org Dendritic Signaling in Tauopathy Somogyi and Wolf FIGURE 11 \| Comparisons of mutant human tau-induced alterations in dendritic signaling. DISCUSSION We predicted unaltered synaptic input pattern recognition/discrimination in layer III pyramidal neurons of the tau-mutant animal. This conservation of synaptic input pattern recognition in TG neurons suggests virtually unaltered summation of multiple subthreshold PSPs. Our prediction on unaltered nature of this summation is also supported by more direct computational evidence, such as spatially limited and minor alterations in voltage/current transfers and in shapes of somatic EPSPs, and the nearly uniform, location-independent increase in delays of individual PSPs shown by our simulations. This way, the size of somatic signals and eﬀective time window for their summation, which are the key factors of synaptic integration, may remain generally unaﬀected, which may, in turn, lead to unaltered synaptic summation. Synaptic Input Pattern Recognition Is Preserved in Tauopathy Percentage of intervals where signiﬁcant alterations in dendritic signaling were detected in TG neurons along absolute and normalized scales of comparison graphs as well as grand summary combining alterations detected along both scales. Upper and lower extensions of bars show alterations in apical and basal dendritic arbors, respectively. Data shown here are based on comparison graphs presented in Figures 5–10. The total number of intervals with at least 3–3 data points for WT and TG neurons was taken as 100%. FIGURE 11 \| Comparisons of mutant human tau-induced alterations in dendritic signaling. Percentage of intervals where signiﬁcant alterations in dendritic signaling were detected in TG neurons along absolute and normalized scales of comparison graphs as well as grand summary combining alterations detected along both scales. Upper and lower extensions of bars show alterations in apical and basal dendritic arbors, respectively. Data shown here are based on comparison graphs presented in Figures 5–10. The total number of intervals with at least 3–3 data points for WT and TG neurons was taken as 100%. diﬀerence in these critical parameters (Mann–Whitney test, p > 0.161 for all cases). This ﬁnding suggests that synaptic input pattern recognition/discrimination remains unaltered in advanced tauopathy (Figure 12). and assuming unaltered axial resistance of the cytoplasm, any modiﬁcation in subthreshold dendritic impulse propagation must be entirely due to tau-induced morphological changes in these TG neurons (Figure 13). Frontiers in Neuroscience \| www.frontiersin.org October 2021 \| Volume 15 \| Article 721773 Unaltered Speciﬁc Membrane Resistance and Capacitance in rTg4510 Pyramidal Neurons Lower and upper boundaries of the boxes indicate the 25th and 75th percentiles; a line within each box marks the median value; whiskers below and above the boxes show the 10th and 90th percentiles; lower and upper closed circles represent the 5th and 95th percentiles. Statistical analysis showed no signiﬁcant (ns.) alteration in the predictors of synaptic input pattern recognition/discrimination capabilities of TG neurons (Mann–Whitney test, p > 0.161, p > 0.46, p > 0.68, and p > 0.93 for apical average and variance and basal average and variance of electrotonic distances, respectively). FIGURE 12 \| Synaptic input pattern recognition/discrimination is unchanged in advanced tauopathy. Means and variances of electronic distances of modeled dendritic synapses (injection sites) from the cell body were used to quantify and compare levels of synaptic input pattern recognition/discrimination capabilities in WT (blue), TG (red) neurons. Apical (upper panels) and basal (lower panels) dendritic arbors were analyzed separately. Lower and upper boundaries of the boxes indicate the 25th and 75th percentiles; a line within each box marks the median value; whiskers below and above the boxes show the 10th and 90th percentiles; lower and upper closed circles represent the 5th and 95th percentiles. Statistical analysis showed no signiﬁcant (ns.) alteration in the predictors of synaptic input pattern recognition/discrimination capabilities of TG neurons (Mann–Whitney test, p > 0.161, p > 0.46, p > 0.68, and p > 0.93 for apical average and variance and basal average and variance of electrotonic distances, respectively). and tau proteins, this may indicate that pathological network functions such as seizures and epileptiform activities seen in patients with AD (Amatniek et al., 2006; Lozsadi and Larner, 2006; Scarmeas et al., 2009) and in model animals of AD (Palop et al., 2007; Bezzina et al., 2015) are not primarily due to intrinsic alterations of signaling in neurons of aﬀected networks but linked to network-level pathological changes, for example, loss/rearrangement or modulation of synaptic connections, all of which have been described. predicted by our simulations is that a relatively bigger fraction of dendritic synapses has increased voltage transfers for their PSPs (Figures 6, 7), increasing the weighted arithmetic mean of steady-state voltage transfer rate of PSPs signiﬁcantly (i.e., reducing average attenuations of signals). Other area-weighed transfers remained unaltered in tau-mutant neurons. Although the increase in arithmetic mean of steady-state voltage transfers was statistically signiﬁcant, this alteration represented only a 5% rise relative to WT level. Unaltered Speciﬁc Membrane Resistance and Capacitance in rTg4510 Pyramidal Neurons When we were building computer models of WT and TG neurons with their reconstructed morphology, we estimated speciﬁc membrane resistances (Rm) and capacitances (Cm) of these model neurons by varying Rm values until electrophysiologically measured neuron resistance is reached and then by varying Cm until experimental membrane time constant was matched in the model neuron. These Rm and Cm values are therefore dependent on both reconstructed morphology and measured physiological properties of respective neurons. The mean speciﬁc membrane resistance and capacitance of model TG neurons showed no change relative to control. This ﬁnding has multiple important implications. In an earlier article, we had shown that synaptic input pattern recognition/discrimination did not get altered in layer III pyramidal neurons of the Tg2576 amyloid mouse model of AD either (Somogyi et al., 2016), where the human amyloid precursor protein with the Swedish mutation was overexpressed, leading to Aβ accumulation. These ﬁndings on the absence of alterations in synaptic input pattern recognition under the eﬀect of pathological Aβ and tau, which are two important proteins of many neurodegenerative diseases and both of which are present in AD, may have important message as to the future directions of research toward therapeutics: Once integration of synaptic signals do not get altered by the pathological Aβ First, to our best knowledge, this is the ﬁrst attempt so far to estimate the eﬀects of a human mutant tau protein on resistance and capacitance of neuronal membranes. Second, predicted unaltered leakage resistance of neuronal membrane is in agreement with the lack of tau-induced membrane permeabilization and channel formations at physiological pH values, reported earlier in a study combining direct biophysical measurements and atomic force microscopy in artiﬁcial membranes (Patel et al., 2015). Third, in the absence of tau-dependent alterations of passive membrane properties, October 2021 \| Volume 15 \| Article 721773 14 Dendritic Signaling in Tauopathy Somogyi and Wolf FIGURE 12 \| Synaptic input pattern recognition/discrimination is unchanged in advanced tauopathy. Means and variances of electronic distances of modeled dendritic synapses (injection sites) from the cell body were used to quantify and compare levels of synaptic input pattern recognition/discrimination capabilities in WT (blue), TG (red) neurons. Apical (upper panels) and basal (lower panels) dendritic arbors were analyzed separately. Unaltered Speciﬁc Membrane Resistance and Capacitance in rTg4510 Pyramidal Neurons Thus, the biological signiﬁcance of this increase alone may be small; however, it is in line with previous suggestions on tau-mediated hyperexcitability of neurons in mutant mice (Roberson et al., 2007; Ittner et al., 2010; Devos et al., 2013; Holth et al., 2013) and in humans with tauopathy (Amatniek et al., 2006; Vossel et al., 2013). All this makes the prediction on increased average transfers to be p Measuring Speciﬁc Membrane Capacitance and Resistance p Measuring Speciﬁc Membrane Capacitance and Resistance Some of the predictions of our simulation study might be tested experimentally. One of these testable predictions is the unaltered nature of the speciﬁc membrane capacitance and resistance in P301L tau-mutant pyramidal neurons. Gentet et al. (2000) developed a direct measurement method to determine speciﬁc membrane capacitance in a wide variety of neurons, including neocortical pyramidal cells in rodents. In this method, 300-µm- thick brain slices were used, and following compensation of Voltage and Current Transfers Our computations revealed spatially limited and relatively small alterations in distance dependence of subthreshold voltage and current transfers of propagating dendritic PSPs (Figures 5–7) in response to tau burden in mutant mice. Another alteration October 2021 \| Volume 15 \| Article 721773 Frontiers in Neuroscience \| www.frontiersin.org 15 Dendritic Signaling in Tauopathy Somogyi and Wolf FIGURE 13 \| Morphofunctional effects of human P301L mutant tau protein on layer III pyramidal neurons in the rTg4510 mouse model of advanced tauopathy. Mutation in tau protein leads to aggregation and formation of NFTs. Mutant tau protein detaches from microtubules leading to destabilization of the cytoskeleton and atrophy of dendrites. On the other hand, mutant tau protein does not alter passive membrane properties, such as speciﬁc membrane resistance and capacitance of neurons. Therefore, detected alterations in subthreshold dendritic signaling of individual PSPs are entirely due to alterations in morphology of tau-mutant neurons. Alterations in voltage and current transfers as well as in rise times and half-widths of somatic EPSPs are limited spatially to PSPs produced by only a fraction of synapses, and these alterations are relatively small in size. Time delays between synaptic activity and somatic voltage response show general signiﬁcant increase in TG neurons, and sizes of these increases are nearly independent of the dendritic location of synapses. Restricted alterations in transfers/rise times/half-widths and the uniform increase in delays of PSPs may leave synaptic integration unchanged, leading to unaltered input pattern recognition/discrimination in advanced tauopathy. WT neurons in blue, TG neurons in red colors, and neurons with both colors are in the process of getting altered. FIGURE 13 \| Morphofunctional effects of human P301L mutant tau protein on layer III pyramidal neurons in the rTg4510 mouse model of advanced tauopathy. Mutation in tau protein leads to aggregation and formation of NFTs. Mutant tau protein detaches from microtubules leading to destabilization of the cytoskeleton and atrophy of dendrites. On the other hand, mutant tau protein does not alter passive membrane properties, such as speciﬁc membrane resistance and capacitance of neurons. Therefore, detected alterations in subthreshold dendritic signaling of individual PSPs are entirely due to alterations in morphology of tau-mutant neurons. Alterations in voltage and current transfers as well as in rise times and half-widths of somatic EPSPs are limited spatially to PSPs produced by only a fraction of synapses, and these alterations are relatively small in size. Voltage and Current Transfers Time delays between synaptic activity and somatic voltage response show general signiﬁcant increase in TG neurons, and sizes of these increases are nearly independent of the dendritic location of synapses. Restricted alterations in transfers/rise times/half-widths and the uniform increase in delays of PSPs may leave synaptic integration unchanged, leading to unaltered input pattern recognition/discrimination in advanced tauopathy. WT neurons in blue, TG neurons in red colors, and neurons with both colors are in the process of getting altered. pipette capacitance in the cell-attached conﬁguration, nucleated patches were pulled from pyramidal neurons. These patches were clamped at −60 mV; −5 mV steps were applied, and capacitive current transients were recorded and averaged and then ﬁtted by a single exponential function with an added constant. Time constant and amplitude were used to estimate capacitance of the membrane patch. Surface area of the nucleated patch was measured from images captured by diﬀerential interference contrast infrared microscopy. Finally, speciﬁc membrane capacitance could be determined by dividing the capacitance of the membrane patch by its surface area. This method is potentially also suitable for measuring speciﬁc membrane resistance of neurons directly. By measuring and comparing membrane properties in WT and TG neurons with the above method, our prediction on unaltered passive membrane properties could be put into experimental test. partially responsible for increased excitability more feasible. This possibility is further supported by the consistency in direction of statistically signiﬁcant alterations in voltage transfers, which were, at all distances, increases rather than decreases in model TG neurons. In addition, P301L mutant tau expression has also been shown to increase glutamate release and reduce glutamate clearance in rTg4510 mice and therefore suggested to underlie neuronal hyperexcitability of neurons (Hunsberger et al., 2015). Our ﬁnding on increased eﬃciency of transfers of voltage transients from dendritic synaptic sites to the soma in the same animal model of tauopathy is another, previously unknown, factor that may contribute to the higher intrinsic excitability of tau-mutant neurons. Possible Experimental Paradigms to Conﬁrm the Hypotheses Generated by Our Computer Model Another, potentially suitable method to measure passive membrane properties of neurons was published by Wright et al. (1996). This is a white noise approach based on zero-mean Gaussian white noise current stimuli, which was successfully applied for estimating passive electrical properties of dentate granule cells in whole-cell patch conﬁguration. Possible Consequences of Mutant-Tau-Driven Alterations at Neuronal Circuitry Level Recording membrane potential dynamics by microelectrodes provide an excellent temporal resolution, but the number of recording sites (spatial resolution) is limited, and dendrites with small calibers remain largely inaccessible. However, new imaging techniques may provide a less invasive, suitable combination of spatial and temporal resolution to analyze membrane potential changes with high throughput at subcellular level in the foreseeable future. Currently, calcium imaging suﬀers from the problem of slow kinetic of calcium transients relative to the underlying membrane potential dynamics. This problem is further complicated by the diﬃculty of relating calcium transients to voltage changes as reviewed by Kulkarni and Miller (2017). Direct voltage imaging techniques have recently shown several new inventions to improve spatiotemporal resolution and brightness of the signal, including the use of novel ﬂuorescent proteins (Jin et al., 2012; St-Pierre et al., 2014), opsins (Hochbaum et al., 2014; Gong et al., 2015), second-harmonic generation (Reeve et al., 2013), and nanomaterials (Marshall and Schnitzer, 2013; Park et al., 2013). Some of these techniques may soon provide a way to measure voltage transfers and time delays of propagating subthreshold dendritic signals in ﬁner, more distal dendrites of neurons. Our simulations predicted subthreshold voltage and current transfers with only spatially localized, small-sized alterations and increased intraneuronal signal delays with very little location dependence in tau-mutant neurons. Statistically signiﬁcant alterations in rise times and half-width of somatic EPSPs were also rarely detected when the dendritic site of PSP generation was varied. These predictions combined with unaltered predictors of synaptic input pattern recognition/discrimination forecast unaltered synaptic integration. However, in reality, neurons are building blocks of neural networks with rich and diverse connection patterns. If the spatiotemporal pattern of synaptic input changes, it may lead to altered action potential output, even if integrative properties of the postsynaptic neuron are unchanged. Spatiotemporal activation pattern of synaptic input may change because of loss/alterations of synaptic connections and also because of the diﬀerent temporal pattern of synaptic activation of existing connections. Loss and alteration of synaptic contacts have been described; alteration in temporal pattern is likely in mutant-tau-protein–aﬀected cortical networks. On the other hand, intraneuronal signaling delays can add up in a series of coupled neurons causing a substantially delayed ﬁring in upstream neurons of networks. Probing Transfers and Delays of PSPs Currently, calcium imaging suﬀers from the problem of slow kinetic of calcium transients relative to the underlying membrane potential dynamics. This problem is further complicated by the diﬃculty of relating calcium transients to voltage changes as reviewed by Kulkarni and Miller (2017). Direct voltage imaging techniques have recently shown several new inventions to improve spatiotemporal resolution and brightness of the signal, including the use of novel ﬂuorescent proteins (Jin et al., 2012; St-Pierre et al., 2014), opsins (Hochbaum et al., 2014; Gong et al., 2015), second-harmonic generation (Reeve et al., 2013), and nanomaterials (Marshall and Schnitzer, 2013; Park et al., 2013). Some of these techniques may soon provide a way to measure voltage transfers and time delays of propagating subthreshold dendritic signals in ﬁner, more distal dendrites of neurons. From voltage–time responses recorded this way at dendritic sites and at the soma, while steady-state or sinusoidal current is injected to the dendrite, voltage transfers as a function of distance could be derived. In addition, by using the time course of the injected current, delays between somatic voltage response and current injection could be calculated, just like in our simulations. In another analysis, coincidence detection in pyramidal neurons was investigated by injecting computer-generated current trains simulating synaptic currents arriving from two input sources (Grande et al., 2004). Delay was systematically varied between the two simulated synaptic inputs, while whole- cell recordings were made from the soma to assess ﬁring rate as a function of phase delay between the two inputs. In these experiments, double dendritic electrodes with minimal distance separation should be applied to inject current independently and measure dendritic depolarization avoiding ﬁltering and adding an oﬀset during current injections (Williams and Stuart, 2002). In such experimental attempts several other special cautions must also be taken as summarized in a detailed review on dendritic patch clamp recordings by Davie et al. (2006). Similar comparative experiments on cortical slices from WT and TG mice could shed some light on the presence or absence of tau-induced alterations in synaptic integration. Possible Consequences of Mutant-Tau-Driven Alterations at Neuronal Circuitry Level In case of feedback or converging/diverging connections, such accumulation of delays in action potential generation may be diﬀerent in converging pathways and may therefore alter temporal pattern of synaptic inputs to downstream neurons. Such a mechanism may lead to devastating degree of alteration in the activity of the whole neural network, even if intraneuronal determinants of synaptic integration and spatial pattern of synaptic inputs remain unaltered. Frontiers in Neuroscience \| www.frontiersin.org Probing Transfers and Delays of PSPs Another prediction by our computational work is that slowdown of somatopetal signal propagation and voltage and current transfers of PSPs in dendrites of tau-mutant neurons remain October 2021 \| Volume 15 \| Article 721773 Frontiers in Neuroscience \| www.frontiersin.org 16 Dendritic Signaling in Tauopathy Somogyi and Wolf virtually unaltered. This prediction could be investigated experimentally by simultaneous somatic and dendritic recordings by patch electrodes, while current is injected at a dendritic location. Essentially, the experiments carried out by Williams and Stuart (2002) on neocortical neurons of brain slices could be repeated. They investigated attenuations of artiﬁcial EPSPs evoked by current injections through an electrode at various dendritic sites, whereas another somatic electrode recorded the somatic voltage response. neuron. Delay between the two sets of inputs was varied. During induction of synaptic inputs, recordings were made from the soma to record the output of the postsynaptic neuron. Simultaneous recordings from the soma and dendrites allowed estimation of the approximate location of the activated synapses. This way, the authors could study integration and coincidence detection of two sets of synaptic inputs to pyramidal neurons. In this analysis, the output was measured by mean spiking probability in the postsynaptic neuron. so at c vo tage espo se. From voltage–time responses recorded this way at dendritic sites and at the soma, while steady-state or sinusoidal current is injected to the dendrite, voltage transfers as a function of distance could be derived. In addition, by using the time course of the injected current, delays between somatic voltage response and current injection could be calculated, just like in our simulations. In these experiments, double dendritic electrodes with minimal distance separation should be applied to inject current independently and measure dendritic depolarization avoiding ﬁltering and adding an oﬀset during current injections (Williams and Stuart, 2002). In such experimental attempts several other special cautions must also be taken as summarized in a detailed review on dendritic patch clamp recordings by Davie et al. (2006). Recording membrane potential dynamics by microelectrodes provide an excellent temporal resolution, but the number of recording sites (spatial resolution) is limited, and dendrites with small calibers remain largely inaccessible. However, new imaging techniques may provide a less invasive, suitable combination of spatial and temporal resolution to analyze membrane potential changes with high throughput at subcellular level in the foreseeable future. Signiﬁcance of Subthreshold Membrane Models Models Distribution of voltage-dependent conductances in plasma membrane of layer III pyramidal neurons of the frontal lobe has not been studied in WT and rTg4510 mice in a comparative manner. Therefore, we had to restrict our analysis to subthreshold signaling. However, dendrites of neocortical pyramidal neurons are known to be endowed with voltage- dependent conductances, although most of these data are from layer V pyramidal neurons because they are easier to access experimentally because of their thicker dendrites. Layer III pyramidal neurons have been reported to have actively back-propagating action potentials and bear voltage- dependent sodium channels (Waters et al., 2003; Waters and Helmchen, 2006), whose activity is accompanied by inﬂux of calcium ions (Svoboda et al., 1999). Hyperpolarization-activated, cyclic nucleotide-gated channels are likely to have important contribution to signaling properties in pyramidal neurons of rTg4510 mice, as their activation is related to the signiﬁcantly increased sag potentials and excitability in TG neurons (Crimins et al., 2012). This is likely because somatic voltage changes, in response to current injections at dendritic sites, are aﬀected by Ih channels in layer V pyramidal neurons (Williams and Stuart, 2000). While this previous experimental work investigated the eﬀect of Ih channels on somatic response in healthy neurons, our simulations predicted alterations in dendritic signaling due to tau-driven dendritic atrophy but in the lack of Ih channels. Absence of voltage-dependent channels in our computer models is a clear limitation. Future computational studies on tau-mutant neurons should also consider the eﬀect of Ih and other voltage- dependent channels on dendritic signaling, once reliable data on these channels from TG neurons become available. g ( ) Our neuron models of rTg4510 neurons accounted for the loss of spines and other morphological alterations of dendrites but did not account for the anomalous diﬀusion in spiny dendrites. Spine loss alone, without considering other dendritic alterations and anomalous diﬀusion, is expected to speed up dendritic signaling toward the soma (Agmon-Snir and Segev, 1993). Reason for this is the decrease in dendritic surface area due to loss of spines that aﬀects eﬀective speciﬁc membrane resistances and capacitances in our cable models. The fact that our cable models of TG neurons, in the absence of anomalous diﬀusion, showed a slowdown of dendritic signal propagation suggests that tau- driven, but non–spine-related morphological alterations (e.g., in diameters or topology of dendrites, etc.) play a role in slowdown of signaling in rTg4510 pyramidal neurons. Testing Synaptic Integration Comparing synaptic input pattern recognition/discrimination in WT and TG neurons by direct laboratory experiments is diﬃcult. However, this ability of neurons is strongly interrelated with more testable features of synaptic integration, such as coincidence detection and eﬀect of input timing of subthreshold synaptic inputs on action potential output. Timing and precision of spike initiation in layer V pyramidal cells of the rat somatosensory cortex have been investigated in brain slices (Berger and Luscher, 2003). In this experiment, a pair of extracellular bipolar electrodes was used to induce two independent or partially overlapping sets of excitatory and inhibitory PSPs in the same pyramidal This mutant human tau-protein–induced toxic mechanism, which leads to delayed and/or completely degraded network function, is speculative but it is based on predictions October 2021 \| Volume 15 \| Article 721773 Frontiers in Neuroscience \| www.frontiersin.org 17 Dendritic Signaling in Tauopathy Somogyi and Wolf of healthy hippocampal CA3 pyramidal neurons in rats (Henze et al., 1996). In another study, by using passive, anatomically realistic compartmental models, Kabaso et al. (2009) computed voltage attenuations in neocortical layer II/III pyramidal neurons of young and old monkeys. These authors found that somatopetal and somatofugal voltage attenuations generally were reduced (voltage transfers were increased) because of morphological changes in older neurons. Major changes in voltage attenuations occurred in apical rather than in basal dendritic arbors. Increased voltage transfers in older neurons were concluded to potentially lead to their increased excitability, and these single-cell level alterations may contribute to age-related cognitive decline. In this context, it is interesting to note that we also found a statistically signiﬁcant increase in area-weighed mean steady-state voltage transfers in pyramidal neurons of tau-mutant mice, but unlike in aging, we found that in tauopathy voltage transfers were altered more in basal than in apical arbors. of our simulations and results in pathological network activity known to exist both in animals and humans with tauopathies. Relation to Previous Modeling Studies g One major prediction of our simulations is the slowdown of dendritic PSP propagation in TG neurons. This slowdown may have multiple reasons. Considering anomalous axial diﬀusion, a theoretical study also predicted a slowdown of PSP propagation along dendrites in neurons with reduced spine densities, which is characteristic in many neurodegenerative disorders, including Frontotemporal Dementia (FTD) (Henry et al., 2008). This study used fractional cable model to account for anomalous axial diﬀusion of ions and molecules in dendrites. Such diﬀusion is involved in electrochemical signaling of neurons; thus, alterations in this diﬀusion may have an impact on electrotonic and ﬁring properties of neurons and also on delays of PSPs propagating along dendrites (Langlands et al., 2009). Degree of this anomaly in axial diﬀusion is associated with the density/number/shape of spines, as spines trap and release molecules as revealed by experimental studies that visualized diﬀusion in spiny dendrites of hippocampal CA1 pyramidal neurons and Purkinje cells (Santamaria et al., 2006, 2011). Beyond spines, alterations in speed of signaling may also be caused by changes in other, non–spine-related morphological features of dendrites and also by changes in intracellular and extracellular diﬀusion due to deposition of amyloid plaques, a hallmark of AD (Mueggler et al., 2004; Banks and Fradin, 2005). Amyloid plaques do not appear in rTg4510 mice, but dendritic atrophy is well-documented at the age of 9 months (Rocher et al., 2010; Crimins et al., 2012). Finally, the importance of general dendritic morphology in determination of functional properties of pyramidal neurons is further emphasized by another model study that found dendritic diameters to aﬀect ﬁring rate of neurons more than active membrane properties in certain scenarios (Weaver and Wearne, 2008). Frontiers in Neuroscience \| www.frontiersin.org Signiﬁcance of Subthreshold Membrane Models As the net eﬀect of morphological alterations is an increase rather than a decrease of delays in our TG neuron models, and passive membrane properties are not aﬀected by mutant tau protein, this indicates that the increase in delays may be caused by non–spine-related morphological alterations, whose eﬀect overweighed the eﬀect of decreased spine density in TG neurons. Indeed, e.g., changes in diameters of dendrites can alter propagation the speed of signals (Agmon-Snir and Segev, 1993), and alterations in dendritic diameters of rTg4510 mice have been described (Rocher et al., 2010; Crimins et al., 2011). Regarding descriptors of dendritic signaling, other than delays, the eﬀects of morphology on amplitudes and rise times of somatic EPSPs have also been studied in computational models October 2021 \| Volume 15 \| Article 721773 Frontiers in Neuroscience \| www.frontiersin.org 18 Dendritic Signaling in Tauopathy Somogyi and Wolf distinct impacts on dendritic signaling, on the other hand, their synergistic eﬀects (Rapoport et al., 2002; Blurton-Jones and Laferia, 2006; Atsmon-Raz and Miller, 2015) on membranes and morphology cannot be excluded when they act simultaneously as in patients with AD. Probably the most relevant of these synergistic eﬀects to our study is that presence of Aβ peptide leads to increased phosphorylation and aggregation of tau protein and therefore facilitates tau pathology (Gotz et al., 2001; Lewis et al., 2001; Blurton-Jones and Laferia, 2006; He et al., 2018). Hence, one possible logical step in this series of computer simulation studies would be analysis of alterations in dendritic signaling properties under simultaneous eﬀects of Aβ and mutant tau proteins in neurons of double-TG animals. On the other hand, the possible importance of subthreshold depolarizations and their summation in layer III pyramidal neurons is emphasized by the sparse ﬁring activity combined with large ﬂuctuations in membrane potentials observed below the ﬁring threshold in layer III pyramidal neurons (Sakata and Harris, 2009; Gentet et al., 2010; Crochet et al., 2011). Finally, although we studied propagation of PSPs with passive membrane restriction, some of our results may be relevant to certain aspects of the dendritic signaling, when voltage- gated ion channels do open (active membrane). Our prediction on unaltered synaptic input pattern recognition/discrimination in TG neurons remains valid in case of active membrane as well. ETHICS STATEMENT Ethical review and approval was not required for the animal study because this study did not directly involve any human or animal subjects since our theoretical study was based on cellular morphological and electrophysiological data that are from the NeuroMorpho.org database and from published manuscript. Signiﬁcance of Subthreshold Membrane Models This is because the two metrics (mean and variance of electrotonic distances of synapses) we used to predict possible changes in synaptic input pattern recognition/discrimination had been shown to be valid predictors of synaptic input pattern recognition/discrimination in neurons with active membrane properties too (de Sousa et al., 2015). Further, regarding attenuation of passively back-propagating action potentials, somatopetal current transfers we calculated in passive membrane model are identical to values of somatofugal voltage transfers (Zador et al., 1995); hence, somatopetal current transfers are related to attenuation of back-propagating action potentials too. DATA AVAILABILITY STATEMENT Program codes are available upon reasonable request to corresponding author or via ModelDb database. FUNDING The authors acknowledge ﬁnancial support from the University of Debrecen, Hungary under the scheme of general research support. EW was grateful to former support by the Campus Hungary No: B2/2R/6825 of TÁMOP 4.2.4. B/2-11-1-2012-0001 and AS acknowledges former support by the TÁMOP 4.2.4. A/2- 11-1-2012-0001 National Excellence Program—Elaborating and operating an inland student and researcher personal support system convergence program. The project was subsidized by the European Union and co-ﬁnanced by the European Social Fund. AUTHOR CONTRIBUTIONS g g Somogyi et al. (2016) presented a similar analysis on how Aβ aﬀects neuronal membranes and dendritic signaling in Tg2576 amyloid mouse model of AD in the absence of mutant tau. This earlier and our recent analyses suggest the two key proteins to aﬀect layer III neocortical pyramidal neurons remarkably diﬀerently, leaving synaptic input pattern recognition unaltered in both cases. It was found that Aβ decreases membrane resistance and increases membrane capacitance (Somogyi et al., 2016), whereas in our current article, we report mutant tau protein not to change membrane resistance and capacitance in TG neurons. Aβ was shown to aﬀect the morphology and membrane properties of pyramidal neurons in a way that morphological and membrane alterations compensate each other’s pathological eﬀects and subthreshold dendritic signaling, and the input pattern recognition/discrimination remains virtually unaltered (Somogyi et al., 2016). Such a compensation of dendritic atrophy by parallel alterations in membrane properties is not possible in rTg4510 mice because of the unaltered nature of passive membrane properties. This means that, in the tau-TG animal, conservation of synaptic input pattern recognition at control level must be due to compensatory eﬀects among the various alterations in morphology (e.g., lengths, diameters, topology, spine densities) of dendrites. 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https://openalex.org/W3082820473	https://journal.uny.ac.id/index.php/pythagoras/article/download/32573/pdf	Indonesian	null	Analisis situasi didaktis dalam pembelajaran matematika berbantuan ICT pada siswa SMP	Pythagoras/Phythagoras	2,020	cc-by	7,399	https://doi.org/10.21831/pg.v15i1.32573 How to Cite: How to Cite: Prabowo, A., & Juandi, D. (2020). Analisis situasi didaktis dalam pembelajaran matematika berbantuan ICT pada siswa SMP. Pythagoras: Jurnal Pendidikan Matematika, 15(1), 1–12. https://doi.org/10.21831/pg.v15i1.32573 Available online at: http://journal.uny.ac.id/index.php/pythagoras PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020, 1-12 Available online at: http://journal.uny.ac.id/index.php/pythagoras PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020, 1-12 ABSTRACT Article History: Received: 19 June 2020 Revised: 06 July 2020 Accepted: 14 July 2020 Penelitian ini bertujuan untuk mengidentifikasi apakah mahasiswa calon guru matematika mampu membangun situasi didaktis matematis pada siswa SMP dalam pembelajaran mate- matika berbasis ICT (Information and Communications Technology). Penelitian ini adalah pene- litian deskriptif kualitatif. Subjek penelitian adalah mahasiswa calon guru yang telah praktik mengajar di sekolah. Subjek menggunakan aplikasi yang dibuat sendiri oleh subjek. Aplikasi yang dibuat berupa Augmented Reality (realitas bertumbuh), yaitu teknologi yang menggabungkan benda maya dua dimensi dan atau pun tiga dimensi ke dalam sebuah lingkungan nyata tiga dimensi lalu memproyeksikan benda-benda maya tersebut dalam waktu nyata. Hasil penelitian menunjukkan karakteristik situasi didaktis dalam pembelajaran matematika berbantuan ICT pada siswa SMP yang dibangun oleh mahasiswa calon guru adalah sebagai berikut: situasi didaktis matematis yang dibangun belum mengonstruksi pengetahuan siswa secara mandiri; situasi didaktis matematis yang dibangun cenderung berasal dari informasi yang disampaikan guru, bukan dari media ICT yang digunakan; dan ICT yang dilibatkan dalam pembelajaran belum mampu mengelaborasikan proses kognitif siswa dalam jejak pembelajarannya. Keywords: Keywords: Situasi didaktis, Calon guru matematika, Didactic situation, Prospective mathematics teacher, Information and Commu- nications Technology (ICT) Keywords: Situasi didaktis, Calon guru matematika, Didactic situation, Prospective mathematics teacher, Information and Commu- nications Technology (ICT) This study aims to identify whether prospective mathematics teacher students are able to build mathematical didactic situations in junior high school students in ICT-based mathematics learning. This research is descriptive qualitative research. The research subjects were prospective teachers who had practiced teaching at school. The subjects used applications made by themselves. Those applications were in the form of augmented reality (reality grows), namely technology that combines two-dimensional and or three-dimensional virtual objects into a real three-dimensional environment and then projecting these virtual objects in real-time. The results showed the characteristics of didactic situations in ICT-assisted mathematics learning in junior high school students built by prospective teacher students were as follows: didactic mathematical situations that were built had not constructed students' knowledge independently; the built mathematical didactic situation tends to originate from the information conveyed by the teacher, not from the ICT media used; and ICT involved in learning has not been able to elaborate the cognitive processes of students in their learning footprint. This is an open access article under the CC-BY-SA license This is an open access article under the CC-BY-SA license This is an open access article under the CC-BY-SA license How to Cite: Prabowo, A., & Juandi, D. (2020). Analisis situasi didaktis dalam pembelajaran matematika berbantuan ICT pada siswa SMP. Pythagoras: Jurnal Pendidikan Matematika, 15(1), 1–12. https://doi.org/10.21831/pg.v15i1.32573 isis situasi didaktis dalam pembelajaran matematika berbantuan ICT pada siswa SM Ardhi Prabowo 1 ᵠ * , Dadang Juandi 2 1 Jurusan Matematika, Universitas Negeri Semarang, Semarang, Indonesia 2 Departemen Pendidikan Matematika, Universitas Pendidikan Indonesia, Bandung, Indonesia ᵠ Saat ini sedang tugas belajar di Universitas Pendidikan Indonesia * Corresponding Author. E-mail: ardhiprabowo@mail.unnes.ac.id PENDAHULUAN Untuk mempelajari dan memahami konsep matematika, seorang siswa dapat membangun pengetahuan dalam pikirannya berdasarkan sebuah konsep pedagogi tingkat tinggi. Untuk mengetahui seberapa jauh konsep matematika tersebut dipahami, asesmen adalah salah satu alat yang dapat digunakan untuk mengetahui hal tersebut (Dubinsky, 2000). Salah satu hal yang dapat mendorong proses membangun dalam pikirannya adalah kemampuan siswa dalam melakukan analisis teoretis (Dubinsky, 2000). Dalam teori APOS (Actions, Processes, dan Objects serta mengorganisasikannya dalam Skema), pengetahuan matematika pada hakikatnya merupakan Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 2 Ardhi Prabowo, Dadang Juandi PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 2 Ardhi Prabowo, Dadang Juandi kecenderungan yang dimiliki oleh siswa untuk merespons situasi masalah matematis yang dihadapinya melalui refleksi atas masalah tersebut serta solusinya dalam suatu konteks sosial. Refleksi tersebut, menurut Dubinsky (2000), dilakukan melalui konstruksi aksi, proses, dan objek matematis serta mengorganisasikan hal tersebut dalam skema yang dapat digunakan dalam kaitannya dengan situasi masalah yang dihadapi. Dalam pendapat yang lain, pengetahuan matematika dapat dibangun dari berlatih dan melalui proses melakukan yang didasarkan pada aktivitas yang berhubungan dengan matematika (Harel, 2011). Aktivitas tersebut diperoleh dari sebuah kegiatan pembelajaran yang mendorong siswa untuk melakukan, mengamati, mengukur, menggambar, dan diakhiri dengan menyimpulkan (Fink, 2003). Aktivitas siswa tersebut merupakan wujud dari aksi mental siswa yang sedang melakukan proses belajar. Bagaimanapun cara siswa belajar, pendidikan matematika menekankan keterlibatan aktif peserta didik dalam membangun pengetahuan mereka sendiri (Fried, 2006; Watson & Mason, 2005). Jalan untuk mencapai pengetahuan (Ways of Understanding, WoU), proses berpikir (Ways of Thinking, WoT), dan awal mula pengetahuan digambarkan dalam siklus triadik sebagaimana tampak pada Gambar 1. Masalah: Bagaimana saya memahami materi X, dari situasi yang diberikan Guru? AKSI MENTAL: Menduga, menyimpulkan, memperkirakan, menyusun, membuktikan, dsb. Ways of Thinking: Dengan mencoret-coret dikertas buram, berdiam, merenung, menulis di papan tulis, mempraktikkan. Ways of Understanding: Pambuktian Aksioma, teorema, Laporan pengamatan, Gambar Geometris, dsb. mewarnai Memfasilitasi proses memperbaiki Sebagai input Menyelesaikan Masalah (Solusi) Saya sudah paham materi X menyelesaikan 1 2 3 4 5 Gambar 1. Siklus triadik belajar matematika (diadaptasi dari Suryadi, 2019) Masalah: Bagaimana saya memahami materi X, dari situasi yang diberikan Guru? AKSI MENTAL: Menduga, menyimpulkan, memperkirakan, menyusun, membuktikan, dsb. mewarnai memperbaiki Sebagai input 1 2 1 Masalah: Bagaimana saya memahami materi X, dari situasi yang diberikan Guru? 2 AKSI MENTAL: Menduga, menyimpulkan, memperkirakan, menyusun, membuktikan, dsb. PENDAHULUAN Selanjutnya mencari solusi atas masalah yang sedang dihadapi dilakukan dengan menggunakan kecerdasan, alat, dan menyelesaikan masalah di mana saja. Dengan demikian, anak akan belajar dari proses memecahkan masalah tersebut (Polya, 1962). Artinya, dimana pun dan kapan pun, masalah tetap akan ada. Siswa harus ditanamkan, bahwa memecahkan masalah dapat terjadi kapan pun dan dimana pun. Masalah yang harus dipecahkan, tidak harus selalu terkait dengan pelajaran di kelas. PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 3 Ardhi Prabowo, Dadang Juandi PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 3 Ardhi Prabowo, Dadang Juandi semakin banyak data atau informasi yang diterima atau diperoleh (Silver, 1994). Adapun dalam pendekatan penemuan, situasi didaktik dimulai dari sebuah proses enaktif. Proses enaktif adalah proses belajar dimana siswa diberi kesempatan dalam memanipulasi objek konkret secara langsung. Penemuan adalah sebuah hadiah atas proses tersebut (Bruner, 1961). Lebih lanjut, dalam pembelajaran berbasis masalah, situasi didaktis dimulai dengan menyajikan masalah kepada siswa untuk diselesaikan oleh siswa tersebut. Menyelesaikan masalah berarti mencari jalan keluar dari kesulitan, memulai dengan menghadapi hambatan, lalu mencapai sasaran yang tidak dapat dicapai dengan segera. Selanjutnya mencari solusi atas masalah yang sedang dihadapi dilakukan dengan menggunakan kecerdasan, alat, dan menyelesaikan masalah di mana saja. Dengan demikian, anak akan belajar dari proses memecahkan masalah tersebut (Polya, 1962). Artinya, dimana pun dan kapan pun, masalah tetap akan ada. Siswa harus ditanamkan, bahwa memecahkan masalah dapat terjadi kapan pun dan dimana pun. Masalah yang harus dipecahkan, tidak harus selalu terkait dengan pelajaran di kelas. Ada beberapa contoh situasi didaktis yang dapat ditemui dengan mudah di sekitar guru. Contoh yang paling mudah tentu situasi didaktis yang ditemukan di Buku Siswa, seperti yang telah tersaji pada Gambar 2. (a) (b) Gambar 2. Contoh situasi didaktis yang terdapat dalam (a) buku siswa (As’ari et al., 2017b) dan (b) buku lembar kerja (Prabowo & Ahmad, 2015) Gambar 2 menunjukkan bahwa situasi didaktis terdiri atas dua bagian, yaitu informasi dan pertanyaan. Bagian informasi berisi cerita yang dapat dilengkapi dengan gambar atau bahkan bisa berupa rangkaian gambar, PENDAHULUAN Sebagai input mewarnai memperbaiki 3 4 Ways of Thinking: Dengan mencoret-coret dikertas buram, berdiam, merenung, menulis di papan tulis, mempraktikkan. Ways of Understanding: Pambuktian Aksioma, teorema, Laporan pengamatan, Gambar Geometris, dsb. Memfasilitasi proses 5 Menyelesaikan Masalah (Solusi) Saya sudah paham materi X Gambar 1. Siklus triadik belajar matematika (diadaptasi dari Suryadi, 2019) Berdasarkan siklus yang tersaji pada Gambar 1, untuk belajar matematika, siswa menerima masalah, melakukan aksi mental, berpikir, dan mengonstruksi pengetahuan. Konstruksi belajar yang semacam ini meyakini bahwa belajar matematika itu bersifat perorangan. Hal ini berarti bahwa kemandirian belajar siswa adalah salah satu hal utama yang diperlukan untuk belajar matematika (Harel, 2011; Suryadi, 2019). Berdasarkan siklus belajar pada Gambar 1, langkah pada kotak 1 merupakan langkah yang sangat penting karena proses belajar seorang siswa berangkat dari masalah yang dibangun oleh guru. Kondisi sekitar, teks bacaan, lingkungan kelas, lembar kerja siswa, bahkan cerita guru bisa menjadi sumber masalah yang dapat dibangun untuk membangkitkan aksi mental siswa. Cerita, kondisi, dan situasi terkait matematika tersebut menghubungkan siswa dengan matematika. Hubungan tersebut disebut dengan hubungan didaktis (Suryadi, 2019). Situasi yang mem- bangkitkan aksi mental dalam hubungan didaktis siswa dan matematika inilah yang kemudian disebut dengan situasi didaktis matematis. Ada banyak cara untuk membangun situasi didaktis. Dalam pembelajaran inkuiri, situasi didaktis dalam pembelajaran matematika dapat dimulai dengan membangun rasa ingin tahu siswa (Risnanosanti, 2009). Pendekatan inkuiri dimulai dengan guru menyajikan suatu kejadian yang menimbulkan teka-teki, sehingga memo- tivasi siswa untuk mencari pemecahannya. Rasa ingin tahu dapat menarik siswa untuk belajar lebih mendalam tentang suatu konsep yang sedang dipelajarinya. Rasa ingin tahu juga lebih terbukti berhasil membentuk guru, sehingga pembelajaran matematika menjadi efektif (Husni, 2014). Semakin tinggi rasa ingin tahu seseorang, berarti Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 3 Ardhi Prabowo, Dadang Juandi semakin banyak data atau informasi yang diterima atau diperoleh (Silver, 1994). Adapun dalam pendekatan penemuan, situasi didaktik dimulai dari sebuah proses enaktif. Proses enaktif adalah proses belajar dimana siswa diberi kesempatan dalam memanipulasi objek konkret secara langsung. Penemuan adalah sebuah hadiah atas proses tersebut (Bruner, 1961). Lebih lanjut, dalam pembelajaran berbasis masalah, situasi didaktis dimulai dengan menyajikan masalah kepada siswa untuk diselesaikan oleh siswa tersebut. Menyelesaikan masalah berarti mencari jalan keluar dari kesulitan, memulai dengan menghadapi hambatan, lalu mencapai sasaran yang tidak dapat dicapai dengan segera. (b) Gambar 2. Contoh situasi didaktis yang terdapat dalam (a) buku siswa (As’ari et al., 2017b) dan (b) buku lembar kerja (Prabowo & Ahmad, 2015) Gambar 2 menunjukkan bahwa situasi didaktis terdiri atas dua bagian, yaitu informasi dan pertanyaan. Bagian informasi berisi cerita yang dapat dilengkapi dengan gambar atau bahkan bisa berupa rangkaian gambar, Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 4 Ardhi Prabowo, Dadang Juandi sedangkan bagian pertanyaan berisi dengan pertanyaan yang mendorong aksi mental siswa (berbingkai merah). Pertanyaan tersebut hanya bisa dijawab jika siswa menduga, melakukan, mengabstraksi, menyimpulkan, dan menemukan solusi. Pertanyaan selanjutnya adalah: “Bagaimanakah penerapannya? Apakah bisa dilaksanakan dalam pembelajaran kelompok? Bagaimana ICT (Information and Communications Technology) membantu siswa mengonstruksi pengetahuannya?” K t il b it i did kti t ti t b t bi l d i b b i h l G bi PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 4 Ardhi Prabowo, Dadang Juandi PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 4 Ardhi Prabowo, Dadang Juandi sedangkan bagian pertanyaan berisi dengan pertanyaan yang mendorong aksi mental siswa (berbingkai merah). Pertanyaan tersebut hanya bisa dijawab jika siswa menduga, melakukan, mengabstraksi, menyimpulkan, dan menemukan solusi. Pertanyaan selanjutnya adalah: “Bagaimanakah penerapannya? Apakah bisa dilaksanakan dalam pembelajaran kelompok? Bagaimana ICT (Information and Communications Technology) membantu siswa mengonstruksi pengetahuannya?” sedangkan bagian pertanyaan berisi dengan pertanyaan yang mendorong aksi mental siswa (berbingkai merah). Pertanyaan tersebut hanya bisa dijawab jika siswa menduga, melakukan, mengabstraksi, menyimpulkan, dan menemukan solusi. Pertanyaan selanjutnya adalah: “Bagaimanakah penerapannya? Apakah bisa dilaksanakan dalam pembelajaran kelompok? Bagaimana ICT (Information and Communications Technology) membantu siswa mengonstruksi pengetahuannya?” Keterampilan membangun situasi didaktis matematis tersebut bisa muncul dari berbagai hal. Guru bisa memperoleh dari pengalaman selama mengikuti pendidikan dan pelatihan, kegiatan rutin MGMP, atau bahkan sudah dikondisikan sejak perkuliahan. Dalam perkuliahan pendidikan, mahasiswa calon guru sudah mendapatkan pemahaman tentang telaah kurikulum, media, dan bahkan sudah mempraktikkan peer teaching bersama rekan- rekan mahasiswa lainnya (Cahyono et al., 2018; UNNES, 2019). Rangkaian perkuliahan tersebut seharusnya akan membentuk pengetahuan tentang bagaimana cara mengajar matematika ke siswa sekolah. Memang dalam perkuliahan lebih banyak terjadi dialektik daripada praktik. Meskipun demikian, bagi pembelajar dewasa (maha- siswa), cara tersebut, dengan memahami kurikulum prodi pendidikannya, akan mendorong mahasiswa untuk mencapai pengetahuan sebagaimana dirancang dalam kurikulum yang ada tersebut (Chaves, 2008). Jika siswa tidak mampu menerima informasi awal yang dibangun oleh guru, maka langkah pertama, yaitu mendorong aksi mental siswa, sudah tidak berjalan dengan baik. Yang terjadi kemudian adalah guru kembali menjelaskan makna dari masalah dan situasi yang dibangun. Siswa sebenarnya tidak lagi memerlukan transfer informasi dari guru, melainkan lebih melihat kepada matematika apa yang ada dalam kehidupan dan bagaimana mengaplikasikannya (Ernest, 1994). Hal itulah yang mendasari lahirnya matematika konstruktivis. Oleh sebab itu, kajian terhadap kemampuan guru dalam membangun situasi didaktis matematis, lingkungan belajar matematis, atau permasalahan sesuai dengan jejak pembelajaran siswa, dengan bantuan ICT menjadi penting untuk dilakukan. Kajian tentang calon guru memang selalu menarik. Dalam riset sebelumnya, calon guru menarik perhatian peneliti untuk dilihat aspek kemampuannya dalam mengenali bukti pemahaman konsep siswa (Bartell et al., 2013). Cara mahasiswa calon guru bertanya, berdasarkan pengalaman dan implementasinya dalam kurikulum juga sudah diteliti sebelumnya (Dawson, 2006). Pada intinya, proses mengajar di kelas sebenarnya adalah transformasi calon guru dari belajar menuju mengajar (Assis et al., 2018). Yang membedakan dengan penelitian sebelumnya adalah bahwa penelitian ini mengkaji situasi didaktis yang dibangun oleh calon guru khususnya yang dibangun melalui pemanfaatan ICT. Hal ini tentu menjadi suatu hal yang menarik untuk dikaji. Berdasarkan penjelasan tersebut, penelitian ini akan mengidentifikasi apakah mahasiswa calon guru matematika mampu membangun situasi didaktis matematis pada siswa SMP dalam pembelajaran matematika berbasis ICT. METODE Penelitian ini adalah penelitian deskriptif dengan pendekatan kualitatif. Secara khusus, penelitian ini ditujukan untuk mendeskripsikan fenomena situasi didaktis yang dibangun oleh mahasiswa calon guru dalam pembelajaran matematika SMP berbantuan ICT. Fenomena tersebut kemudian dikaji berdasarkan: (1) apakah situasi didaktis yang dibangun konstruktif? dan (2) apakah ICT yang digunakan dapat membantu mahasiswa calon guru dalam membangun situasi didaktis yang konstruktif? Untuk menyusun deskriptif kualitatif dari fokus kajian, maka penelitian ini dilaksanakan dalam tahapan yang terdiri atas: (1) menganalisis perangkat pembelajaran matematika yang digunakan termasuk buku pelajaran dan Lembar Kegiatan (LK) yang digunakan; (2) menganalisis ICT yang digunakan dalam pembelajaran; (3) melakukan konfirmasi dan verifikasi temuan dengan mewawancarai subjek penelitian; dan (4) menyusun deskriptif kualitatif hasil penelitian. Cara ini sudah pernah dilaksanakan dalam penelitian sebelumnya (misalnya Fadholi et al., 2015; Wahono & Budiarto, 2014), dan pada penelitian ini teknik pelaksanaannya dimodifikasi untuk menyesuaikan dengan tujuan yang ditetapkan dalam penelitian ini. Dalam rangkaian besar studi didaktis, analisis situasi didaktis ini sebenarnya dilakukan dalam rangka meyakinkan peneliti apakah ada hambatan dalam perkuliahan di Lembaga Pendidikan Tenaga Kependidikan (LPTK). Model penemuan hambatan ini juga pernah dilakukan dalam penelitian didaktis sebelumnya (misalnya Romdhani & Suryadi, 2017; Sulistiawati et al., 2015; Yunarti, 2014). Penelitian ini dilakukan mulai dari bulan Februari sampai dengan bulan Juni 2020. Rangkaian penelitian dimulai dengan penemuan masalah, penentuan data dan sumber data, pengumpulan data, analisis data, serta diakhiri dengan penyusunan laporan penelitian dan artikel hasil penelitian. Penelitian dilakukan di Jurusan Mate- matika FMIPA Universitas Negeri Semarang. Pengambilan data dilaksanakan pada rentang tanggal 24 Februari sampai dengan 6 Maret 2020. Data yang terkumpul berupa dokumen perangkat pembelajaran yang meliputi Buku Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 5 Ardhi Prabowo, Dadang Juandi Guru, Rencana Pelaksanaan Pembelajaran (RPP), Lembar Kerja Peserta Didik (LKPD), dan media pembelajaran; dokumen hasil wawancara; dan artikel hasil penelitian yang relevan. Guru, Rencana Pelaksanaan Pembelajaran (RPP), Lembar Kerja Peserta Didik (LKPD), dan media pembelajaran; dokumen hasil wawancara; dan artikel hasil penelitian yang relevan. Subjek penelitian ini adalah tiga orang mahasiswa calon guru dengan kode S1, S2, dan S3. Selanjutnya, dalam pembahasan, yang dimaksud dengan guru dalam konteks ini adalah mahasiswa calon guru yang telah melak- sanakan praktik mengajar di kelas. METODE Subjek S1 telah melakukan pembelajaran di SMP untuk proses pengambilan data skripsi, subjek S2 sedang proses bimbingan dengan dosen dan guru pamong untuk proses mengajar di sekolah dalam rangka penyusunan skripsi, dan subjek S3 telah selesai melaksanakan tugas PPL di SMP. Subjek S1 dan S2 mengemas pembelajaran matematika berbantuan ICT berupa aplikasi android yang memanfaatkan augmented reality, sedangkan subjek S3 mengemas pembelajaran matematika dengan bantuan aplikasi presentation slide. Ketiga subjek penelitian ini dipilih karena memenuhi syarat sebagai berikut: (1) telah berpengalaman menga- jar di sekolah, khususnya di tingkat SMP baik dalam kegiatan Praktik Pengalaman Lapangan (PPL) maupun pengam- bilan data untuk skripsi; (2) mudah diajak berkomunikasi; (3) terbuka dan tidak ragu mengakui kesalahan; (4) berani mempertahankan pendapat yang dianggap benar berdasarkan referensi yang cukup; dan yang paling utama (5) perangkat pembelajaran yang dimiliki sudah lengkap dan telah diimplementasikan di sekolah. Untuk memaknai data yang terkumpul, peneliti membandingkan antara seluruh dokumen perangkat pem- belajaran, buku guru, Lembar Kerja Peserta Didik (LKPD), dengan artikel penelitian yang relevan. Hasil komparasi perangkat dengan penelitian yang relevan tersebut kemudian dikonfirmasi dengan subjek penelitian. Jika subjek memenuhi syarat dari peneliti, maka hasil konfirmasi disusun dalam bentuk narasi kualitatif. Jika tidak memenuhi, maka data akan direduksi. Hasil pemaknaan akan memunculkan kriteria, menemukan hambatan, atau menguat- kan penelitian sebelumnya. Reduksi data Tahap awal penelitian ini memfokuskan kepada tahapan reduksi data. Wawancara kepada subjek penelitian telah dilakukan pada 26 Februari dan 4 Maret 2020 (lihat Prabowo, 2020). Wawancara dilakukan di Laboratorium Matematika UNNES. Pernyataan yang diperoleh di luar waktu wawancara merupakan informasi tambahan yang dapat digunakan untuk memperkuat temuan penelitian. Dari tiga subjek penelitian, diperoleh fakta bahwa baik S1, S2, dan S3, ketiganya telah memahami tujuan pembelajaran matematika di sekolah dan telah melaksanakan praktik mengajar dengan siswa SMP dalam kegiatan PPL. Ketiga subjek telah memperoleh mata kuliah terkait media pembelajaran dan dasar-dasar proses pembelajaran matematika. Dengan demikian, ketiga subjek diyakini telah memiliki dasar pengetahuan terkait pembelajaran dan media pembelajaran dalam bentuk ICT. Setelah dilakukan wawancara, peneliti memutuskan hanya menggunakan data yang berasal dari subjek S1 dan S3 saja, dengan pertimbangan subjek S2 telah membuat aplikasi berbasis augmented reality, namun belum diimplementasikan dalam pembelajaran di kelas. Sesuai jadwal, subjek S2 akan melaksanakan praktik mengajar di kelas untuk pengambilan data mulai awal Maret hingga April. Akan tetapi, adanya pandemi Covid-19 memaksa subjek S2 menunda praktik pengambilan data. Berdasarkan kriteria subjek penelitian butir ke-5, berarti subjek S2 tidak memenuhi syarat. ICT hanya berperan membantu instruksi guru agar tidak cepat hilang dalam memori siswa. Dalam pembelajaran yang biasa dilakukan, instruksi dan petunjuk kepada siswa dilakukan secara lisan. ICT yang dilakukan S2 hanya bersifat instruktif saja. Teknik reduksi data semacam ini juga dilakukan dalam beberapa penelitian sebelumnya (misalnya Sulistiawati et al., 2015; Yunarti, 2014). Situasi didaktis matematis berdasarkan perangkat pembelajaran Ada dua hal yang menjadi fokus peneliti dalam menganalisis perangkat pembelajaran dari dua subjek riset, yaitu: (1) melihat apakah situasi didaktis yang direncanakan sudah bersifat konstruktif dan (2) melihat sejauh mana ICT dapat membantu guru dalam membangun situasi didaktis matematis dalam perencanaan pembelajarannya. Dari dua perangkat pembelajaran yang dihasilkan oleh subjek S1 dan S3, dapat disimpulkan bahwa situasi didaktis matematis yang dibangun oleh kedua subjek tersebut tidak konstruktif. Hal ini tampak dalam perangkat RPP yang dilengkapi dengan LKPD sebagaimana tersaji pada Tabel 1. Tabel 1. Cuplikan RPP dan LKPD yang dikembangkan oleh subjek S3 Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 6 Ardhi Prabowo, Dadang Juandi Cuplikan LKPD Cuplikan RPP Peserta didik diberi motivasi atau rangsangan untuk memusatkan perhatian pada topik materi Posisi Titik terhadap Titik Asal (0,0) dengan cara: A. Melihat tayangan PPT. A. Melihat tayangan PPT. B. Mengamati (1) Lembar kerja materi Posisi Titik Terhadap Titik Asal (0,0) dan (2) Pemberian contoh-contoh materi Posisi Titik Terhadap Titik Asal (0,0) untuk dapat dikembangkan peserta didik, dari media interaktif, dsb. C. Membaca. D. Menulis. Menulis resume dari hasil pengamatan dan bacaan terkait Posisi Titik Terhadap Titik Asal (0,0). E. Mendengar. Pemberian materi Posisi Titik terhadap Titik Asal (0,0) oleh guru. F. Menyimak. Penjelasan pengantar kegiatan secara garis besar/global tentang materi pelajaran. Tabel 1 menunjukkan cuplikan kegiatan inti dalam pembelajaran materi tempat kedudukan yang disusun oleh subjek S3. Pada Tabel 1 tampak bahwa langkah E dan F pada cuplikan RPP menunjukkan langkah yang tidak produktif. Siswa hanya mendengar dan menyimak penjelasan guru. Langkah tersebut tidak mendorong keman- dirian siswa untuk mengonstruksi pengetahuannya. Langkah B tampak mendorong siswa untuk bekerja dengan LKPD. Namun demikian, jika diperhatikan pada cuplikan LKPD yang ada, maka tampak bahwa guru memberi contoh dan lalu siswa mengerjakan hal yang sama. Aktivitas mental yang terjadi hanyalah mengamati dan menduga bahwa isian di bawahnya adalah sama, namun tidak mengonstruksi pengetahuan. Siswa menduplikasi atau meniru dari peragaan yang disajikan oleh guru. Subjek S1 dalam merencanakan pembelajaran lebih jelas dan lebih tampak aktivitas siswa yang harus dilakukan. Hal ini tampak pada Fase 1 dalam kegiatan inti pada cuplikan RPP yang disajikan pada Tabel 2. Tabel 2. Situasi didaktis matematis berdasarkan perangkat pembelajaran Cuplikan RPP dan LKPD oleh subjek S1 Cuplikan RPP Cuplikan LKPD Fase 1 : Observasi untuk menemukan masalah (10 Menit) 1) Peserta didik dibagikan LKPD Kubus dan Balok 2) Setiap kelompok membuka aplikasi augmented reality mengguna- kan smartphone 3) Guru menginstruksikan peserta didik untuk mengisi LKPD yang telah dibagikan sesuai dengan petunjuk di aplikasi. 4) Peserta didik mencermati instruksi dan pertanyaan-pertanyaan yang disajikan di LKPD (mengamati). 5) Peserta didik mengerjakan LKPD untuk mengetahui unsur-unsur luas permukaan dan volume kubus dan balok. Subjek S1 dalam menyusun rencana pembelajaran sudah berfokus pada siswa. Pada fase 1 (lihat Tabel 2), tampak jelas aktivitas siswa dalam pembelajaran. Proses berpikir siswa juga tampak meningkat dari setiap tahapan belajar. Proses mengonstruksi pengetahuan tampak pada tahap kelima. Akan tetapi, ketika dikonfirmasi dengan LKPD, langkah aktivitas siswa tampak bertentangan dengan prinsip konstruktif. Siswa tidak menemukan dan mengon- struksi apa yang dimaksud dengan unsur-unsur bangun ruang, melainkan lebih kepada mengingat apa yang sudah dipahami. Tabel 1 dan Tabel 2 menunjukkan bahwa situasi didaktis matematis yang dibangun, berdasarkan RPP dan LKPDyangdisusunolehguru masihbelummendorongkonstruksipengetahuansiswa Siswacenderungmengikuti Tabel 2. Cuplikan RPP dan LKPD oleh subjek S1 Subjek S1 dalam menyusun rencana pembelajaran sudah berfokus pada siswa. Pada fase 1 (lihat Tabel 2), tampak jelas aktivitas siswa dalam pembelajaran. Proses berpikir siswa juga tampak meningkat dari setiap tahapan belajar. Proses mengonstruksi pengetahuan tampak pada tahap kelima. Akan tetapi, ketika dikonfirmasi dengan LKPD, langkah aktivitas siswa tampak bertentangan dengan prinsip konstruktif. Siswa tidak menemukan dan mengon- struksi apa yang dimaksud dengan unsur-unsur bangun ruang, melainkan lebih kepada mengingat apa yang sudah dipahami. Subjek S1 dalam menyusun rencana pembelajaran sudah berfokus pada siswa. Pada fase 1 (lihat Tabel 2), tampak jelas aktivitas siswa dalam pembelajaran. Proses berpikir siswa juga tampak meningkat dari setiap tahapan belajar. Proses mengonstruksi pengetahuan tampak pada tahap kelima. Akan tetapi, ketika dikonfirmasi dengan LKPD, langkah aktivitas siswa tampak bertentangan dengan prinsip konstruktif. Siswa tidak menemukan dan mengon- struksi apa yang dimaksud dengan unsur-unsur bangun ruang, melainkan lebih kepada mengingat apa yang sudah dipahami. Subjek S1 dalam menyusun rencana pembelajaran sudah berfokus pada siswa. Pada fase 1 (lihat Tabel 2), tampak jelas aktivitas siswa dalam pembelajaran. Proses berpikir siswa juga tampak meningkat dari setiap tahapan belajar. Proses mengonstruksi pengetahuan tampak pada tahap kelima. Akan tetapi, ketika dikonfirmasi dengan LKPD, langkah aktivitas siswa tampak bertentangan dengan prinsip konstruktif. Situasi didaktis matematis berdasarkan perangkat pembelajaran Siswa tidak menemukan dan mengon- struksi apa yang dimaksud dengan unsur-unsur bangun ruang, melainkan lebih kepada mengingat apa yang sudah dipahami. Tabel 1 dan Tabel 2 menunjukkan bahwa situasi didaktis matematis yang dibangun, berdasarkan RPP dan LKPD yang disusun oleh guru, masih belum mendorong konstruksi pengetahuan siswa. Siswa cenderung mengikuti apa yang dilakukan guru dan belum didorong untuk mengonstruksi pengetahuannya secara mandiri, sebagaimana contoh LKPD pada Gambar 2. Kemampuan membangun situasi didaktis ini sebenarnya telah dilatihkan dalam Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 7 Ardhi Prabowo, Dadang Juandi perkuliahan, namun temuan di lapangan menunjukkan adanya hambatan belajar dalam mengaplikasikan apa yang telah dilatihkan selama perkuliahan. Hasil konfirmasi (lihat Prabowo, 2020) dengan subjek S1 berhasil menemukan fakta bahwa dalam menyusun perencanaan pembelajaran, S1 menggunakan Buku Guru untuk mata pelajaran matematika yang disusun oleh As’ari et al. (2017a). Pada materi tersebut, penyusun buku memulai dengan membangun konsep tentang bangun ruang dan kumpulan susunan apakah yang membentuk suatu bangun ruang. Jika mengikuti buku guru, pembelajaran yang dilakukan oleh subjek S1 akan sangat menarik karena berangkat dari hal yang paling sederhana, yaitu jaring-jaring. Akan tetapi, subjek S1 lebih memilih memulai materi dengan meminta siswa untuk mengingat kembali unsur-unsur bangun ruang. Adapun proses pembelajaran yang dilakukan oleh subjek S3 dengan cara mengulang-ulang proses berpikir siswa pada level yang sama merupakan repetisi bukan iterasi. Repetisi tersebut diharapkan akan membentuk perilaku yang berbeda dari anak sebagai tanda telah belajar. Subjek S3 berharap bahwa ketika anak bertemu dengan pertanyaan serupa, maka ia akan langsung bisa menjawab. Kelemahan dari teknik repetisi atau mengulang ini adalah siswa kebingungan ketika bertemu dengan masalah yang berbeda dari contoh yang diberikan oleh guru. Dari hasil konfirmasi ditemukan fakta bahwa subjek S3 dalam menyusun perangkat dibantu oleh perangkat pembelajaran yang telah dihasilkan oleh kakak tingkat. ICT yang digunakan dalam pembelajaran Subjek S1 dan S3, keduanya telah memanfaatkan teknologi dalam pelaksanaan pembelajaran di kelas. Subjek S1 menggunakan aplikasi augmented reality, sedangkan subjek S3 menggunakan aplikasi presentation slide. Aplikasi yang digunakan oleh subjek S1 merupakan aplikasi berbasis android yang memanfaatkan teknologi augmented reality, yaitu menyajikan bentuk nyata dalam tayangan layar HP. Prinsip kerjanya, aplikasi yang dibuat oleh S1 jika diarahkan ke barcode tertentu akan memindai barcode tersebut yang telah diisi data tentang bangun ruang sisi datar. Data tersebut menayangkan bangun ruang bentuk digital yang dapat dimanipulasi dengan menggerakkan smartphone. Tangkapan layar untuk aplikasi augmented reality yang dikembangkan oleh subjek S1 dapat dilihat pada Gambar 3. Gambar 3. Tangkapan layar aplikasi augmented reality Pada Gambar 3, setelah memindai barcode (lihat tanda panah) yang diletakkan pada titik tertentu, aplikasi akan memunculkan gambar bangun ruang. Gambar tersebut seolah nyata karena jika smartphone diputar, maka tampilan kubus dilayar juga akan berputar. A lik i dib t l h bj k S1 i i d l h lik i t bil b A lik i b b i d id Gambar 3. Tangkapan layar aplikasi augmented reality setelah memindai barcode (lihat tanda panah) yang diletakkan pada titik tertentu aplikasi akan Gambar 3. Tangkapan layar aplikasi augmented reality Gambar 3. Tangkapan layar aplikasi augmented reality Gambar 3. Tangkapan layar aplikasi augmented reality Pada Gambar 3, setelah memindai barcode (lihat tanda panah) yang diletakkan pada titik tertentu, aplikasi akan memunculkan gambar bangun ruang. Gambar tersebut seolah nyata karena jika smartphone diputar, maka tampilan kubus dilayar juga akan berputar. Pada Gambar 3, setelah memindai barcode (lihat tanda panah) yang diletakkan pada titik tertentu, aplikasi akan memunculkan gambar bangun ruang. Gambar tersebut seolah nyata karena jika smartphone diputar, maka tampilan kubus dilayar juga akan berputar. Aplikasi yang dibuat oleh subjek S1 ini adalah aplikasi yang terbilang baru. Aplikasi berbasis android sebelumnya sudah banyak, namun yang memunculkan augmented reality baru subjek S1 yang dapat mewujud- kannya di Program Studi (Prodi). Dalam RPP, S1 cukup jelas melibatkan aplikasi ini dalam perencanaan. Di RPP pula tampak bahwa siswa akan diajak belajar di luar kelas dan siswa juga difasilitasi untuk berlomba mencari barcode yang diletakkan di tempat tertentu, sekaligus memecahkan masalah yang akan di aplikasi tersebut. Yang dilakukan oleh subjek S1 ini serupa dengan metode Jelajah Alam Sekitar yang biasa diaplikasikan pada mata pelajaran IPA. Aplikasi yang dibuat oleh subjek S1 ini adalah aplikasi yang terbilang baru. PEMBAHASAN Pertama, peneliti akan membahas tentang pembelajaran dan situasi didaktis yang dibangun oleh guru. Yang dilakukan oleh subjek S1 dengan memulai pelajaran melalui sesuatu yang telah dikuasai siswa sebenarnya sesuai dengan teori belajar Vygotsky. Teori ini menyatakan bahwa untuk memulai belajar, siswa harus memulainya dengan sesuatu yang telah dikuasainya (Vygotsky, 1978). Adapun yang dilakukan oleh subjek S3 dengan mengulang-ulang proses berpikir siswa pada level yang sama adalah suatu pembiasaan yang diharapkan akan memunculkan perubahan perilaku. Perubahan perilaku inilah yang merupakan tanda bahwa siswa belajar (Thorndike, 1914). Namun, dalam implementasinya, subjek S1 belum dapat mendorong kemandirian belajar siswa. Prinsip dasar belajar matematika adalah bahwa pengetahuan dapat dicapai oleh siswa sendiri (Harel, 2011; Suryadi, 2019). Walaupun dalam perjalanan mencapai pengetahuan siswa tersebut melakukan diskusi, konfirmasi penge- tahuan yang diperoleh, dan bertanya; namun untuk menjadi paham, siswa sendirilah yang harus mencapainya. Tahapan berpikir hingga mencapai pengetahuan melahirkan jejak pembelajaran siswa (Sztajn et al., 2012). Dalam setiap tahap, pada jejak pembelajaran, guru seharusnya dapat memulainya dengan membangun situasi didaktis matematis. Pada praktiknya, jejak pembelajaran tersebut dapat direncanakan dan dibangun oleh guru dengan cara menjabarkan kompetensi dasar menjadi indikator penunjang dan inti (Dahlan, 2014). Temuan bahwa, baik subjek S1 dan S3 sudah memahami cara menjabarkan hal tersebut dari perkuliahan yang diambil, menun- jukkan adanya hambatan belajar dalam perkuliahan. Hambatan belajar tersebut bisa menjadi hambatan ontogeni, yaitu kurang siapnya mahasiswa menerima ilmu baru; hambatan didaktis, yaitu ketidakpaduan antara model, modul, dan produk yang diharapkan; atau hambatan epistemologi, yaitu kesenjangan antara teori dan praktis karena kurangnya praktik (Brousseau, 2002). Penelitian ini belum mampu mengelompokkan kepada jenis hambatan manakah kasus pada penelitian ini. Kedua, peneliti akan membahas tentang ICT yang digunakan dalam pembelajaran. Pembelajaran yang dilakukan oleh subjek S1 adalah pembelajaran matematika berbantuan ICT. ICT yang digunakan berupa aug- mented reality (tampilan tangkapan layar aplikasi dapat dilihat pada Gambar 2). Pembelajaran berbantuan ICT mengandung arti bahwa kegiatan belajar siswa tidak seluruhnya berada dalam dunia elektronik. ICT digunakan untuk menunjang pembelajaran yang dapat berupa melakukan visualisasi atas benda abstrak yang sedang dipelajari oleh siswa. Pembelajaran yang dilakukan oleh subjek S1 menampakkan model kubus yang dilengkapi dengan unsur-unsur bangun ruang. Unsur-unsur tersebut sulit dilihat pada objek nyata. Dengan aplikasi ini, subjek S1 menampilkan objek tiga dimensi hasil pindai marker. Objek tiga dimensi yang muncul dalam aplikasi ini bersifat dinamis. Siswa dapat menggerakkan HP berputar mengelilingi objek sehingga seluruh bagian objek terlihat semua seperti bentuk nyata. ICT yang digunakan dalam pembelajaran Aplikasi berbasis android sebelumnya sudah banyak, namun yang memunculkan augmented reality baru subjek S1 yang dapat mewujud- kannya di Program Studi (Prodi). Dalam RPP, S1 cukup jelas melibatkan aplikasi ini dalam perencanaan. Di RPP pula tampak bahwa siswa akan diajak belajar di luar kelas dan siswa juga difasilitasi untuk berlomba mencari barcode yang diletakkan di tempat tertentu, sekaligus memecahkan masalah yang akan di aplikasi tersebut. Yang dilakukan oleh subjek S1 ini serupa dengan metode Jelajah Alam Sekitar yang biasa diaplikasikan pada mata pelajaran IPA. Aplikasi yang digunakan subjek S3 dalam pembelajaran tidaklah seistimewa aplikasi yang digunakan oleh subjek S1. Subjek S3 hanya menggunakan presentation slide untuk menayangkan LKPD di depan kelas. Dalam RPP, S3 juga tidak secara eksplisit menjelaskan kapan media berbasis ICT ini digunakan Subjek S3 benar-benar Aplikasi yang digunakan subjek S3 dalam pembelajaran tidaklah seistimewa aplikasi yang digunakan oleh subjek S1. Subjek S3 hanya menggunakan presentation slide untuk menayangkan LKPD di depan kelas. Dalam RPP, S3 juga tidak secara eksplisit menjelaskan kapan media berbasis ICT ini digunakan. Subjek S3 benar-benar menerapkan ICT hanya sebagai bantuan untuk menayangkan LKPD di depan kelas. Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 8 Ardhi Prabowo, Dadang Juandi Apakah ICT yang digunakan membantu guru dalam membangun situasi didaktis matematis? Dari hasil wawancara (lihat Prabowo, 2020), diketahui bahwa aplikasi augmented reality digunakan cukup lama di dalam pembelajaran. Bahkan subjek S1 berani menjamin bahwa siswa senang belajar menggunakan aplikasi yang dibuatnya. Namun demikian, melalui konfirmasi lebih lanjut, ditemukan fakta bahwa konstruksi pengetahuan siswa tidak sepenuhnya dari aplikasi augmented reality tersebut. Subjek S1 menjawab bahwa kemungkinan siswa paham karena penjelasan guru. Hal ini berarti ada temuan bahwa walaupun aplikasi yang digunakan selama proses pembelajaran bersifat menarik, bagus, modern, dan dinamis, konstruksi pengetahuan siswa justru dari penjelasan guru. Aplikasi yang digunakan belum mampu membantu siswa untuk membangun pengetahuannya sendiri. Tentu saja dalam kaitannya dengan konstruksi pengetahuan siswa, aplikasi yang digunakan oleh subjek S3 dalam pembelajaran jelas tidak mampu mendorong konstruksi pengetahuan siswa. Dalam RPP subjek S3, jelas bahwa langkah konstruksi siswa ada pada proses menyimak penjelasan guru (lihat Tabel 1). PEMBAHASAN Dari rangkaian pembelajaran tersebut, S1 melihat bahwa siswa sangat antusias belajar menggunakan aplikasi yang dibuatnya. Antusias siswa tampak dari respon siswa yang terheran-heran, “Kok bisa?” (lihat Prabowo, 2020, data wawancara menit ke-21). Setelah mendapati hasil bahwa siswa sangat antusias dalam belajar, subjek S1 melanjutkan pembelajaran dengan nyaman karena siswa sudah termotivasi belajar sejak awal. Membangun rasa ingin tahu adalah salah satu proses memulai pembelajaran (Risnanosanti, 2009). ICT yang digunakan subjek S1 dalam pembelajaran terbukti mampu mendorong rasa penasaran atau ingin tahu siswa. Rasa ingin tahu siswa tersebut disebabkan karena aplikasi yang digunakan relatif baru bagi mereka dan unik. Dua kriteria itu mendorong remaja usia dini untuk tertarik mengikuti pelajaran (Saputro, 2018). Setelah aplikasi tersebut dapat membangun rasa ingin tahu, siswa semakin tertarik karena kedinamisan aplikasi. Siswa bisa menggerakkan benda dalam aplikasi seperti menggerakkan benda nyata. Ketertarikan tersebut disebabkan karena Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 9 Ardhi Prabowo, Dadang Juandi keterlibatan langsung siswa dalam menggunakan aplikasi di pembelajaran, baik secara individu dengan perangkat (Ackermann, 2001) maupun individu dengan rekannya (Vygotsky, 1978). keterlibatan langsung siswa dalam menggunakan aplikasi di pembelajaran, baik secara individu dengan perangkat (Ackermann, 2001) maupun individu dengan rekannya (Vygotsky, 1978). Penelitian-penelitian sebelumnya menyimpulkan bahwa pembelajaran berbantuan ICT dapat meningkatkan motivasi, hasil belajar, dan minat belajar siswa, bahkan sikap siswa terhadap matematika (Fahmi & Marsigit, 2014; Kartika, 2014; Putra, 2015; Zulkarnaen, 2017). Berbeda dengan penelitian sebelumnya, dalam penelitian ini dite- mukan fakta bahwa peran guru lebih besar dalam membantu siswa mencapai pengetahuan daripada peran ICT yang digunakan di dalam pembelajaran. Media berbasis ICT yang dikembangkan oleh subjek S1 dapat membangun rasa penasaran dan motivasi belajar siswa. Akan tetapi, media tersebut belum dapat membantu siswa dalam menaiki tangga kognitifnya. Siswa terbantu dengan media augmented reality tersebut setelah berhasil menaiki anak tangga hasil bantuan yang diberikan oleh guru. Dengan demikian, berdasarkan uraian penjelasan di atas, dapat dikatakan bahwa kriteria media ICT yang baik untuk digunakan dalam pembelajaran adalah media yang mampu membangun situasi didaktis matematis siswa. Kedua subjek dalam penelitian ini, S1 dan S3, belum menghasilkan media ICT yang mampu membangun situasi didaktis matematis siswa Ketiga, peneliti akan mengelaborasi antara ICT yang digunakan dalam pembelajaran dan situasi didaktis yang dibangun oleh guru. Dari konfirmasi data dan wawancara, ditemukan fakta bahwa ICT yang digunakan oleh subjek S1 benar-benar membantu guru dalam melaksanakan pembelajaran. PEMBAHASAN Subjek S1 mengklaim bahwa media augmented reality yang digunakan siswa lebih baik daripada media fisik. Hal ini karena media yang dibuat oleh subjek S1 dapat memvisualkan berbagai bangun ruang dari berbagai sudut pandang. Berbeda dengan media fisik yang hanya bisa menampilkan satu bangun ruang. Namun demikian, media augmented reality yang dibuat tersebut belum mampu membangun situasi didaktis matematis siswa. Peran guru dalam membangun situasi didaktis terjadi di awal pembelajaran. Pada saat siswa kesulitan menerjemahkan makna pertanyaan dalam LKPD, guru kemudian mengambil alih peran media dan LKPD dengan menjelaskan maksud dari pertanyaan. Media yang digunakan oleh subjek S3 tidak lebih membantu pembelajaran jika dibandingkan dengan media yang digunakan oleh subjek S1. Media berupa presentation slide yang digunakan oleh subjek S3 lebih berperan sebagai pengganti kertas yang mana hanya menayangkan LKPD di dalam slide, walaupun siswa sebenarnya telah menerima LKPD dalam bentuk cetak. Hal ini menunjukkan bahwa media berbasis ICT yang menarik pun belum tentu mampu membangun situasi didaktis matematis. Melalui konfirmasi kepada dua subjek, kedua subjek tersebut menyatakan bahwa telah mengikuti perkuliahan media di prodi. Perkuliahan media tersebut bahkan sudah dua kali diikuti oleh subjek S1 dan S3. Mata Kuliah Media Pendidikan Matematika 2 berfokus kepada media yang berbasis ICT. Subjek S1 bahkan mengambil mata kuliah media yang ketiga yang merupakan mata kuliah pilihan yang berfokus kepada media berbasis daring. Dengan pengalaman mengikuti perkuliahan tersebut, subjek S1 dan S3 ternyata belum dapat membangun media yang mampu mendorong siswa untuk melakukan aksi mental secara mandiri. Dengan kata lain, media yang dihasilkan belum mampu membangun situasi didaktis matematis. Hal ini berarti ada hambatan dalam perkuliahan yang telah dialami oleh kedua subjek. Sebagaimana dijelaskan sebelumnya bahwa penelitian ini belum mampu mengelompokkan kepada jenis hambatan manakah yang terjadi dalam perkuliahan yang diikuti oleh subjek S1 dan S3. Untuk mengetahui jenis hambatan ini, diperlukan sumber daya yang lebih luas seperti kurikulum prodi, dosen pengampu, dan evaluasi prodi. Menemukan hambatan ini sebenarnya bagian awal dari sebuah riset didaktis untuk menemukan desain baru sebagaimana yang telah dilaksanakan dalam penelitian sebelumnya (misalnya Evayanti, 2017; Romdhani & Suryadi, 2017; Sulistiawati et al., 2015; Yunarti, 2014). Dari penjelasan sebelumnya, dapat disampaikan bahwa penelitian ini masih dapat dikembangkan untuk penelitian lebih lanjut. Penelitian ini belum berhasil mengungkap jenis hambatan belajar yang terjadi di mahasiswa calon guru. Selain itu, subjek riset yang berbeda dari S1 dan S3, akan dapat menghasilkan temuan yang berbeda pula. PEMBAHASAN Penelitian lebih lanjut juga dapat menghubungkan temuan riset dengan jejak pembelajaran siswa. Pengem- bangan dari penelitian ini akan dapat menghasilkan desain perkuliahan yang mampu menghasilkan mahasiswa dengan kemampuan menyusun media pembelajaran berbasis ICT. Media pembelajaran berbasis ICT yang dimak- sud adalah media pembelajaran berbasis ICT yang mampu membangun situasi didaktis matematis siswa. Secara praktis, hasil riset ini akan dapat dimanfaatkan oleh peneliti dan dosen pengampu mata kuliah media sebagai bagian dari refleksi dan perbaikan penyusunan rencana perkuliahan di masa yang akan datang. Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 10 Ardhi Prabowo, Dadang Juandi SIMPULAN Berdasarkan hasil penelitian dan pembahasan yang disajikan dalam artikel ini, dapat disimpulkan bahwa karakteristik situasi didaktis dalam pembelajaran matematika berbantuan ICT pada siswa SMP yang dibangun oleh mahasiswa calon guru adalah sebagai berikut: (1) situasi didaktis matematis yang dibangun belum mampu mengonstruksi pengetahuan siswa secara mandiri; (2) situasi didaktis matematis yang dibangun cenderung berasal dari informasi yang disampaikan guru, bukan dari media ICT yang digunakan; dan (3) ICT yang digunakan dalam pembelajaran belum mampu mengelaborasikan proses kognitif siswa dalam jejak pembelajarannya. Sebagai tindak lanjut, kami sarankan untuk melakukan penelitian lanjutan guna: (1) mengelompokkan jenis hambatan yang ditemukan dalam penelitian ini; dan (2) menyusun desain perkuliahan yang mampu mendorong mahasiswa calon guru untuk merancang situasi didaktis matematik yang konstruktif berdasarkan kurikulum sekolah yang berlaku. DAFTAR PUSTAKA https://doi.org/10.4324/9780203454206 Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 11 Ardhi Prabowo, Dadang Juandi - 11 Evayanti, M. (2017). Desain didaktis konsep garis dan sudut berdasarkan realistic mathematics education (RME) pada pembelajaran matematika sekolah menengah pertama (SMP) [Unpublished master’s thesis]. Universitas Pendidikan Indonesia. http://repository.upi.edu/33717/ Fadholi, T., Waluya, B., & Mulyono. (2015). Analisis Pembelajaran matematika dan kemampuan literasi serta karakter siswa SMK. Unnes Journal of Research Mathematics Education, 4(1), 42–48. https://journal.unnes.ac.id/sju/index.php/ujmer/article/view/6906 Fahmi, S., & Marsigit, M. (2014). Pengembangan multimedia macromedia flash dengan pendekatan kontekstual dan keefektifannya terhadap sikap siswa pada matematika. Pythagoras: Jurnal Pendidikan Matematika, 9(1), 90–98. https://doi.org/10.21831/pg.v9i1.9071 Fink, L. D. (2003). A self-directed guide to designing courses for significant learning. https://www.bu.edu/sph/files/2011/06/selfdirected1.pdf Fried, M. N. (2006). Mathematics as a constructive activity: Learners generating examples (Book Review). ZDM (Zentralblatt Für Didaktik Der Mathematik), 38(2), 209–211. https://doi.org/10.1007/bf02655890 Harel, G. (2011). What is mathematics? A pedagogical answer to a philosophical question. In B. Gold & R. Simons (Eds.), Proof and other Dilemmas (pp. 265–290). Spectrum. https://doi.org/10.5948/upo9781614445050.018 Husni, M. A. (2014). Keefektifan pembelajaran matematika dengan problem posing dan problem solving ditinjau dari prestasi dan curiosity. Pythagoras: Jurnal Pendidikan Matematika, 9(1), 11–21. https://doi.org/https://doi.org/10.21831/pg.v9i1.9058 Kartika, H. (2014). Pembelajaran matematika berbantuan software matlab sebagai upaya meningkatkan kemampuan komunikasi matematis dan minat belajar siswa SMA. Jurnal Pendidikan UNSIKA, 2(1), 24–35. https://doi.org/10.22342/jpm.10.2.3637.93-108 Polya, G. (1962). Mathematical discovery, on understanding, learning, and teaching problem solving (Combined ed.). John Wiley & Sons. Prabowo, A., & Ahmad, A. (2015). Lembar kerja yang bermakna. In U. Sukandi (Ed.), Modul pelatihan good practice school (pp. 93–104). USAID PRIORITAS. Putra, F. G. (2015). Eksperimentasi model pembelajaran kooperatif tipe teams games tournament (TGT) berbantuan software cabri 3D ditinjau dari kemampuan koneksi matematis siswa. Al-Jabar: Jurnal Pendidikan Matematika, 6(2), 143–154. https://doi.org/10.24042/ajpm.v6i2.43 Risnanosanti, R. (2009). Membangun suatu situasi-didaktis dalam pembelajaran inkuiri untuk meningkatkan kemampuan berpikir kreatif siswa. In Prosiding Seminar Nasional Penelitian, Pendidikan dan Penerapan MIPA. (pp. M.501-M.506). Universitas Negeri Yogyakarta. https://eprints.uny.ac.id/12301/1/M_Pend_27_ Risnanosanti.pdf Romdhani, W., & Suryadi, D. (2017). Desain didaktis konsep pecahan untuk kelas III sekolah dasar. EduHumaniora: Jurnal Pendidikan Dasar Kampus Cibiru, 8(2), 198–210. https://doi.org/10.17509/eh.v8i2.5142 Saputro, K. Z. (2018). Memahami ciri dan tugas perkembangan masa remaja. Aplikasia: Jurnal Aplikasi Ilmu-Ilmu Agama, 17(1), 25–32. https://doi.org/10.14421/aplikasia.v17i1.1362 Silver, E. A. (1994). On mathematical problem posing. For the Learning of Mathematics, 14(1), 19-28. https://www.jstor.org/stable/40248099 Sulistiawati, S., Suryadi, D., & Fatimah, S. (2015). DAFTAR PUSTAKA Ackermann, E. (2001). Piaget’s constructivism, Papert’s constructionism: What’s the difference. Future of Learning Group. https://learning.media.mit.edu/content/publications/EA.Piaget%20_%20Papert.pdf As’ari, A. R., Tohir, M., Valentino, E., Imron, Z., & Taufiq, I. (2017a). Buku guru matematika SMP/MTs kelas VIII edisi revisi (A. Lukito, A. Mahmudi, & D. Hidayat, Eds.). Kementerian Pendidikan dan Kebudayaan Republik Indonesia. As’ari, A. R., Tohir, M., Valentino, E., Imron, Z., & Taufiq, I. (2017a). Buku guru matematika SMP/MTs kelas VIII edisi revisi (A. Lukito, A. Mahmudi, & D. Hidayat, Eds.). Kementerian Pendidikan dan Kebudayaan Republik Indonesia. As’ari, A. R., Tohir, M., Valentino, E., Imron, Z., & Taufiq, I. (2017b). Matematika SMP kelas VIII semester 1 edisi revisi. (A. Lukito, A. Mahmudi, Turmudi, Y. Marpaung, Y. Satria, Widowati, & D. Hidayat, Eds.). Kementerian Pendidikan dan Kebudayaan Republik Indonesia. Assis, C., Gitirana, V., & Trouche, L. (2018). The metamorphosis of resource systems of prospective teacher: From studying to teaching. In V. Gitirana, T. Miyakawa, M. Rafalska, S. Soury-Lavergne, & L. Trouche (Eds.), Proceedings/Actes Re(S)Source 2018 International Conference (pp. 39–42). Ecole Normale Superieure de Lyon (ENS de Lyon). Bartell, T. G., Webel, C., Bowen, B., & Dyson, N. (2013). Prospective teacher learning: Recognizing evidence of conceptual understanding. Journal of Mathematics Teacher Education, 16(1), 57–79. https://doi.org/10.1007/s10857-012- 9205-4 Brousseau, G. (2002). Epistemological obstacles, problems, and didactical engineering. In N. Balacheff, M. Cooper, R. Sutherland, & V. Warfield (Eds.), Theory of didactical situations in mathematics (Didactique des mathématiques), 1970–1990 (pp. 79–117). Kluwer Academic Publishers. https://doi.org/10.1007/0-306-47211-2_6 Bruner, J. (1961). The act of discovery. Harvard Educational Review, 31, 21–32. https://digitalauthorshipuri.files.wordpress.com/2015/01/the-act-of-discovery-bruner.pdf Cahyono, E., Isnarto, I., Ridlo, S., Handoyo, E., Yulianto, A., & Sugianto. (2018). Kurikulum UNNES 2018 program studi sarjana pendidikan (Rustono & E. Subkhan, Eds.). UNNES Press. Chaves, C. (2008). Adult learners and the dialectical process: A validating constructivist approach to learning tranfer and application. Online Journal for Workforce Education and Development, 3(1), 1–14. https://opensiuc.lib.siu.edu/ojwed/vol3/iss1/2/ Dahlan, J. A. (2014). Kurikulum dan pengembangannya. In Analisis Kurikulum (pp. 1.1-1.34). Universitas T Dawson, K. (2006). Teacher inquiry: A vehicle to merge prospective teachers’ experience and reflection during curriculum-based, technology-enhanced field experiences. Journal of Research on Technology in Education, 38(3), 265–292. https://doi.org/10.1080/15391523.2006.10782460 Dubinsky, E. (2000). Using a theory of learning in college mathematics courses. TaLUM, the Teaching and Learning Undergraduate Mathematics, 10–16. http://www.math.wisc.edu/~wilson/Courses/Math903/UsingAPOS.pdf Ernest, P. (Ed.). (1994). Constructing mathematical knowledge: Epistemology and mathematics education. Routledge. DAFTAR PUSTAKA Desain didaktis penalaran matematis untuk mengatasi kesulitan belajar siswa SMP pada luas dan volume limas. Kreano: Jurnal Matematika Kreatif-Inovatif, 6(2), 135–146. https://doi.org/10.15294/kreano.v6i2.4833 Suryadi, D. (2019). Landasan filosofis penelitian desain didaktis (DDR) (E. Gapura, Ed.). Gapura Press. Sztajn, P., Confrey, J., Wilson, P. H., & Edgington, C. (2012). Learning trajectory-based instruction: Toward a theory of teaching. Educational Researcher, 41(5), 147–156. https://doi.org/10.3102/0013189X12442801 Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X PYTHAGORAS: Jurnal Pendidikan Matematika, 15 (1), 2020 - 12 Ardhi Prabowo, Dadang Juandi Thorndike, E. L. (1914). Educational psychology, Vol 3: Mental work and fatigue and individual differences and their causes. Teachers College. https://doi.org/10.1037/13796-000 ES. (2019). Kurikulum UNNES program studi pendidikan matematika jenjang S1. UNNES. (2019). Kurikulum UNNES program studi pendidikan matematika jenjang S1. Vygotsky, L. S. (1978). Mind and society: The development of higher psychological processes (M. Cole, V. John- Steiner, S. Scribner, & E. Souberman, Eds.). Harvard University Press. Wahono, T. K., & Budiarto, M. T. (2014). Kecerdasan visual-spasial siswa SMP dalam menyelesaikan soal geometri ruang ditinjau dari perbedaan kemampuan matematika. Jurnal Ilmiah Pendidikan Matematika, 3(1), 158– 164. http://ejournal.unesa.ac.id/index.php/mathedunesa/article/view/7322 Watson, A., & Mason, J. (2005). Mathematics as a constructive activity: Learners generating examples. Mathematics as a constructive activity: Learners generating examples. Routledge. https://doi.org/10.4324/9781410613714 Yunarti, T. (2014). Desain Didaktis Teori Peluang SMA. Jurnal Pendidikan MIPA Universitas Lampung, 15(1), 15–20, http://jurnal.fkip.unila.ac.id/index.php/jpm/article/view/5479/3415 Zulkarnaen, R. (2017). Penerapan pendekatan realistik berbantuan ICT terhadap kemampuan penalaran matematis siswa kelas VII. Euclid, 3(2), 578–585. https://doi.org/10.33603/e.v3i2.334 Data Wawancara Data Wawancara Prabowo, A. (2020). Podcast #1. Jadi, saat mengajar, situasi matematika yang baik itu .... Retrieved from https://www.youtube.com/watch?v=KL_JmSDhWro&t=669s data taken at February 26, 2020. Prabowo, A. (2020). Podcast #1. Jadi, saat mengajar, situasi matematika yang baik itu .... Retrieved from https://www.youtube.com/watch?v=KL_JmSDhWro&t=669s data taken at February 26, 2020. Copyright © 2020, Pythagoras, Print ISSN: 1978-4538, Online ISSN: 2527-421X
https://openalex.org/W3119937561	https://www.researchsquare.com/article/rs-86723/v1.pdf	English	null	Bacteriophage cocktail supplementation improves growth performance, gut microbiome and production traits in broiler chickens	Journal of Animal Science and Biotechnology/Journal of animal science and biotechnology	2,021	cc-by	8,580	Research License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Journal of Animal Science and Biotechnology on April 16th, 2021. See the published version at https://doi.org/10.1186/s40104-021-00570-6. Page 1/17 Abstract Background: Effective antibiotic alternatives are the urgent need of poultry industry to control disease outbreaks. Phage therapy mainly utilizes lytic phages to kill their respective bacterial hosts and can be attractive solution to combating the emergence of antibiotic resistance in livestock. Methods: Five hundred and four one-day--old broilers (Ross 308) were allotted into 1 of 4 treatment groups according to a completely randomized design. Dietary treatments consisted of CON (basal diet), PC (CON + 0.025% Methods: Five hundred and four one-day--old broilers (Ross 308) were allotted into 1 of 4 treatment groups according to a completely randomized design. Dietary treatments consisted of CON (basal diet), PC (CON + 0.025% Avilamax®(antibiotics), TR1 (CON + 0.05% bacteriophage), and TR2 (CON + 0.10 % bacteriophage) groups. Results: A significant linear effect on body weight gain (BWG) was observed during day 1-7, day 22-35, and overall experiment in bacteriophage (BP) supplemented groups. The BWG tended to be higher (P = 0.08) and the feed intake (FI) was increased (P = 0.017) in birds fed PC than CON diets. A greater (P = 0.016) BWG and trends in increased FI (P = 0.06) were observed during the overall experiment period in birds fed PC than CON diet. A trend in linear (P = 0.0833) increment in excreta Lactobacillus counts was observed in birds fed graded level of BP supplemented diets. At the genus level, the relative abundance of Lactobacillus was decreased in PC (65.28%), while it was similar in TR1, 2, (90.65%, 86.72%, 81.44%) compared to CON (90.19%). At the species level, relative abundance of Lactobacillus salivarus was higher in TR1 (40.15%) and TR2 (38.58%) compared with CON (20.04%) and PC (18.05%). A linear reduction in the weight of Bursa of Fabricus (P = 0.022) and spleen (P = 0.052) was seen in birds fed increasing level of BP diets and a trend in increment (P = 0.059) in the weight of gizzard was observed in birds fed PC than BP diets. Linear and quadratic responses were observed in redness of breast muscle color in birds fed graded level of BP. Conclusions:The increase in dietary BP supplementation linearly increased BWG, Lactobacillus counts and enhanced beneficial microbiota in the gut, and 0.05% BP addition was sufficient for supporting immune organs, bursa and spleen. Background In response to the increase in the demand of livestock products such as meat, milk and egg by a global growing population, livestock producers are compelled to significantly increase these products. Thus, large scale intensive farming system is continuing to rise. Unfortunately, such production systems can promote disease transmission very easily due to their low genetic diversity and high stocking density leading to concomitant production and economic losses [1, 2]. Zoonotic pathogens associated with poultry and pigs such as Salmonella spp., E. coli, Campylobacter spp., Clostridium spp. and Listeria spp have been reported by European Food Safety Authority (EFSA) to be often resistant to several antibiotics [3, 4]. In this context, alternative approaches have become urgent. One option would be the application of lytic bacteriophage to combat the bacterial diseases in livestock [5]. Bacteriophages are viruses that infect and use bacterial resources for their own reproduction. They are very common in all environments and have a high specificity to bacteria at infection [6]. In a review, Domingo et al. [7] suggested that bacteriophages have narrow spectrum activity against bacteria, in opposite to broad spectrum activity of antibiotics against bacteria. Bacteriophages being specific for particular bacteria, the phage therapy is considered to be safe and effective in comparison to antibiotics which is partially manifested as their ability to infect only one species, serotype or strain. This mechanism of action does not inhibit the proliferation of commensal intestinal flora [8, 9]. In a study, Fiorentin et al. [10] noted that the application of single oral cocktail of phages at a dosage of 1011 pfu decreased the occurrence of Salmonella Enteritidis strains by 3.5 log units. Fiorentin et al. [10] noted that the application of single oral cocktail of phages at a dosage of 1011 pfu decreased the occurrence of Salmonella Enteritidis strains by 3.5 log units. In addition, other studies have also reported a successful reduction in the Salmonella spp counts in chicken internal organs and excreta [11] as well as in poultry products [12, 13] with bacteriophage application. Furthermore, it has been Page 2/17 reported that bacteriophage supplementation improved feed efficiency, liver weight and reduced pathogens in broiler chickens [14] and improved egg production and egg quality in laying hens [15]. Background The inclusion of phage as a feed additive may potentially provide an integrated solution to modulate the gut microbiome in chicken by reducing specific pathogenic microbial population thereby promoting the proliferation of beneficial microbiota resulting in improved gut health [16]. Under bacterial challenge, bacteriophage has shown to be effective in several studies [17–19], however reports on the usage of bacteriophage cocktail through dietary application in birds without bacterial challenge is scarce. Thus, the objective of the current study was to assess the effect of two different concentrations of cocktail bacteriophage on the performance and production characteristics as well as gut microbiome of broiler chickens raised under normal physiological condition (without inducing infection via bacterial challenge). Experimental design, animals and diets Bacteriophages used in the present study was a commercial product from CJ Cheiljedang Corp. Seoul, South Korea, consisting of Salmonella gallinarum, Salmonella typhimurium, S. Enteritidis, Escherichia coli at the concentrations of 1.0 × 108 pfu/g each and Clostridium perfringens (1.0 × 106 pfu/g). A total of 504 1-d-old broilers (ROSS 308) with the initial BW 42.9 ± 1.0 g were used in a 35-d experiment. Chicks were randomly divided into the four experimental groups, and each group had 7 replicate cages, with 18 broilers per cage. The bacteriophage cocktail was administrated by replacing the same amount of corn. The treatment groups were as follows: i) CON group (Control/ basal diet without BP supplementation), ii) PC group (CON + 0.25 g antibiotics; AVILAMIX®/kg feed), iii) TR1 group (CON + 0.5 g bacteriophage/kg feed), and iv) TR2 group (CON + 1.0 g bacteriophage/kg feed). Broiler chickens were raised in a temperature-controlled room with stainless steel pens of identical size (1.75 × 1.55 m2). Room temperature was maintained at 33 ± 1 °C for the first 3 d, and then gradually reduced by 3 °C a week until reaching 24 °C and maintained for the remainder of the experiment and the relative humidity was around 60%. The basal diet was formulated to meet or exceed all the nutrient requirements of broilers as recommended by National Research Council [20], and supplied in mash form. There were two nutritional phases, including starter (1 to 21 d), and finisher phase (22 to 35 d), and the ingredients and analyzed nutrient composition of the basal diet are shown in Table 1. Artificial light was provided 24 h/d by the use of fluorescent lights. All diets were fed in mash form with feed and water being provided ad libitum throughout the experimental period. Sampling And Measurements Sampling And Measurements Growth performance Broilers were weighed by cage and feed consumption was recorded at day 0, 7, 21 and 35. This information was then used to calculate body weight gain (BWG) average feed intake (FI), and feed conversion ratio (FCR). b l Broilers were weighed by cage and feed consumption was recorded at day 0, 7, 21 and 35. This information was then used to calculate body weight gain (BWG) average feed intake (FI), and feed conversion ratio (FCR). Nutrient Digestibility Ileal Mucosa Microbiome For gut microbiome analysis, six ileal mucosal samples from each group (CON, PC, TR1 and TR2) were collected at day 35 from randomly selected 24 broilers. Briefly, birds were sacrificed by cervical dislocation and exsanguination. After autopsy, the intestinal tract was excised and the intestinal content was removed followed by washing the intestinal segment with distilled water. Then ileal segment (distal ileum) was cut about 10–15 cm proximally to caeca and separated from the intestine and then rinsed in PBS and the mucosal layer was scraped with a glass slide. Mucosal scrapings were collected into a 50 ml conical tube and stored in an ice box and then transferred to Macrogen Inc., (Seoul, Republic of Korea) for gene sequencing. Genomic DNA extraction from the mucosal samples and the preparation of library of amplicons consisting of 16S rRNA gene and sequencing was done by Illumina MiSeq platform at Macrogen Inc. (Seoul, Republic of Korea) using MiSeq sequencing including barcoded 16S rRNA amplicons. The 16S rRNA gene sequences were processed using the Mothur software to remove low-quality sequences [23]. Briefly, sequences that did not match the PCR primers were eliminated from de-multiplexed sequence reads. The sequences containing ambiguous base calls and sequences with a length less than 100 bp to were trimmed minimize the effects of random sequencing errors. Chimeric sequences were further deleted using the UCHIME algorithm implemented in Mothur. QIIME (Quantitative Insights into Microbial Ecology) software package (version 1.9.1) was used for de novo operational taxonomic unit (OTU) clustering with an OTU definition at an identity cutoff 97% [24]. Taxonomic assignment was performed using the naïve Bayesian RDP classifier and the Greengenes reference database. Beta-diversity was measured using unweighted UniFrac distance metrics using QIIME. The unweighted UniFrace considers the community membership (presence or absence of OTUs) [25]. Principal coordinate analysis (PCoA) plots were generated based on the unweighted UniFrac distance metrics. Nutrient Digestibility The apparent total tract digestibility of DM, N and energy was comparable between CON and PC treatments. In addition, inclusion of graded level of bacteriophage to the CON diet did not affect the digestibility of nutrients in birds as shown in Table 3. Excreta Microbial Counts Excreta Microbial Counts Page 3/17 Page 3/17 Page 3/17 For excreta microbial counts, excreta samples were collected from all 7 cages each treatment at day 35. The excreta samples were kept frozen at − 20 °C until microbiota analysis for the enumeration of Salmonella, Escherichia coli (E. coli), Clostridium spp and Lactobacillus. After thawing, viable counts of bacteria in the excreta were then determined by plating serial 10-fold dilutions (in 10 g/L peptone solution) in respective media. The selective medium used for isolation of Salmonella was Salmonella Shigella (Difco, USA), for E. coli, Mac Conkey (Difco, USA), for Clostridia spp. Cooked Meat Medium (Oxoid, UK) and for Lactobacillus, Lactobacilli medium III (Medium 638, DSMZ, Braunschweig, Germany). The Lactobacilli MRS agar plates were incubated for 48 h at 39 °C, and the MacConkey agar and Salmonella Shigella agar plates were incubated for 24 h at 37 °C whereas Cooked Meat Medium agar plates were incubated at 30 °C for 24 h under anaerobic conditions. The colony counts were then enumerated and results are presented as log10-transformed data. Meat Quality For physicochemical properties of the breast meat, at least one bird per pen (n = 10) from each treatment were selected randomly at day 35 and were individually weighed and killed by cervical dislocation and exsanguinated. The breast muscle (pectoralis major), Bursa of Fabricius, liver, spleen, and abdominal fat were then removed and weighed. Organ weights were expressed as a relative percentage to the whole body weight. The breast muscle Hunter lightness (L), redness (a), and yellowness (b) values were determined using a Minolta CR410 chromameter (Konica Minolta Sensing Inc., Osaka, Japan). The pH of the breast muscle sample was measured by a calibrated, glass-electrode pH meter (Testo 205, Testo, Germany). The water-holding capacity (WHC) was analyzed according to the methods described by Kauffman et al. [26]. Drip loss was measured using approximately 2 g of meat sample according to the plastic bag method described by Honikel [27]. Statistical Analysis Page 4/17 Data were analyzed using the GLM procedure of SAS (version 9.4; SAS Inst., Inc., Cary, NC) in a completely randomized design. Pen served as the experimental unit. Pre-planned contrast was used to test the following: 1) the individual effect of CON versus PC diets 2) the overall effect of Bacteriophage supplementation versus PC diet (PC vs TR1, TR2). Furthermore, linear and quadratic polynomial contrasts were used to examine responses to supplemental graded levels of Bacteriophage at 0%, 0.05% and 0.1%. Variability in the data was expressed as the standard error of means (SEM) and P ≤ 0.05 was considered to be statistically significant and P < 0.1 as trends. Data were analyzed using the GLM procedure of SAS (version 9.4; SAS Inst., Inc., Cary, NC) in a completely randomized design. Pen served as the experimental unit. Pre-planned contrast was used to test the following: 1) the individual effect of CON versus PC diets 2) the overall effect of Bacteriophage supplementation versus PC diet (PC vs TR1, TR2). Data were analyzed using the GLM procedure of SAS (version 9.4; SAS Inst., Inc., Cary, NC) in a completely randomized design. Pen served as the experimental unit. Pre-planned contrast was used to test the following: 1) the individual effect of CON versus PC diets 2) the overall effect of Bacteriophage supplementation versus PC diet (PC vs TR1, TR2). Furthermore, linear and quadratic polynomial contrasts were used to examine responses to supplemental graded levels of Bacteriophage at 0%, 0.05% and 0.1%. Variability in the data was expressed as the standard error of means (SEM) and P ≤ 0.05 was considered to be statistically significant and P < 0.1 as trends. Furthermore, linear and quadratic polynomial contrasts were used to examine responses to supplemental graded levels of Bacteriophage at 0%, 0.05% and 0.1%. Variability in the data was expressed as the standard error of means (SEM) and P ≤ 0.05 was considered to be statistically significant and P < 0.1 as trends. For gut microbiome, analysis of similarities (ANOSIM) to determine whether the microbial compositions between the treatment and control groups were significantly different was done using QIIME software package (version 1.9.1) and was based on the unweighted UniFrac distance metrics. Excreta Microbial Enumeration The effect of dietary bacteriophage supplementation on excreta microbiota counts in broiler chicken is presented in Table 4. The Lactobacillus counts tended to be higher (P = 0.058) in birds fed BP supplemented diet than the birds fed PC diet. However, the concentrations of E. coli, Clostiridium perfringens, and Salmonella were comparable between CON and PC diets or PC and TR1 and TR2 diets. A trend in linear (P = 0.0833) increment in Lactobacillus counts was observed in birds fed graded level of BP supplemented basal diet. Growth performance As shown in Table 2, the BWG tended to be higher (P = 0.089) in birds fed TR1 and TR2 diet during day 1–7 and overall experiment period compared with birds fed CON diet. A significant linear effect on BWG was observed during days 1–7, 22–35, and overall experiment in birds fed the diet supplemented with graded level of BP. During day 1–7, there were no significant differences between PC and CON diet on the growth performance parameters. However, the BWG tended to be higher (P = 0.08) during day 8–22 in birds fed PC diet than CON diet. During day 8–22, the FI was significantly increased (P = 0.017) in birds fed PC than CON diets and FI tended to be higher (P = 0.0796) in birds fed PC than the diet supplemented with BP. A significantly greater (P = 0.016) BWG and trends in increased FI (P = 0.06) were observed during the overall experiment period in birds fed PC diet than CON diet. Gastrointestinal Microbiome At the species level, while Lactobacillus salivarius and Lactobacillus aviarius represented the 2 most abundant species in all groups, its relative abundance increased from an average of 18.86% in CON to 40.13% in TR1 and 37.80% in TR2 in L. salivarius (Fig. 3c), 10.04% in CON to 15.60% in TR1 and 15.87% in TR2 in L. aviarius (Fig. 3d). Meat Quality And Organ Weight The effect of bacteriophage supplementation on organ weight and meat quality in broilers is shown in Table 5. Except for the significant reduction in relative weight of Bursa of Fabricus in birds fed PC than CON diets, none of the other meat quality and organ weight parameters were affected between CON and PC diets. The relative weight of gizzard showed trends in increment in birds fed PC than TRI and TR2 diets. A linear reduction in weight of bursa of fabricus (P = 0.026) and spleen (P = 0.052) relative to body weight were seen in birds fed diets supplemented with increasing level of bacteriophage. Linear and quadratic responses were observed in redness of breast muscle color for birds fed graded level of bacteriophage. Gastrointestinal Microbiome To evaluate the effect of BP on the gut microbiota of broiler chicken, the mucosa-attached microbiome in the ileum were analyzed by deep sequencing. Sequencing of the 16S rRNA genes in the mucosal samples produced a total of 1,121,448 reads after quality-filtering, with a mean sequence number of 36,473 ± 37,381 reads per sample. Analysis of similarities (ANOSIM) of unweighted UniFrace distances indicated that each group was clustered significantly different excluding control group (P < 0.05) suggesting that microbiota of the PC and TR1, 2 groups were significantly different. The unweighted UniFrac PCoA plot visually confirmed the distinct separation of microbial communities between groups (Fig. 1). Comparisons of the relative abundances of the gut microbiota compositions between 4 groups at the phylum and genus levels are shown in Fig. 2. At the phylum level, the bacterial sequences from the CON samples were composed predominatly of the phyla Firmicutes (94.56%), Bacteroidetes (3.89%), Proteobacteria (1.38%) and 4 other phyla that collectively comprised 0.17% of the total sequences analyzed (Fig. 2a). PC group consisted largely of phyla Firmicutes (80.86%), Bacteroidetes (15.09%), Proteobacteria (2.78%), Deferribacteres (1.03%) and 4 other phyla which collectively comprised of 0.24% of the total sequences analyzed (Fig. 2a). In TR1 group, Firmicutes (94.57%) and Bacteroidetes (3.76%) were composed as predominant, the rest 6 phyla were comprised of 1.67% of the total sequences (Fig. 2a). In Page 5/17 Page 5/17 TR2 group, Firmicutes (91.81%), Proteobacteria (5.45%) and Bacteroidetes (2.42%) were predominant, while other 5 phyla were composed of 0.32% of the total sequences analyzed (Fig. 2a). TR2 group, Firmicutes (91.81%), Proteobacteria (5.45%) and Bacteroidetes (2.42%) were predominant, while other 5 phyla were composed of 0.32% of the total sequences analyzed (Fig. 2a). At the genus level, Lactobacillus was the most enriched genera in all mucosal samples (Fig. 2b). And its relative abundance was decreased in PC (65.28%), while it was similar in TR1, 2, (90.65%, 86.72%, 81.44%) compared to CON (90.19%). The relative abundance of Prevotella increased from an average of 1.15% in CON to 2.56% in TR1 and 1.37% in TR2 (Fig. 3a) and Bifidobacteria also increased from an average of 0.01% in CON to 0.70% in TR1 and 0.14% in TR2 (Fig. 3b). Discussion The emergence of multidrug-resistant bacterial pathogens and the imposition of ban on the usage of antimicrobials in animal production have led to a resurgence of interest in phage therapy [28]. Research on reducing zoonotic pathogens with the application of BP as a viable option in food animals has also focused on reducing the impact of infections in the animals themselves [29] thereby improving the production and performance of animals. In the present study, commercially available BP consisting of Salmonella gallinarum, S. typhimurium, S. Enteritidis, E. coli and Clostridium perfringes was assessed for its suitability as feed additive for the enhancement of performance and production of broiler chickens under normal physiological state (without bacterial challenge). In agreement with the findings of Kim et al. [30] who demonstrated that FI and FCR were unaffected by supplementing the broilers diet with anti-SE bacteriophage, the inclusion of BP as feed additive at 0.05% and 0.1% levels in the present study showed no effects on FI and FCR throughout the trail, except for a trend in linear reduction in FCR during day 22– 35. However, the present study showed significant linear effect in increasing the BWG with the increase in BP levels during the initial starter and finisher phases and overall experiment period indicating that BP supplementation had no detrimental effect on feed consumption but promoted the BWG. In contrast, Huff et al. [31] suggested that the BWG were not affected by the inclusion of bacteriophage in broiler chickens without bacterial challenge; and Wang et al. [14] noted that the supplementation of BP consisting of mixture of Salmonella gallinarum, S. typhimurium, and S. Enteritidis at the ratio of 3:3:4 at the dose level of 0.05% improved FCR during day 1–14 in broiler chickens. In broiler production, increase in body weight is an important parameter since lower body weight equates with increased cost for broiler meat production [30]. The increase in BWG when BP was used as a feed additive instead of antibiotics in animal feed might be due to the inhibitive or lytic effect on harmful bacteria replication in the gastrointestinal tract of broiler chickens [32]. Moreover as expected, the inclusion of sub-therapeutic dose of antibiotic as positive control in the diet of broiler chickens production [30]. Discussion The increase in BWG when BP was used as a feed additive instead of antibiotics in animal feed might be due to the inhibitive or lytic effect on harmful bacteria replication in the gastrointestinal tract of broiler chickens [32]. Moreover as expected, the inclusion of sub-therapeutic dose of antibiotic as positive control in the diet of broiler chickens Page 6/17 Page 6/17 led to higher BWG and FI than the birds fed basal diet without antibiotics which agrees with several other studies [33–35] suggesting that improvement in BWG might be due to increase in FI. led to higher BWG and FI than the birds fed basal diet without antibiotics which agrees with several other studies [33–35] suggesting that improvement in BWG might be due to increase in FI. The supplementation of antibiotics or bacteriophage to the basal diet did not have significant effect on nutrient digestibility. In line with the findings of Wang et al. [14], the ATTD of nutrients was not affected by the supplementation of increasing levels of BP. Further experiments are needed to confirm the lack of response of antibiotics or BP on nutrient digestibility. Salmonella is the major cause of food borne diseases worldwide with chickens as the main reservoir. Other zoonotic pathogens include Clostridium, Campylobacter, E. coli. For the control of these pathogens in poultry, bactericidal bacteriophages may provide a natural, nontoxic, feasible and non-expensive component. Previous works indicated that Salmonellae can be controlled by the use of bacteriophages [18, 36, 37, 38]. Early studies with E. coli also demonstrated that phage therapy can be as efficient as antibiotics [39, 40]. The reduction in E. coli and Salmonella counts in the excreta of broiler chickens by the application of bacteriophage has been reported [14]. Conversely, in the present study, dietary supplementation of BP did not have significant effect on the pathogenic bacteria such as E. coli, Salmonella as well as Clostridium counts isolated from the caecal digesta. However, a trend in linear increase in Lactobacillus count was observed in birds fed BP diets. The possible reason for non-significant effect of BP on nutrient digestibility and pathogenic food borne bacterial counts among the treatments might be the birds were raised in hygienic environment and were not experimentally challenged with bacteria due to which gastro intestinal tract might not have been colonized by harmful micro-organisms thereby maintaining gut in healthy state. Discussion The gastrointestinal microbiota plays a crucial role in gut associated host immune system. Moreover, the physiological development, health, and productivity is also influenced by gut microbiota. Poultry diets have tremendous impact on the gut microbiome in regard to diversity and composition [41]. The manipulation of the microbial community through the inclusion of feed additives such as phage is feasible in order to enhance chicken growth and control either human or animal pathogens. Several studies have reported the use of bacteriophages as a feed additive in animals in order to control bacteria transmitted by foodstuffs. These models include the use of phages to control Salmonella and Campylobacter in broiler chickens [8, 42]. Microbiome analysis showed that Firmicutes, Bacteroidetes, and Proteobacteria are the predominant phyla in the avian gut [43], which is also supported by the results from our study. In TR1 group, Firmicutes and Bacteroidetes were composed as predominant, whereas in TR2 group besides, Firmicutes, and Bacteroidetes the members of phylum Proteobacteria was also predominant. The presence of members of phylum Proteobacteria in TR2 may indicate that BP dose of 0.1% may not be favorable as increase in Proteobacteria may be associated to increase in E. coli. In PC group, in addition to Firmicutes, Bacteroidetes, Proteobacteria, members of phylum Deferribacteres (1.03%) was also present. Members of phylum Firmicutes was reduced whereas members of Proteobacteria and Bacteroidetes were increased in pigs receiving antibiotic treatment. The composition of microbiota at the genus and species level was modified, decreasing the abundance of Lactobacillus at the genus level in PC as compared with CON, TR1 and TR2 as well as an increase in the relative abundance of Prevotella and Bifidobacteria in phage treated groups compared with CON and PC groups. The genus Lactobacillus plays a crucial role in the homeostasis of the gastrointestinal tract of metazoans [44]. At the species level, the Lactobacillus salivarus population in ileum mucosa in phage treated groups was twofold as compared with CON and PC suggesting the efficacy of the phage in promoting the beneficial bacteria which eventually contributes in improved performance and gut health. With regards to meat quality, a significant quadratic response in the redness and lightness values of meat color was observed with the increase in the level of bacteriophage. Conclusions Collectively, the data from the present study indicate that the application of increasing level of bacteriophage cocktail to the diet of commercially raised broiler chickens could promote body weight gain as well as enhance gut microbiota diversity and increase excreta Lactobacillus counts without having significant difference in Salmonella, E. coli and Clostridium counts as compared with broilers fed antibiotic supplemented diet. Furthermore, it was observed that lower levels of bacteriophage cocktail addition were sufficient for supporting bursa and spleen which are immune organs. These findings suggest that phage therapy is effective and a safe alternative feed additive for raising broilers under intensive farming systems. Author’s contributions IHK, HBK, DKK, JYK and SWK designed the research; JMA, JHC and other lab members assisted with sampling, laboratory analyses and data generation. JMA, SDU and HBK analysed the data. SDU, IHK and HBK wrote the manuscript. All authors read and approved the final manuscript. List Of Abbreviations ADFI, average daily feed intake; ADG, average daily gain; ATTD, apparent total tract digestibility; BP, Bacteriophage; BW, body weight; DM, dry matter; FCR, feed conversion ratio; GE, gross energy; N, nitrogen Availability of data and materials All data generated or analyzed during this study are available from the corresponding All data generated or analyzed during this study are available from the corresponding author on request. or analyzed during this study are available from the corresponding author on request. Acknowledgments The authors are grateful to the lab members of the Department of Animal Resource and Science of Dankook University for their valuable assistance in conducting the experiments. We acknowledge CJ Cheiljedang Corp. Seoul, South Korea, for providing the financial support to conduct this research. Discussion Although, the meat color is closely associated with the meat pH [45], we found that the pH of breast muscle was not different among treatments indicating that change in color was not due to pH. In partial agreement to our finding, Wang et al. [14] demonstrated that meat pH as well as meat color were not Page 7/17 affected by the addition of bacteriophage in the basal diet of broiler chickens. Besides pH, other reported factors affecting color inside the muscle include myoglobin content, muscle fiber orientation and the space between the muscle fibers [46]. Further studies on these factors with bacteriophage application could help explain the changes in color observed. With regards to organ weight, a tendency to increase in the relative weight of gizzard in PC than NC and BP groups was observed. The possible reason for increase in the relative weight of gizzard in PC compared with NC may be due to increase in FI in PC groups. The weight of spleen and Bursa of Fabricius relative to the percentage of body weight in NC was higher than PC group during day 35. However, the inclusion of the increasing level of bacteriophage to NC diet linearly reduced the weight of spleen and Bursa of Fabricius relative to the percentage of the body weight indicating BP at 0.05% is better among the levels tested. As the spleen and bursa are associated with immune function (as lymphoid organs) this may explain that BP level higher than 0.05% may not be effective in improving the immune functions. References 1. Jones BA, Grace D, Kock R, Alonso S, Rushton J, Said MY, et al. Zoonosis emergence linked to agricultural intensification and environmental change. Proc Natl Acad Sci. 2013;110(21):8399- 1. Jones BA, Grace D, Kock R, Alonso S, Rushton J, Said MY, et al. Zoonosis emergence linked to agricultural intensification and environmental change. Proc Natl Acad Sci. 2013;110(21):8399- 2. 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New York: CABI Publishing; p. 159-75. 46. Barbut S. 2015. The Science of Poultry and Meat Processing. Accessed May 2020. http://www.poultryandmeatprocessing.com Tables Tables Page 11/17 Table 1 Table 1 Table 1 Ingredients composition and analyzed nutrient content of basal diets (as fed-basis). Phase1 Starter Finisher Ingredients, % Corn 55.84 61.57 Soybean meal 20.50 18.67 Corn gluten meal 14.73 10.35 Wheat bran 2.00 3.00 Soybean oil 3.00 3.00 Tri-calcium phosphate 1.81 1.29 Limestone 0.94 1.13 Salt 0.46 0.41 DL-Methionine (98%) 0.19 0.09 L-Lysine (98%) 0.23 0.19 Mineral mix2 0.10 0.10 Vitamin mix3 0.10 0.10 Choline 0.10 0.10 Calculated composition Metabolizable energy, kcal/kg 3184 3191 Analyzed composition Crude protein, % 22.79 19.90 Crude fat, % 5.51 5.63 Ash,% 5.59 5.06 Ca, % 0.92 0.85 Available P, % 0.40 0.29 Lysine, % 1.06 0.98 Methionine, % 0.45 0.36 1 Starter diet provided during d 1 to 21; Finisher diet provided during d 22 to 35. 2Provided per kg of complete diet: 11,025 IU vitamin A; 1103 IU vitamin D3; 44 IU vitamin E; 4.4 mg vitamin K; 8.3 mg riboflavin; 50 mg niacin; 4 mg thiamine; 29 mg d-pantothenic; 166 mg choline; 33 µg vitamin B12. 3Provided per kg of complete diet:12 mg Cu(as CuSO4.5 H2O); 85 mg Zn (as ZnSO4); 8 mg Mn (as MnO 2); 0.28 mg I (as KI); 0.15 mg Se (as Na2SeO3.5H2O). Tables Page 12/17 Table 2 The effect of bacteriophage cocktail supplementation on growth performance in broilers1 Items PC CON TR1 TR2 SEM2 P- value 0% 0.05% 1.0% PC vs CON CON vs TR1, TR2 PC vs TR1, TR2 Linear Quadratic d 1 to 7 BWG, g 114.33 109.55 111.37 116.92 2.092 0.1235 0.0896 0.9437 0.0383 0.5101 FI, g 138.68 135.74 139.2 141.19 3.049 0.5077 0.2476 0.6833 0.1746 0.8240 FCR 1.213 1.241 1.253 1.21 0.025 0.4302 0.749 0.5538 0.2499 0.2511 d 8 to 21 BWG, g 706.35 675.18 683.16 685.25 11.94 0.0813 0.5447 0.1471 0.6080 0.8671 FI, g 1017.19 969.53 985.43 990.12 12.93 0.0178 0.2644 0.0796 0.2770 0.7263 FCR 1.441 1.443 1.444 1.4471 0.032 0.9749 0.9422 0.9134 0.9344 0.9874 d 22 to 35 BWG, g 977.95 927.46 958.56 982.54 20.62 0.1006 0.1052 0.773 0.0379 0.8647 FI, g 1774.22 1741.25 1744.61 1755.91 27.8 0.4127 0.4943 0.4907 0.6542 0.8881 FCR 1.820 1.883 1.826 1.791 0.044 0.3226 0.1821 0.8333 0.0840 0.7902 Overall BWG, g 1798.63 1712.19 1753.09 1784.71 23.00 0.016 0.0593 0.3053 0.0288 0.8576 FI, g 2930.06 2846.52 2869.25 2887.22 30.64 0.0698 0.4902 0.1842 0.2470 0.9359 FCR 1.63 1.665 1.638 1.619 0.023 0.2881 0.2052 0.902 0.1370 0.8915 1 Abbreviation: CON, Basal diet without antibiotics or bacteriophage; PC, CON + 0.025% AVILAMIX®; TR1, CON + 0.05% Bacteriophage; TR2, CON + 0.10% Bacteriophage. 2 Standard error of means. Table 2 Values represent the means of 7 pens with 18 chickens per pen Page 13/17 Page 13/17 Page 13/17 Table 3 The effect of bacteriophage cocktail supplementation on apparent total tract nutrient digestibility in broilers1 Items,% PC CON TR1 TR2 SEM2 P- value 0% 0.05% 1.00% PC vs CON CON vs TR1, TR2 PC vs TR1, TR2 Linear Quadratic Day 35 Dry matter 71.50 69.85 70.26 70.68 0.899 0.2104 0.5788 0.3625 0.4715 0.9976 Nitrogen 71.03 68.63 69.99 70.24 1.660 0.3209 0.4747 0.6592 0.4716 0.7731 Energy 71.76 69.92 70.55 70.86 0.918 0.1745 0.4923 0.3638 0.4338 0.8755 1 Abbreviation: CON, Basal diet without antibiotics or bacteriophage; PC, CON + 0.025% AVILAMIX®; TRT1, CON + 0.05% Bacteriophage; TRT2, CON + 0.10% Bacteriophage. 2 Standard error of means. Values represent the means of 7 pens with 18 chickens per pen. Table 4 The effect of bacteriophage cocktail supplementation on excreta microbial counts in broilers1 Items, log 10 cfu/ml PC CON TR1 TR2 SEM2 P- value 0% 0.05% 1.00% PC vs CON CON vs TR1, TR2 PC vs TR1, TR2 Linear Quadratic Day 35 Lactobacillus 8.93 8.99 9.115 9.206 0.096 0.6578 0.1551 0.058 0.0833 0.8632 E.coli 5.524 5.552 5.566 5.706 0.129 0.8789 0.5985 0.4835 0.3863 0.6798 Clostridium perfringens 5.512 5.601 5.543 5.529 0.142 0.6607 0.7111 0.8911 0.6689 0.8789 Salmonella 4.096 4.168 4.128 4.118 0.114 0.6581 0.7493 0.8478 0.733 0.9058 1 Abbreviation: CON, Basal diet without antibiotics or bacteriophage; PC, CON + 0.025% AVILAMIX®; TR1, CON + 0.05% Bacteriophage; TR2, CON + 0.10% Bacteriophage. 2 Standard error of means. Values represent the means of 7 pens with 18 chickens per pen. Table 3 The effect of bacteriophage cocktail supplementation on apparent total tract nutrient digestibility in broilers1 Items,% PC CON TR1 TR2 SEM2 P- value 0% 0.05% 1.00% PC vs CON CON vs TR1, TR2 PC vs TR1, TR2 Linear Quadratic Day 35 Dry matter 71.50 69.85 70.26 70.68 0.899 0.2104 0.5788 0.3625 0.4715 0.9976 Nitrogen 71.03 68.63 69.99 70.24 1.660 0.3209 0.4747 0.6592 0.4716 0.7731 Energy 71.76 69.92 70.55 70.86 0.918 0.1745 0.4923 0.3638 0.4338 0.8755 1 Abbreviation: CON, Basal diet without antibiotics or bacteriophage; PC, CON + 0.025% AVILAMIX®; TRT1, CON + 0.05% Bacteriophage; TRT2, CON + 0.10% Bacteriophage. 2 Standard error of means. Values represent the means of 7 pens with 18 chickens per pen. Page 13/17 Table 4 The effect of bacteriophage cocktail supplementation on excreta microbial counts in broilers1 Items, log 10 cfu/ml PC CON TR1 TR2 SEM2 P- value 0% 0.05% 1.00% PC vs CON CON vs TR1, TR2 PC vs TR1, TR2 Linear Quadratic Day 35 Lactobacillus 8.93 8.99 9.115 9.206 0.096 0.6578 0.1551 0.058 0.0833 0.8632 E.coli 5.524 5.552 5.566 5.706 0.129 0.8789 0.5985 0.4835 0.3863 0.6798 Clostridium perfringens 5.512 5.601 5.543 5.529 0.142 0.6607 0.7111 0.8911 0.6689 0.8789 Salmonella 4.096 4.168 4.128 4.118 0.114 0.6581 0.7493 0.8478 0.733 0.9058 1 Abbreviation: CON, Basal diet without antibiotics or bacteriophage; PC, CON + 0.025% AVILAMIX®; TR1, CON + 0.05% Bacteriophage; TR2, CON + 0.10% Bacteriophage. 2 Standard error of means. Values represent the means of 7 pens with 18 chickens per pen. Table 3 The effect of bacteriophage cocktail supplementation on apparent total tract nutrient digestibility in broilers1 Items,% PC CON TR1 TR2 SEM2 P- value 0% 0.05% 1.00% PC vs CON CON vs TR1, TR2 PC vs TR1, TR2 Linear Quadratic Day 35 Dry matter 71.50 69.85 70.26 70.68 0.899 0.2104 0.5788 0.3625 0.4715 0.9976 Nitrogen 71.03 68.63 69.99 70.24 1.660 0.3209 0.4747 0.6592 0.4716 0.7731 Energy 71.76 69.92 70.55 70.86 0.918 0.1745 0.4923 0.3638 0.4338 0.8755 1 Abbreviation: CON, Basal diet without antibiotics or bacteriophage; PC, CON + 0.025% AVILAMIX®; TRT1, CON + 0.05% Bacteriophage; TRT2, CON + 0.10% Bacteriophage. 2 Standard error of means. Values represent the means of 7 pens with 18 chickens per pen. Table 3 Table 3 Values represent the means of 7 pens with 18 chickens per pen. Table 4 The effect of bacteriophage cocktail supplementation on excreta microbial counts in broilers1 Items, log 10 cfu/ml PC CON TR1 TR2 SEM2 P- value 0% 0.05% 1.00% PC vs CON CON vs TR1, TR2 PC vs TR1, TR2 Linear Quadratic Day 35 Lactobacillus 8.93 8.99 9.115 9.206 0.096 0.6578 0.1551 0.058 0.0833 0.8632 E.coli 5.524 5.552 5.566 5.706 0.129 0.8789 0.5985 0.4835 0.3863 0.6798 Clostridium perfringens 5.512 5.601 5.543 5.529 0.142 0.6607 0.7111 0.8911 0.6689 0.8789 Salmonella 4.096 4.168 4.128 4.118 0.114 0.6581 0.7493 0.8478 0.733 0.9058 1 Abbreviation: CON, Basal diet without antibiotics or bacteriophage; PC, CON + 0.025% AVILAMIX®; TR1, CON + 0.05% Bacteriophage; TR2, CON + 0.10% Bacteriophage. 2 Standard error of means. Values represent the means of 7 pens with 18 chickens per pen. Page 14/17 Page 14/17 Page 14/17 Table 5 Table 5 The effect of bacteriophage cocktail supplementation on meat quality and organ weight in broilers1 Items PC CON TR1 TR2 SEM2 P- value 0% 0.05% 1.00% PC vs CON CON vs TR1, TR2 PC vs TR1, TR2 Linear Quadratic pH value 7.49 7.52 7.54 7.54 0.0287 0.3845 0.6189 0.1392 0.6821 0.8963 Breast muscle color Lightness (L) 56.51 57.81 54.97 59.07 1.011 0.3689 0.5285 0.6803 0.3749 0.0096 Redness (a) 12.81 13.02 14.05 11.85 0.457 0.7455 0.9000 0.8030 0.0598 0.0050 Yellowness (b) 10.80 10.73 11.71 11.65 0.67 0.9449 0.2592 0.2932 0.3568 0.5366 WHC, % 54.79 55.26 54.69 54.97 2.465 0.8931 0.8886 0.9880 0.9279 0.8783 Drip loss, % d 1 3.43 3.96 3.58 3.77 0.304 0.2314 0.4583 0.5141 0.6853 0.4802 d 3 5.29 5.48 5.58 5.34 0.126 0.3022 0.9057 0.2832 0.4853 0.3250 d 5 9.17 9.74 9.65 9.16 0.374 0.2913 0.4701 0.6140 0.2868 0.6650 d 7 13.93 14.66 14.20 14.10 0.414 0.2284 0.3322 0.6660 0.3787 0.7361 Relative organ weight, % Breast muscle 24.28 22.97 23.46 24.07 0.592 0.1290 0.2815 0.4844 0.2076 0.9382 Liver 2.74 2.72 2.72 2.62 0.122 0.8969 0.7486 0.6388 0.5869 0.7342 Bursa of Fabricius 0.12 0.13 0.12 0.11 0.006 0.0464 0.0226 0.9944 0.0262 0.6886 Abdominal fat 1.19 1.11 1.21 1.21 0.067 0.4196 0.2395 0.7995 0.3781 0.5717 Spleen 0.18 0.19 0.18 0.17 0.006 0.1444 0.0667 0.8625 0.0521 0.8386 Gizzard 1.13 1.12 1.05 1.05 0.031 0.8796 0.0847 0.0596 0.1135 0.2432 1 Abbreviation: CON, Basal diet without antibiotics or bacteriophage; PC, CON + 0.025% AVILAMIX®; TR1, CON + 0.05% Bacteriophage; TR2, CON + 0.10% Bacteriophage. 2 St d d f Values represent the means of 10 chickens that are randomly selected (including at least 1 from each pen) Figures Page 15/17 Page 15/17 Figure 1 Principal coordinates analysis (PCoA) plots based on unweighted UniFrac distance metrics showing difference in microbial community structure between CON, Basal diet (green), PC, CON + 0.025% Avilamix (red), TR1, CON + 0.05% Bacteriophage (blue), and TR2, CON + 0.10% Bacteriophage (orange) group. Figure 1 Principal coordinates analysis (PCoA) plots based on unweighted UniFrac distance metrics showing difference in microbial community structure between CON, Basal diet (green), PC, CON + 0.025% Avilamix (red), TR1, CON + 0.05% Bacteriophage (blue), and TR2, CON + 0.10% Bacteriophage (orange) group. Page 16/17 Figure 2 Figure 2 Page 16/17 Page 16/17 Taxonomic classification of the 16S rRNA gene sequences at the (a) phylum and (b) genus levels in the gut microbiome of broiler fed CON, Basal diet without antibiotics/bacteriophage; PC, CON + 0.025% Avilamix®; TR1, CON + 0.05% Bacteriophage; TR2, CON + 0.10% Bacteriophage. Taxonomic classification of the 16S rRNA gene sequences at the (a) phylum and (b) genus levels in the gut microbiome of broiler fed CON, Basal diet without antibiotics/bacteriophage; PC, CON + 0.025% Avilamix®; TR1, CON + 0.05% Bacteriophage; TR2, CON + 0.10% Bacteriophage. Figure 3 The bar plot identifying the difference in taxa between the gut microbiome of broiler fed CON, Basal diet without antibiotics or bacteriophage; PC, CON + 0.025% Avilamix®; TR1, CON + 0.05% Bacteriophage; TR2, CON + 0.10% Bacteriophage groups at the genus (a, b) and species (c, d) level. The numbers on each bar indicates the normalized abundance of each strains. Figure 3 The bar plot identifying the difference in taxa between the gut microbiome of broiler fed CON, Basal diet without antibiotics or bacteriophage; PC, CON + 0.025% Avilamix®; TR1, CON + 0.05% Bacteriophage; TR2, CON + 0.10% Bacteriophage groups at the genus (a, b) and species (c, d) level. The numbers on each bar indicates the normalized abundance of each strains. Page 17/17 Page 17/17 Page 17/17
https://openalex.org/W4323971677	https://zenodo.org/records/7723365/files/IJCSTR2023000111.pdf	English	null	ALGORITHM FOR CLASSIFYING DOCUMENTS OF A SCIENTIFIC AND EDUCATIONAL ORGANIZATION USING MACHINE LEARNING METHODS	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	4,537	1 Graduate Student, Tashkent University of Information Technologies named after Muhammad al-Khwarizmi, Tashkent, Uzbekistan, (zebiniso_777@mail.ru) 2 Assistant of the Department of Information systems and technologies, Jizzakh branch of the National University of Uzbekistan named after Mirzo Ulugbek, Jizzakh, Uzbekistan, (ergashev@jbnuu.uz) International Journal of Contemporary Scientific and Technical Research Journal home page: http://ijournal.uz/index.php/jartes International Journal of Contemporary Scientific and Technical Research Journal home page: http://ijournal.uz/index.php/jartes ALGORITHM FOR CLASSIFYING DOCUMENTS OF A SCIENTIFIC AND EDUCATIONAL ORGANIZATION USING MACHINE LEARNING METHODS Zebiniso Abduvalieva1 Sirojiddin Ergashev2 Tashkent University of Information Technologies named after Muhammad al-Khwarizmi, Jizzakh branch of the National University of Uzbekistan named after Mirzo Ulugbek ABSTRACT Intelligent analysis is used in almost all areas of technology. Machine learning does not stand still and is constantly evolving. Given the transition in modern society to electronic document management, the main assumption in them is that the training and test data must be in the same feature space and follow the same distribution. In real applications, this is not always the case. In this case, the role of transfer learning can be distinguished since transfer learning does not make the same distributional assumptions as traditional machine learning and reduces dependencies on the target task and training data, and has a wider knowledge migration. The article proposes a transfer learning algorithm for document categorization based on clustering. An experiment is also used to test the algorithm. The experiment shows that the algorithm proposed in this article has its advantages. transfer learning, machine learning; classification of documents; ; data mining, based spatial clustering transfer learning, machine learning; classification of documents; ; data mining, based spatial clustering 2181-3884/©2023 in Jizzakh branch of the National University of Uzbekistan. DOI: 10.5281/zenodo.7723365 This is an open access article under the Attribution 4.0 International (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/deed.ru) 2181-3884/©2023 in Jizzakh branch of the National University of Uzbekistan. DOI: 10.5281/zenodo.7723365 This is an open access article under the Attribution 4.0 International (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/deed.ru) 2181-3884/©2023 in Jizzakh branch of the National University of Uzbekistan. DOI: 10.5281/zenodo.7723365 This is an open access article under the Attribution 4.0 International (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/deed.ru) 1 Graduate Student, Tashkent University of Information Technologies named after Muhammad al-Khwarizmi, Tashkent, Uzbekistan, (zebiniso_777@mail.ru) 2 Assistant of the Department of Information systems and technologies, Jizzakh branch of the National University of Uzbekistan named after Mirzo Ulugbek, Jizzakh, Uzbekistan, (ergashev@jbnuu.uz) International Journal of Contemporary Scientific and Technical Research (IJCSTR) SPECIAL ISSUE / ISSN 2181-3884 KALIT SO‘ZLAR: Intellektual tahlil texnologiyaning deyarli barcha sohalarida qo'llaniladi. Mashinani o'rganish bir joyda turmaydi va doimo rivojlanib boradi. Zamonaviy jamiyatda elektron hujjat aylanishiga o'tishni hisobga olgan holda, ulardagi asosiy taxmin shundan iboratki, o'quv va test ma'lumotlari bir xil xususiyat maydonida bo'lishi va bir xil taqsimotga amal qilishi kerak. Haqiqiy ilovalarda bu har doim ham shunday emas. Bunday holda, transfer o'rganishning rolini ajratib ko'rsatish mumkin, chunki transferni o'rganish an'anaviy mashinani o'rganish kabi bir xil taqsimlash taxminlarini qilmaydi va maqsadli vazifa va o'quv ma'lumotlariga bog'liqlikni kamaytiradi va kengroq bilim migratsiyasiga ega. Maqolada klasterlash asosida hujjatlarni turkumlashtirish uchun uzatishni o'rganish algoritmi taklif etiladi. Algoritmni sinab ko'rish uchun tajriba ham qo'llaniladi. Tajriba shuni ko'rsatadiki, ushbu maqolada taklif qilingan algoritm o'zining afzalliklariga ega. transferni o'rganish, mashinani o'rganish; hujjatlarni tasniflash; ma'lumotlarni qazib olish, fazoviy klasterlash ANNOTATSIYA KALIT SO‘ZLAR: I. INTRODUCTION Most machine learning and data mining algorithms usually assume that the training and test data have the same feature space and data distribution, but in a real application, these two factors often change, so the trained model becomes outdated very easily.When the existing training data is outdated and there is very little new data, or labeling the new data is expensive, you might consider using the existing training data but a different distribution with test data to help the new data learn what transfer learning is. Cluster analysis comes from many areas of research, including data mining, statistics, biology, and machine learning. The main clustering methods include partitioning methods, hierarchical methods, density-based methods, grid-based methods, and model-based methods. In the algorithm considered and used below, D is a set (collection) of text documents of the department, W is a set (dictionary) of all words used in them, C is a set of document categories fixed in advance. Each document d ∈ D is a sequence of words слов (w1, . . . , wnd )w1,… ,wd from dictionary W, where d is the length of the document in words. The same word can be repeated many times in a document. The categorization problem is the problem of assigning a boolean value to each pair {d, c} ∈D ∗C.. Boolean value 1 means document d belongs to category c, while a value of 0 means the opposite. More formally, the categorization problem is the problem of recovering an unknown objective function Φ: D ∗ C →{1,0}. 104 International Journal of Contemporary Scientific and Technical Research (IJCSTR) SPECIAL ISSUE / ISSN 2181-3884 In many document categories, the training data set from the source field is always out of date, but if some existing stale data is similar to the test data in the target field, then we might consider using clustering technology to find them to help train targets and goals. In this article, the density-based spatial clustering (DSP) algorithm is applied to classify documents in higher education institutions. In this case, the documentation of the Department of "System and Software" at the Institute of Tashkent University of Information Technologies. The essence of classification is to reduce and filter characters and repeat words. Information in documents is treated as character labels and has no additional meaning. One of the difficulties of categorizing documents is the high dimension of the feature element space. I. INTRODUCTION Characteristic elements in the categorization of documents mainly refer to words obtained as a result of text processing, and the dimension of a functional element is equal to the number of different words. In this article, density-based spatial clustering The problem of non-hierarchical categorization can be considered as a multi-class classification problem, for which the set of classes is the set of categories C, the set of objects is the set of documents D, and the set of precedents is a previously known set of pairs {d,c}, where d ∈ D, c ∈ C . II. MATERIAL AND METHODS There are three approaches to solving the text classification problem: supervised learning, unsupervised learning, and confirmation learning. One of the popular approaches of most interest is classification based on machine learning. With this approach, the training of the classifier (a system of naming objects, each of which corresponds to a unique identifier) is carried out on a set of initial training data in the form of documents with categories assigned In formula (1), the vertical and horizontal coordinates are the keyword list index. aij (i ≠j) indicates the number of words iw and jwoccurring together in the same document. aij (i −1,2, … n) expresses the frequency of words. The distance between words can be determined using the word matching matrix. The higher the frequency of matching two words, the smaller the distance between them. The transformation formula used here is: In formula (1), the vertical and horizontal coordinates are the keyword list index. aij (i ≠j) indicates the number of words iw and jwoccurring together in the same document. aij (i −1,2, … n) expresses the frequency of words. The distance between words can be determined using the word matching matrix. The higher the frequency of matching two words, the smaller the distance between them. The transformation formula used here is: 105 International Journal of Contemporary Scientific and Technical Research (IJCSTR) SPECIAL ISSUE / ISSN 2181-3884 d(wi,wj) = 1 (1+co_words(wi,wj)) (2) (2) co_words(wi,wj)in formula (2) shows the number of words wi, wj in the same document, denominator plus 1 to eliminate the possibility of infinite distance while protecting the standardized requirements.Using formula (1), formula 2aijcan be converted into a distance to obtain a word matching matrix of documents. To initialize the Eps radius, a minimum number of points is considered using a "density-based spatial clustering" algorithm to achieve word clustering. The cluster should be output after clustering and cluster processing. An isolated point is removed. The more closely the words are connected in a cluster, the farther it is. Definitions in the "Density Based Spatial Clustering" algorithm: dense area: for each point in the cluster, the circle with radius contains at least the minimum number of points (Min points). II. MATERIAL AND METHODS The Epsilon neighborhood of a point P in the database is determined by the following formula: The Epsilon neighborhood of a point P in the database is determined by the following formula: N(p) = {qϵD\|dist(p, q) ≤∈} (3) N(p) = {qϵD\|dist(p, q) ≤∈} (3) With feature clustering, the new model of the vector space of documents can be expressed as the i-th cluster. With feature clustering, the new model of the vector space of documents can be expressed as the i-th cluster. If we consider N as the number of documents in collection D. If we consider N as the number of documents in collection D. The weight formula will look like this[4]: wi(d) = TF∗log2(N Nt+β ⁄ ) √∑ TF∗[log2(N Nt+β ⁄ )] n i=1 (5) Formula (5) is directly related to the vector space model, where TF is the word frequency of the characteristic word in the tth feature cluster, and Ntrefers to the number of documents in the collection. The characteristic word appears in the tth characteristic cluster. The feature cluster weight can also be obtained by accumulating each feature word in a member of the cluster. The similarity between two documents can be obtained by calculating the cosine of the angle between the two vectors, assuming that the two documents d1 = (t1,w1, t2, w2, … , tn,wn) и d1 = (t1, x1, t2,x2, … , tn,xn) similarities between formulas: similarities between formulas: sim(d1, d2) = cos ∝= ∑ ϖi∗xi n i=1 (∑ ϖ2 n i=1 ∗∑ xi 2 n i=1 ) 1 2 (6) The categorization task means assigning a boolean value to each pair of fdf {d. c} ∈D ∗C A boolean value of 1 means that document d belongs to the given category, while a value of 0 means the opposite. The categorization problem is the problem of recovering an unknown objective function : Ф: D ∗C →{1,0} 106 International Journal of Contemporary Scientific and Technical Research (IJCSTR) SPECIAL ISSUE / ISSN 2181-3884 All categories can be considered symbolic labels, and their meaning does not have any additional meaning. All categories can be considered symbolic labels, and their meaning does not have any additional meaning. When categorizing documents, first of all, the secondary training data with the target training data are combined for clustering. II. MATERIAL AND METHODS The secondary training data that is not collected together with the training target data in the same cluster is filtered out. The rest is higher than the target data, and it will be trained along with the training target data. This will greatly improve the classification performance. Some definitions will be given for the main characters used in the article. If you set the target pattern space to F′ and F for the secondary pattern space, Y = If you set the target pattern space to F′ and F for the secondary pattern space, Y = {0, 1} for the class space. test data set: Sc = {(x1 c, x2 c, … , xp c)}, xk c ϵF′ , k = 1.2.3, … p. The training dataset consists of two parts : the target training dataset: D′ = {(x1 t, y1 t), (x2 t , y2 t),… , (xn t , yn t )} xi tϵFt , yi tϵ Y(i = 1,2, … n) and a set of secondary training data : Ds = {(x1 s, y1 s), (x2 s, y2 s),… , (xn s, yn s)} xj sϵFs , yj sϵ Y(i = 1,2, … m) weight of samples in D′is: w1 , t w2 t, … , wn t {}; prediction objective function: h(xi): xi →yi , P̃(xi 1)~ is the prior probability of xi 1 в D′. The training dataset consists of two parts : the target training dataset: D′ = {(x1 t, y1 t), (x2 t , y2 t),… , (xn t , yn t )} xi tϵFt , yi tϵ Y(i = 1,2, … n) and a set of secondary training data : Ds = {(x1 s, y1 s), (x2 s, y2 s),… , (xn s, yn s)} xj sϵFs , yj sϵ Y(i = 1,2, … m) weight of samples in D′is: w1 , t w2 t, … , wn t {}; prediction objective function: h(xi): xi →yi , P̃(xi 1)~ is the prior probability of xi 1 в D′. Algorithm steps: Algorithm steps: The training data sets DSand the target data set Dt, the test data set S are fed into the input. II. MATERIAL AND METHODS Output: classified h′ (x′) a) into the input DS and Dt set p̃(xj) = wi/∑ wi ni i=1 ; a) into the input DS and Dt set p̃(xj) = wi/∑ wi ni i=1 ; b) according to the class standard, the training data can be divided into N classes: Di (i = 1,2,3, … N) , Di (i = 1, 2,``` N), где Di where Di means the set of instance classes labeled i; c) for i = 1 to N; c) for i = 1 to N; c) for i = 1 to N; d) The k means clustering algorithm for Di clustering is invoked and returns the clustering results; e) scan Di deleting a cluster of instances in secondary data that were not collected along with the target data; g) derived a classification model from the filtered training data and test data S by calling the KNN algorithm h(xi)(xi ∈Ft ∪ FS); h) calculate the error rate h(xt)on Dt g) derived a classification model from the filtered training data and test data S by calling the KNN algorithm h(xi)(xi ∈Ft ∪FS); g g ( )( ) h) calculate the error rate h(xi t)on Dt δt = ∑p̃ ni j=1 \|(xi t −yi t)\| set β = 1 2 ln(1 −δt)/δt ⁄ ; ( )/ i) update the weight vector of the target training data, the weight of the first K + 1 iterations is i) update the weight vector of the target training data, the weight of the first K + 1 iterations is wi t(k + 1) = {wi t(k)e−β,h(xi t) = yi t wi t(k)e−β,h(xi t) ≠yi t(6) 107 International Journal of Contemporary Scientific and Technical Research (IJCSTR) SPECIAL ISSUE / ISSN 2181-3884 International Journal of Contemporary Scientific and Technical Research (IJCSTR) SPECIAL ISSUE / ISSN 2181-3884 at the entrance: j) hi(xt) {1, ∏ βt −hi t (x) ≥∏ βt −1/2 N t=[N/2] N t=[N/2] 0, otherwise (7) IV. RESULTS The result after CLUSTERING data set before clustering after clustering Students' documents 532 250 Plan 400 190 Scientific works 500 220 Teachers documents 380 230 Departments document 450 341 0 100 200 300 400 500 600 Students' documents Plan Scientific works Teachers documents Departments document Results after clustering before clustering ACKNOWLEDGMENT The work is carried out with the Department of System and Practical Programming of TUIT. based on the documentation of the department. The work is carried out with the Department of System and Practical Programming of TUIT. based on the documentation of the department. III. Application implementation RESULTS The result after CLUSTERING data set before clustering after clustering Students' documents 532 250 Plan 400 190 Scientific works 500 220 Teachers documents 380 230 Departments document 450 341 ACKNOWLEDGMENT The work is carried out with the Department of System and Practical Programming TUIT. based on the documentation of the department. VI. CONCLUSION This article considers the task of classifying documents in the electronic document 0 100 200 300 400 500 600 Students' documents Plan Scientific works Teachers documents Departments document Results after clustering before clustering III. Application implementation III. Application implementation In the experiments, I used the documents of the Department of Systematic Practical Programming of the Tashkent University of Information Technologies. The data set contains categories of documents of the department, including information about the department, about students, articles, etc. Each large class also contains several sub-categories below and includes a total of 500 documents. In the experiment, the main categories are selected: department documents; documents related to teachers; and documents related to students, including the annual report, plans, and rating. In each selected category, there are subcategories. We chose the main categories because the objectives of the class and secondary documents are outdated. For example, the annual report of the department means that we select the report for 2021 and 2022 as target categories, for example, financial (wealth, consumption). The specific data distribution is shown in Table 1. TABLE I. DEPARTMENT DATA DISTRIBUTION Target dataset Initial Initial training data Auxiliary data Students' documents Themes of diploma work and master's theses Written work by part-time students and reviews Documents on student practice Documents on student practice Plan document control/documents to test students' knowledge (bases of written and oral control questions, written and electronic test questions, options for written work, block modules, and so on). report on spiritual and educational work. Scientific works Reviews of scientific works Diplomas, dissertations Teachers documents Documents of cooperation with professional colleges and enterprises of the department articles of teachers of the department / Individual work plans of teachers of the department Department documents Orders of the dean of the faculty and information on their implementation / Decisions of the University Council, Methodological Council, Faculty Academic Council (copy) Certificates and instructions of the rector and vice-rectors of the university on the activities of the department and their implementation The study compared the transfer learning algorithm, which is based on the application of knowledge gained from other studies, with the density-based spatial clustering algorithm. The article proposes a transfer learning algorithm based on clustering. The feature cluster is first achieved by the algorithm, then the algorithm is used for the dataset cluster, 108 International Journal of Contemporary Scientific and Technical Research (IJCSTR) SPECIAL ISSUE / ISSN 2181-3884 and finally, after the weight adjustment strategy, the K-means algorithm is used to classify the documents into different classes. IV. VI. CONCLUSION Ng MK, Wu Q, Ye Y. Collaborative learning with a method based on a joint transition probability graph. In: Proceedings of the 1st International Workshop on Revealing Cross- Domain Knowledge in Web and Social Networks. 2012. [5] 6. W. Dai, Q. Yang, G. Xue and Y.Yu, “Boosting for Transfer learning”, Proc.24 International Conference. Machine Learning, pp.193-200, June 2007. [6] 6. W. Dai, Q. Yang, G. Xue and Y.Yu, “Boosting for Transfer learning”, Proc.24 International Conference. Machine Learning, pp.193-200, June 2007. [6] 7. Xinno Jialing Pan, Yang K. Survey on transfer learning. IEEE TKDE, 2009. [7] 7. Xinno Jialing Pan, Yang K. Survey on transfer learning. IEEE TKDE, 2009. [7] 8. Dai V, Yang Q, Xue GR, Yu Yu. Boosting for transfer learning. Proceedings of the Twenty-fourth International Conference on Machine Learning, 2007: 193 × 200. [8] 8. Dai V, Yang Q, Xue GR, Yu Yu. Boosting for transfer learning. Proceedings of the Twenty-fourth International Conference on Machine Learning, 2007: 193 × 200. [8] 9. Dai Wee, Chen WK, Xue GR, Yang Q, Yu Yu. Transfer learning: Transferring learning to different functional spaces. Advances in Neural Information Processing Systems 21, 2009. [9] 9. Dai Wee, Chen WK, Xue GR, Yang Q, Yu Yu. Transfer learning: Transferring learning to different functional spaces. Advances in Neural Information Processing Systems 21, 2009. [9] 10. Юсупович Ҳ. Ж., Эргашев С. Б. Ў. МAКТAБ ЎҚУВЧИЛAPИДA AXБOPOТ БИЛAН ИШЛAШ КOМПEТEНЦИЯСИНИ PИВOЖЛAНТИPИШИ МОДЕЛИ //JOURNAL OF INNOVATIONS IN SCIENTIFIC AND EDUCATIONAL RESEARCH. – 2022. – Т. 2. – №. 13. – С. 189-194. 10. Юсупович Ҳ. Ж., Эргашев С. Б. Ў. МAКТAБ ЎҚУВЧИЛAPИДA AXБOPOТ БИЛAН ИШЛAШ КOМПEТEНЦИЯСИНИ PИВOЖЛAНТИPИШИ МОДЕЛИ //JOURNAL OF INNOVATIONS IN SCIENTIFIC AND EDUCATIONAL RESEARCH. – 2022. – Т. 2. – №. 13. – С. 189-194. 11. Anvar o‘g‘li A. B., Baxtiyor o‘g‘li E. S. MY. GOV. UZ SAYTI LOYIHASINI ISHLAB CHIQISH. IDEF0, DFD VA IDEF3 DIZAYN METODOLOGIYALARIDAN FOYDALANISH //Journal of new century innovations. – 2022. – Т. 11. – №. 6. – С. 41-46. 12. Naim o’g’li M. D., Baxtiyor o‘g’li E. S. DATA SCIENCE METHODOLOGY IN LEARNING PROGRAMMING //JOURNAL OF INNOVATIONS IN SCIENTIFIC AND EDUCATIONAL RESEARCH. – 2022. – Т. 2. – №. 13. – С. 207-210. 13 Naim o’g’li M D Baxtiyor o‘g’li E S KATTA HAJMDAGI MA'LUMOTLARINI TAHLIL 11. Anvar o‘g‘li A. B., Baxtiyor o‘g‘li E. S. MY. GOV. UZ SAYTI LOYIHASINI ISHLAB CHIQISH. VI. CONCLUSION This article considers the task of classifying documents in the electronic document management system of a scientific and educational institution. A comparative analysis of existing approaches to machine learning was carried out, on the basis of which it was concluded that the use of transfer learning gives a more effective result in the classification of documents. educational institution to improve the quality of classification and. For solving the classification problem it is also necessary to select certain to which the initial set of documents will be distributed, for which the algorithm presented in the article is proposed. Thus, the algorithmic support presented in the article can be used as a theoretical 109 International Journal of Contemporary Scientific and Technical Research (IJCSTR) SPECIAL ISSUE / ISSN 2181-3884 basis for the integration of machine learning methods in the analysis and classification of documents of a scientific and educational institution. REFERENCES basis for the integration of machine learning methods in the analysis and classification of documents of a scientific and educational institution. 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https://openalex.org/W3109613417	https://aip.scitation.org/doi/pdf/10.1063/5.0027994	English	null	Normal mode analysis of spectral density of FMO trimers: Intra- and intermonomer energy transfer	Journal of chemical physics online/The Journal of chemical physics/Journal of chemical physics	2,020	cc-by	22,330	RESEARCH ARTICLE \| DECEMBER 04 2020 RESEARCH ARTICLE \| DECEMBER 04 2020 Normal mode analysis of spectral density of FMO trimers: Intra- and intermonomer energy transfer J. Chem. Phys. 153, 215103 (2020) https://doi.org/10.1063/5.0027994 J. Chem. Phys. 153, 215103 (2020) https://doi.org/10.1063/5.0027994 Articles You May Be Interested In Influence of weak vibrational-electronic couplings on 2D electronic spectra and inter-site coherence in weakly coupled photosynthetic complexes J. Chem. Phys. (August 2015) Do coupling exciton and oscillation of electron-hole pair exist in neutral and charged π -dimeric quinquethiophenes? J. Chem. Phys. (August 2007) 24 October 2024 05:45:55 Simulated two-dimensional electronic spectroscopy of the eight-bacteriochlorophyll FMO complex J. Chem. Phys. (December 2014) ABSTRACT The intermolecular contribution to the spectral density of the exciton-vibrational coupling of the homotrimeric Fenna–Matthews–Olson (FMO) light-harvesting protein of green sulfur bacteria P. aestuarii is analyzed by combining a normal mode analysis of the protein with the charge density coupling method for the calculation of local transition energies of the pigments. Correlations in site energy fluctuations across the whole FMO trimer are found at low vibrational frequencies. Including, additionally, the high-frequency intrapigment part of the spectral density, extracted from line-narrowing spectra, we study intra- and intermonomer exciton transfer. Whereas the intrapigment part of the spectral density is important for fast intramonomer exciton relaxation, the intermolecular contributions (due to pigment-environment coupling) determine the intermonomer exciton transfer. Neither the variations of the local Huang–Rhys factors nor the correlations in site energy fluctuations have a critical influence on energy transfer. At room temperature, the intermonomer transfer in the FMO protein occurs on a 10 ps time scale, whereas intramonomer exciton equilibration is roughly two orders of magnitude faster. At cryogenic temperatures, intermonomer transfer limits the lifetimes of the lowest exciton band. The lifetimes are found to increase between 20 ps in the center of this band up to 100 ps toward lower energies, which is in very good agreement with the estimates from hole burning data. Interestingly, exciton delocalization in the FMO monomers is found to slow down intermonomer energy transfer, at both physiological and cryogenic temperatures. © 2020 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license 24 October 2024 05:45:55 © 2020 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0027994., s AFFILIATIONS Institut für Theoretische Physik, Johannes Kepler Universität Linz, Altenberger Str. 69, 4040 Linz, Austria Note: This paper is part of the JCP Special Topic on Excitons: Energetics and Spatio-temporal Dynamics. a)Author to whom correspondence should be addressed: thomas.renger@jku.at Normal mode analysis of spectral density of FMO trimers: Intra- and intermonomer energy transfer Cite as: J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 Submitted: 1 September 2020 • Accepted: 25 October 2020 • Published Online: 4 December 2020 Cite as: J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 Submitted: 1 September 2020 • Accepted: 25 October 2020 • Published Online: 4 December 2020 Alexander Klinger, Dominik Lindorfer, Frank Müh, and Thomas Rengera) The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp Normal mode analysis of spectral density of FMO trimers: Intra- and intermonomer energy transfer I. INTRODUCTION Excitation energy absorb y the outer chlorosome antenna is transferred through the FMO protein to eaction center complex. The phytyl chains of the pigments were truncated for b er visibility and the protein is shown in ribbon style Note that we have includ The inhomogeneous charge distribution of the protein gen- erates a site energy funnel in the FMO monomer, where those pigments that are closer to the reaction center complex are more redshifted than those facing the baseplate and the outer chloro- some.3–5,7,33,34 Therefore, the low-energy exciton states are localized close to the reaction center complex, and directed energy trans- fer becomes possible. In addition, the inhomogeneous charge dis- tribution of the protein also leads to different coupling constants between the vibrational normal modes of the complex and the elec- tronic excitations of the pigments (site energies).9 In this way, cor- relations between site energy fluctuations are suppressed, and the excess energy of excitons can be dissipated efficiently. In our ear- lier work9 on the spectral density of the monomeric subunit of the FMO protein, we found correlations in site energy fluctuations, but only at vibrational frequencies, which are too small to affect exciton relaxation. One of the aims of the present study is to investigate whether these correlations in site energy fluctuations extend over the whole FMO trimer and, if so, how they influence the energy transfer between different FMO monomers. Due to the C3 symmetry of the complex, we expect similar local exciton-vibrational coupling con- stants of equivalent sites and similar exciton energies in the three monomers. Hence, in principle, the former could lead to correlated site energy fluctuations for the delocalized normal modes, and the respective low vibrational frequencies could match energy differ- ences between exciton states in different monomers. In order to investigate this question, we will extend generalized Förster theory to include correlated site energy fluctuations. The intermolecular (low-frequency) part of the spectral density containing these corre- lations is obtained by combining a normal mode analysis (NMA) of the whole pigment–protein complex with the charge density cou- pling (CDC) method for the local optical transition energies of the pigments, as described earlier.9 Since in this earlier analysis of FMO monomers, it was found that the fluctuations of excitonic cou- plings are two orders of magnitude smaller than those of the site energies, we do not consider the former in the present work. I. INTRODUCTION rather than electronic coherences.21–23,26,27 It is not the protection of inter-exciton state coherences, but the equal strength of near- est neighbor pigment–pigment and local pigment–protein coupling that holds the key for efficient excitation energy transfer.28,29 In this way, the excitons become partially localized over just a few pig- ments, exciton relaxation corresponds to a spatially directed energy transfer toward the low-energy pigments, and the excess energy of excitons can be well dissipated by the vibrations.3,4,25 A protection of inter-exciton state coherences would be detrimental for exciton relaxation.9 The Fenna–Matthews–Olson (FMO) protein mediates energy transfer between an outer antenna system in green sulfur bacte- ria, termed a chlorosome, and the reaction center complex. Since the discovery of its molecular structure in 1975,1 it has served as an important model system for the development of structure-based theories and calculation schemes for excitation energy transfer and optical spectra of light-harvesting complexes.2–17 The FMO pro- tein is one of the first systems to which the newly developed 2D electronic spectroscopy was applied,18 revealing long-lived coherent oscillations19–23 that founded the field of quantum biology24,25 and brought this protein into the attention of a general audience from many different fields. It is becoming more and more clear that the origin of the long-lived part of the oscillations is due to vibrational The FMO protein is a homotrimer, where each monomer binds eight bacteriochlorophyll a (BChl a) pigments (Fig. 1). Seven of the BChls are bound inside a protein bag made of mostly β-sheets, and the eighth is bound at the surface. During biochemical isolation of the FMO protein, the eighth BChl is J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 153, 215103-1 153, 215103-1 The Journal of Chemical Physics The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp monomer with the closest atom-to-atom distance between pigment and protein. This assignment has caused some confusion in the literature since in terms of pigment–pigment distances, the assign- ment would be different. Grouping the pigments according to the strength of their excitonic couplings results in BChls 1–7 and 8′′ in the first domain, BChls 8 and 1′–7′ in the second domain, and BChls 8′ and 1′′–7′′ in the third domain, where a prime (dou- ble prime) denotes assignment to monomer 2 (3) in the crystal structure. FIG. 1. Upper part: Monomeric subunit of the FMO protein viewed parallel to membrane containing the reaction center complex. I. INTRODUCTION The high-frequency part of the spectral density that is due to intrapig- ment vibrations will be assumed site-independent and taken from an analysis of delta-fluorescence line narrowing (ΔFLN) spectra of the FMO protein.35 A comparison of our approach to calculate the spectral density with more advanced quantum mechanics/molecular mechanics (QM/MM) methods from the literature8,10,15,16,36,37 including specific results on the FMO protein will be given in Sec. V A. II. THEORY For the pigments within a monomeric subunit of the FMO protein, the local reorganization energies of the exciton-vibrational coupling are in the same order of magnitude as their nearest neigh- bor excitonic couplings. Therefore, in the basis of localized excited states, there is no small parameter that can be treated perturbatively. Numerically exact methods such as the hierarchical equation of motion (HEOM) approach51,52 exist, which are, however, computa- tionally expensive, in particular, when arbitrary functional forms of the spectral density and low temperatures are investigated. A numer- ically efficient alternative is to transform the exciton-vibrational coupling into the basis of exciton states, defined as the eigenstates of the electronic Hamiltonian taken at the equilibrium position of nuclei in the electronic ground state of the complex. For the present system, the off-diagonal elements of the exciton-vibrational cou- pling in this basis are smaller than the diagonal elements.9,25 We take advantage of this difference by treating the diagonal elements exactly and applying a Markov and secular approximation to the off-diagonal elements.53 The latter approximations results in a Red- field rate constant for the relaxation between different exciton states. HEOM calculations have found54 only small non-secular contribu- tions to the linear absorption spectrum of the FMO monomer, indi- cating55 that the exciton basis is already close to the true eigenbasis that is optically excited. Intermonomer transfer times in the 20 ps–40 ps range were estimated from low-temperature transient absorption and 2D elec- tronic photon echo experiments.42–44 From the homogeneous broadening of the low-energy exciton band, measured in hole burn- ing spectroscopy on FMO complexes of P. aestuarii at 4.2 K, a life- time of 26 ps was obtained at a wavelength of 822.8 nm and one of 99 ps at the longer wavelength of 824.8 nm.47 Similar values were determined for the FMO protein from Chlorobaculum (formerly Chlorobium) tepidum.46 Intermonomer excitation energy transfer at cryogenic temper- atures was studied theoretically by Jankowiak and co-workers48–50 using two models. In the first model, excitation energy transfer between the lowest sites of each monomer was investigated with standard Förster theory.48,50 Averaged intermonomer transfer times of 41 ps and 70 ps were obtained. In the second model, exciton delocalization between the pigments in the FMO monomers was taken into account, and the transfer was described by using gen- eralized Förster theory. II. THEORY Averaged times of 17 ps, 16 ps, and 47 ps were reported for downhill transfer between the low-energy exci- ton states of the different monomers.49,50 These results seem to suggest that the intramonomer delocalization of the low-energy exciton states promotes intermonomer exciton transfer. A direct comparison of the transfer times in the two models is, how- ever, difficult since different spectral densities were used in these calculations. Due to the large distances between pigments in different FMO monomers (Fig. 1), the excitons dynamically localize inside the FMO monomers. Such a localization of exciton states in the light- harvesting complex II of higher plants was investigated56 by com- paring the exact HEOM calculations with the combined modi- fied Redfield/Generalized Förster theory.57 In the latter approach, dynamic localization is taken into account implicitly by assigning exciton domains of strongly coupled pigments and allowing exci- ton delocalization only within these domains. A pigment belongs to an exciton domain if it is coupled stronger than a given cut- off value Vc to at least one other pigment of this domain. In the case of LHC-II, the best agreement between the combined modi- fied Redfield/Generalized Förster theory and the exact HEOM solu- tion was obtained for Vc = 30 cm−1. In general, Vc is expected to be in the same order of magnitude as the local reorganization energy of the exciton-vibrational coupling of the pigments. If two pigments are excitonically coupled much weaker than their local reorganization energies, the system can minimize its free energy by localizing the excited states. For the present system, the inter- monomer excitonic couplings are at least one order of magnitude smaller than the local reorganization energies of the pigments, and the intramonomer nearest neighbor excitonic couplings are in the same order of magnitude but somewhat larger than these reorga- nization energies. Therefore, as noted already above, we identify the exciton domains with the monomeric subunits of the FMO protein. 24 October 2024 05:45:55 FIG. 1. Upper part: Monomeric subunit of the FMO protein viewed parallel to the membrane containing the reaction center complex. Excitation energy absorbed by the outer chlorosome antenna is transferred through the FMO protein to the reaction center complex. The phytyl chains of the pigments were truncated for bet- ter visibility, and the protein is shown in ribbon style. Note that we have included BChl 8 from monomer 3 in the crystal structure30 as BChl 8′′ into monomer 1, as explained in the main text. Lower part: Complete FMO trimer viewed nor- mal to the membrane from the direction of the reaction center complex. Same color code for the pigments as in the upper part. The black horizontal bar at the bottom corresponds to a distance of 20 Å. The graphics were generated using VMD.31 FIG. 1. Upper part: Monomeric subunit of the FMO protein viewed parallel to the membrane containing the reaction center complex. Excitation energy absorbed by the outer chlorosome antenna is transferred through the FMO protein to the reaction center complex. The phytyl chains of the pigments were truncated for bet- ter visibility, and the protein is shown in ribbon style. Note that we have included BChl 8 from monomer 3 in the crystal structure30 as BChl 8′′ into monomer 1, as explained in the main text. Lower part: Complete FMO trimer viewed nor- mal to the membrane from the direction of the reaction center complex. Same color code for the pigments as in the upper part. The black horizontal bar at the bottom corresponds to a distance of 20 Å. The graphics were generated using VMD.31 easily lost. Therefore, in the original crystal structure, only the seven central BChls are present, and the eighth was found later.30,32 The eighth BChl is bound between two FMO monomers, and its central magnesium atom has a ligand from both monomers. The assignment of the three respective BChl 8 pigments in the crys- tal structure30 is such that each of them belongs to the protein Extensive experimental information exists about intramonomer exciton relaxation and intermonomer energy transfer in the FMO protein from time-resolved pump–probe38–43 and 2D elec- tronic spectroscopy44 and from frequency-resolved hole burning spectra.45–47 Intramonomer exciton relaxation occurs mostly on a J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 J. Chem. Phys. 24 October 2024 05:45:55 153, 215103 (2020); doi: 10.1063/5.0027994 153, 215103-2 153, 215103-2 The Journal of Chemical Physics The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp sub-picosecond timescale, where the lifetimes of high-energy exci- ton states are below 100 fs, even at cryogenic temperatures.41,44 The intermonomer transfer is orders of magnitude slower and can be detected best at cryogenic temperatures, where the lifetime of the lowest exciton state in two of the three monomers in the FMO protein is determined by the energy transfer to the monomer with the lowest energy exciton state of the trimer. Note that due to static disorder in site energies, for every realization of disorder, the three lowest exciton states are likely to be located in different monomers of FMO having slightly different energies. At higher tem- peratures, the uphill energy transfer within the FMO monomers most likely out-competes the intermonomer downhill transfer, mak- ing the latter hard to detect. To the best of our knowledge, there is only one room temperature study that inferred an intermonomer transfer time in the FMO protein, suggesting a time constant of 1.4 ps–2 ps.38 and Förster theory are discussed as well. Afterward, we summa- rize the calculation of the spectral density and the computational methods. The theories and calculation schemes are finally applied to the FMO protein with particular focus on the intermonomer transfer. II. THEORY (5) (5) The exciton-vibrational coupling constant gξ(Ma, Na) of delocal- ized states is related to the local coupling constant g(ma) ξ of the modulation of the transition energy of pigment ma by The exciton-vibrational coupling constant gξ(Ma, Na) of delocal- ized states is related to the local coupling constant g(ma) ξ of the modulation of the transition energy of pigment ma by gξ(Ma, Na) = ∑ ma c(Ma) ma c(Na) ma g(ma) ξ . (6) (6) The intermonomer coupling ˆV in Eq. (1) reads The intermonomer coupling ˆV in Eq. (1) reads A. Hamiltonian ˆV = a≠b ∑ Ma,Nb VMaNb∣Ma⟩⟨Nb∣, (7) ˆV = a≠b ∑ Ma,Nb VMaNb∣Ma⟩⟨Nb∣, (7) The Hamiltonian of the FMO trimer is expressed as (7) H = ∑ a Ha + ˆV, (1) (1) with VMaNb = ∑ manb c(Ma) ma c(Nb) nb Vmanb, (8) where Ha contains the Hamiltonian of the exciton domain (FMO monomer) a and ˆV is the excitonic couplings between different domains. We expand the above Hamiltonian in the basis of excited states ∣Ma⟩that are delocalized in the different exciton domains. The exciton state ∣Ma⟩of domain a is given as a linear combination of local excited states ∣ma⟩of this domain, ∣Ma⟩= ∑ma c(Ma) ma ∣ma⟩, where the coefficients c(Ma) ma are obtained from the eigenvectors of the exciton matrix. This matrix contains in the diagonal the local transition energies Ema (site energies) and in the off-diagonal the excitonic couplings Vma,na between the pigments in this domain. The respective eigenenergy ϵMa = ̵hωMa is the vertical excitation energy of the exciton state ∣Ma⟩at the equilibrium position of nuclei in the electronic ground state. (8) 24 October 2024 05:45:55 where Vmanb is the excitonic coupling between pigments ma in domain a and nb in domain b. where Vmanb is the excitonic coupling between pigments ma in domain a and nb in domain b. II. THEORY In the present work, besides re-investigating the above effect, we extend these calculations in the following ways: (i) Possible cor- relations in site energy fluctuations are studied by extending gener- alized Förster theory and using an intermolecular spectral density, calculated by combining an NMA with the charge density coupling method.5,9 (ii) Frequency-resolved time constants of intermonomer transfer are calculated and compared with the results from hole burning spectroscopy.47 (iii) In addition to cryogenic temperatures, we investigate intramonomer exciton relaxation and intermonomer transfer at physiological temperatures. The remainder of this work is organized in the following way. We start with a summary of the Frenkel exciton Hamiltonian and Redfield theory of exciton relaxation. An expression is derived for the generalized Förster rate constant that includes correlations in site energy fluctuations. Certain limiting cases such as a classical descrip- tion of nuclear motion giving rise to a simple analytical expres- sion for the rate constant, the standard generalized Förster theory, J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 153, 215103-3 153, 215103-3 The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp with the exciton energy ̵hωMa and the reorganization energy E(Ma) λ = ∑ξ ̵hωξgξ(Ma, Ma)2. The latter contains the diagonal exciton-vibrational coupling constants gξ(Ma, Ma) in the exciton basis. The off-diagonal elements of these couplings are contained in ˆVa in Eq. (2), reading Concerning intradomain (intramonomer) exciton relaxation in the FMO protein, it has been found that modified Redfield theory and Redfield theory give very similar results.3 We note in pass- ing that, in general, Redfield theory not necessarily is less accurate than modified Redfield theory, as has been reported in a model study,58 where these two theories were compared with the more accurate non-equilibrium modified Redfield theory.58,59 For a large parameter range, Redfield theory was found to be even more accu- rate than modified Redfield theory due to compensation effects between two different approximations. Here, we will describe intra- monomer exciton relaxation by Redfield theory and intermonomer energy transfer by generalized Förster theory, where the diago- nal elements of the exciton-vibrational coupling in the basis of intramonomer exciton states are treated non-perturbatively. We extend the latter theory by taking into account correlations in site energy fluctuations of pigments in different exciton domains (FMO monomers). ˆVa = Ma≠Na ∑ Ma,Na ∑ ξ ̵hωξgξ(Ma, Na)Qξ∣Ma⟩⟨Na∣. B. Spectral density The prefactor 1/ϵeff takes into account the screen- ing of the Coulomb interaction due to the electronic polarizability of the protein and solvent environment and uncertainties of the quantum chemical method used to determine the atomic partial charges of the pigments in an effective way. Here, we approximate ϵeff by an average optical dielectric constant ϵeff = ϵopt ≈2 that can be estimated, e.g., by evaluating the difference in oscillator strength of pigments between the protein and a solvent environment.61 The coupling constants g(m) ξ and g(k) ξ finally enter the intermolecular spectral density Jmk(ω) in Eq. (10) that will be used to calculate rate constants of energy transfer below. Note that if a normal mode ξ is delocalized, the spectral density Jmk(ω), in general, will have non- zero off-diagonal elements, reflecting a correlation (Jmk(ω) > 0) or anticorrelation (Jmk(ω) < 0) in site energy fluctuations, that will be analyzed below. describes the modulation of the transition energy of the pigments by their intramolecular vibrations. We assume the same local coupling constants gintra ξ for all pigments. The numerical values for (gintra ξ ) 2, which equal the Huang–Rhys factors of the different modes ξ, and the respective vibrational frequencies ωξ were taken from an anal- ysis of ΔFLN spectra of the FMO protein.35 We included the first 18 intrapigment modes with frequencies <500 cm−1 from Table I of Ref. 35 in our calculations. The vibrational frequencies of these modes range between ω1 = 46 cm−1 and ω18 = 481 cm−1. The first eight modes exhibit the largest Huang–Rhys factors ranging between (gintra 3 ) 2 = 0.009 and (gintra 8 ) 2 = 0.012. A graphical representation is given in Fig. 2 (green bars).i Note that there are two different definitions of the spectral den- sity J(ω) in the literature. The present one is directly related to the spectral shape of the vibrational sideband seen in linear absorption and fluorescence.53,60 The alternative definition10,36 is obtained from the present one by multiplication with the factor ω2π̵h. B. Spectral density The Journal of Chemical Physics The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp not just over those of a single exciton domain. In this way, we take into account the possibility that the vibrational normal modes of the complex can be delocalized over the whole complex, notwith- standing the fact that the excitons are dynamically localized in cer- tain domains, as discussed above. We will, in fact, find evidence for interdomain delocalization of normal modes. The normal coordi- nates qξ are related to the Cartesian nuclear coordinates Rj of the complex by Rj −R(0) j = M−1/2 j ∑ ξ A(ξ) j qξ, (12) (12) where R(0) j denotes the equilibrium position of atom j in the elec- tronic ground state of the complex, which is obtained from the crystal structure as described in Sec. III. Mj is the mass of atom j, and A(ξ) j is part of the eigenvector of normal mode ξ with eigenfre- quency ωξ obtained from the diagonalization of the Hessian matrix in the NMA. The coupling constants g(m) ξ in Eq. (11) contain the gradients of the local transition energy of pigment m with respect to the nuclear coordinates Rj taken at their equilibrium positions R(0) j . Using our CDC method,5 these gradients can be calculated analytically, resulting in the following expression for the coupling constant:9 FIG. 2. Comparison between the intermolecular spectral density J33(ω) of low- energy pigment BChl 3 (red bars) obtained from the present NMA, the intrapigment part of the spectral density Jintra(ω) extracted from experiment35 (green bars), and the overall spectral density of the lowest exciton state in the FMO protein, extracted from fluorescence line narrowing spectra (black line).62 For comparison, the intermolecular spectral density averaged over all sites JNMA,av(ω) [Eq. (46)] is also shown (dashed red line). g(m) ξ = 1 ϵeffω3/2 ξ √ 2̵h ∑ i,k qi(q(m) k (1) −q(m) k (0)) ∣R(0) i −R(0) k,m∣ (R(0) i −R(0) k,m) ⋅⎛ ⎝ A(ξ) i M1/2 i −A(ξ) k M1/2 k ⎞ ⎠, (13) The intrapigment part 24 October 2024 05:45:55 Jintra(ω) = ∑ ξ (gintra ξ ) 2δ(ω −ωξ) (14) (13) (14) where the sum over i runs over all atoms of the complex, except for those atoms that contribute to the difference charge density of pigment m. B. Spectral density The respective atomic partial charges qi at equilibrium position R(0) i interact with the difference partial charge q(m) k (1) −q(m) k (0) between the excited and the ground state of the kth atom of pigment m at equilibrium position R(0) k,m, which are summed over by index k. In this way, the change in the difference in Coulomb interaction between the excited and the ground state of pigment m and the rest of the complex upon normal mode displacement, giving rise to the fluctuation in the transition energy of the pigment, is determined. The prefactor 1/ϵeff takes into account the screen- ing of the Coulomb interaction due to the electronic polarizability of the protein and solvent environment and uncertainties of the quantum chemical method used to determine the atomic partial charges of the pigments in an effective way. Here, we approximate ϵeff by an average optical dielectric constant ϵeff = ϵopt ≈2 that can be estimated, e.g., by evaluating the difference in oscillator strength of pigments between the protein and a solvent environment.61 The coupling constants g(m) ξ and g(k) ξ finally enter the intermolecular spectral density Jmk(ω) in Eq. (10) that will be used to calculate rate constants of energy transfer below. Note that if a normal mode ξ is delocalized, the spectral density Jmk(ω), in general, will have non- zero off-diagonal elements, reflecting a correlation (Jmk(ω) > 0) or anticorrelation (Jmk(ω) < 0) in site energy fluctuations, that will be analyzed below. where the sum over i runs over all atoms of the complex, except for those atoms that contribute to the difference charge density of pigment m. The respective atomic partial charges qi at equilibrium position R(0) i interact with the difference partial charge q(m) k (1) −q(m) k (0) between the excited and the ground state of the kth atom of pigment m at equilibrium position R(0) k,m, which are summed over by index k. In this way, the change in the difference in Coulomb interaction between the excited and the ground state of pigment m and the rest of the complex upon normal mode displacement, giving rise to the fluctuation in the transition energy of the pigment, is determined. B. Spectral density We split the total spectral density Jtotal mk (ω) of the local exciton- vibrational coupling into an intermolecular part obtained from a NMA of the pigment–protein complex9 and an intrapigment part that is obtained from difference fluorescence line narrowing experi- ments,35 Jtotal mk (ω) = Jmk(ω) + δm,kJintra(ω). (9) (9) g The Hamiltonian Ha in Eq. (1) reads The intermolecular part Ha = H(0) a + ˆVa, (2) (2) Jmk(ω) = ∑ ξ g(m) ξ g(k) ξ δ(ω −ωξ) (10) Jmk(ω) = ∑ ξ g(m) ξ g(k) ξ δ(ω −ωξ) (10) (10) with describes the site energy fluctuations of pigment m induced by the intermolecular vibrations for m = k, and it contains the correla- tions in site energy fluctuations of pigments m and k for m ≠k. The dimensionless coupling constant g(m) ξ originates from the exciton- vibrational coupling Hamiltonian expanded in the basis of local excited states ∣m⟩of the complex,9 H(0) a = ∑ Ma ⎛ ⎝ ̵hω′ Ma + ∑ ξ ̵hωξ 4 (Qξ + 2gξ(Ma, Ma))2 + Tnucl ⎞ ⎠ × ∣Ma⟩⟨Ma∣, (3) (3) where Tnucl denotes the kinetic energy of nuclei and Qξ is the dimen- sionless coordinate of normal mode ξ with eigenfrequency ωξ that is related to the mass-weighted original normal coordinate qξ by Qξ = √ 2ωξ/̵h qξ.60 The energy difference ̵hω′ Ma between the min- ima of the potential energy surfaces of exciton state ∣Ma⟩and the electronic ground state is given as Hex-vib = ∑ m ∑ ξ ̵hωξg(m) ξ √ 2ωξ ̵h qξ∣m⟩⟨m∣, (11) (11) which describes how the local excitation energy of site m varies if the mass-weighted normal coordinate qξ is displaced. Note that the sum over m runs over all local excited states of the complex and ̵hω′ Ma = ̵hωMa −E(Ma) λ (4) (4) J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 153, 215103-4 © Author(s) 2020 ARTICLE scitation.org/journal/jcp FIG. 2. Comparison between the intermolecular spectral density J33(ω) of low- energy pigment BChl 3 (red bars) obtained from the present NMA, the intrapigment part of the spectral density Jintra(ω) extracted from experiment35 (green bars), and the overall spectral density of the lowest exciton state in the FMO protein, extracted from fluorescence line narrowing spectra (black line).62 For comparison, the intermolecular spectral density averaged over all sites JNMA,av(ω) [Eq. (46)] is also shown (dashed red line). 1. Intradomain exciton relaxation: Redfield theory Exciton relaxation within the domains is described by Red- field theory, where the rate constant kRedf Ma→Na for relaxation between exciton states ∣Ma⟩and ∣Na⟩is given as J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 153, 215103-5 153, 215103-5 J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 kRedf Ma→Na = 2πω2 MaNa ∑ mana c(Ma) ma c(Na) ma c(Ma) na c(Na) na × {Jtotal mana(ωMaNa)(1 + n(ωMaNa)) contains the fluctuations of the intradomain site energies and GMaNb(t) = ∫ ∞ −∞dω{(1 + n(ω))e−iωt + n(ω)eiωt} kRedf Ma→Na = 2πω2 MaNa ∑c(Ma) ma c(Na) ma c(Ma) na c(Na) na contains the fluctuations of the intradomain site energies and πω2 MaNa ∑c(Ma) ma c(Na) ma c(Ma) na c(Na) na contains the fluctuations of the intradomain site energies and edf Ma→Na = 2πω2 MaNa ∑c(Ma) ma c(Na) ma c(Ma) na c(Na) na contains the fluctuations of the intradomain site energies and kRedf Ma→Na = 2πω2 MaNa ∑ mana c(Ma) ma c(Na) ma c(Ma) na c(Na) na × {Jtotal mana(ωMaNa)(1 + n(ωMaNa)) + Jtotal mana(ωNaMa)n(ωNaMa)}, (15) B Ei i di ib i f i contains the fluctuations of the intradomain site energies and GMaNb(t) = ∫ ∞ −∞dω{(1 + n(ω))e−iωt + n(ω)eiωt} × ⎧⎪⎪⎨⎪⎪⎩ ∑ ma,nb ∣c(Ma) ma ∣2∣c(Nb) nb ∣2Jmanb(ω) ⎫⎪⎪⎬⎪⎪⎭ (22) n(ωMaNa)) Ma)}, (15) n GMaNb(t) = ∫ ∞ −∞dω{(1 + n(ω))e−iωt + n(ω)eiωt} × ⎧⎪⎪⎨⎪⎪⎩ ∑ ma,nb ∣c(Ma) ma ∣2∣c(Nb) nb ∣2Jmanb(ω) ⎫⎪⎪⎬⎪⎪⎭ (22) (15) (22) with the Bose–Einstein distribution function with the Bose–Einstein distribution function contains the correlations between site energy fluctuations in differ- ent exciton domains (a ≠b), which are described by the intermolec- ular spectral density Jmanb(ω) in Eq. (10). 1 n(ω) = 1 e̵hω/kBT −1, (16) (16) The exciton relaxation-induced inverse dephasing time τ−1 Mc is given as the spectral density Jtotal mana introduced in Eqs. (9)–(14), and the transi- tion frequency ωKaLa = (ϵKa −ϵLa)/̵h between exciton states ∣Ka⟩and ∣La⟩. given as τ−1 Mc = 1 2 Nc≠Mc ∑ Nc kRedf Mc→Nc, (23) (23) Note that the site energy correlations are taken into account by the off-diagonal elements of the spectral density. It was found previ- ously9 that these correlations have practically no influence on exci- ton relaxation in the FMO monomers. What remains to be inves- tigated is whether they can influence intermonomer energy trans- fer in the FMO trimer. 1. Intradomain exciton relaxation: Redfield theory For this purpose, we extend the standard generalized Förster theory in the following. and the shifted transition frequencies are defined as and the shifted transition frequencies are defined as ˜ωMaNb = ˜ωMa −˜ωNb, (24) (24) with 2. Interdomain transfer: Generalized Förster theory with correlations ˜ωKC = ωKC −E(Kc) λ /̵h + Δωc. (25) (25) Here, ωKC is the vertical excitation frequency of exciton state ∣Kc⟩, obtained from the diagonalization of the exciton matrix of domain c, and E(Kc) λ is the reorganization energy introduced above. It is related to the spectral density Jtotal mcnc [Eq. (9)] via Here, ωKC is the vertical excitation frequency of exciton state ∣Kc⟩, obtained from the diagonalization of the exciton matrix of domain c, and E(Kc) λ is the reorganization energy introduced above. It is related to the spectral density Jtotal mcnc [Eq. (9)] via We assume a fast intradomain relaxation leading to equilibra- tion of the excitation energy within the domains prior to energy transfer between different domains. Hence, the overall rate constant of energy transfer between domain a and domain b can be written as E(Kc) λ = ∑ mcnc ∣c(Kc) mc ∣2∣c(Kc) nc ∣2 ∫ ∞ 0 dω ̵hωJtotal mcnc(ω). (26) ka→b = ∑ Ma,Nb fMakGF Ma→Nb, (17) (17) (26) with the Boltzmann factor with the Boltzmann factor Δωc in Eq. (25) is a small additional shift caused by the off-diagonal elements of the exciton-vibrational coupling, fMa = e−̵hωMa /kBT ∑Na e−̵hωNa /kBT . (18) (18) Δωc = Mc≠Kc ∑ Mc ∑ mcnc c(Kc) mc c(Mc) mc c(Kc) nc c(Mc) nc × ℘∫ ∞ −∞dωω2{(1 + n(ω))Jtotal mcnc(ω) + n(−ω)Jtotal mcnc(−ω)} ωKcMc −ω , (27) Here, the generalized Förster rate constant kGF Ma→Nb for transfer between exciton state ∣Ma⟩of domain a and ∣Nb⟩of domain b, obtained as described in Appendix A, is given as Here, the generalized Förster rate constant kGF Ma→Nb for transfer between exciton state ∣Ma⟩of domain a and ∣Nb⟩of domain b, obtained as described in Appendix A, is given as (27) kGF Ma→Nb = ∣VMaNb∣2 ̵h2 ∫ ∞ −∞dt ei˜ωMaNb teFMaNb (t)−FMaNb (0)e −∣t∣( 1 τMa + 1 τNb ) . (19) where ℘denotes the principal part of the integral. Note that the intermolecular spectral density Jtotal mcnc(ω) in Eqs. (15), (21), and (26)l where ℘denotes the principal part of the integral. Note that the intermolecular spectral density Jtotal mcnc(ω) in Eqs. (15), (21), and (26) contains, both the site energy fluctuations (mc = nc) and their corre- lations (mc ≠nc), whereas the interdomain spectral density Jmanb(ω) in Eq. (22) just describes the interdomain correlations in site energy fluctuations of pigment ma in domain a and pigment nb in domain b (b ≠a). © Author(s) 2020 2. Interdomain transfer: Generalized Förster theory with correlations (19) y c c contains, both the site energy fluctuations (mc = nc) and their corre- lations (mc ≠nc), whereas the interdomain spectral density Jmanb(ω) in Eq. (22) just describes the interdomain correlations in site energy fluctuations of pigment ma in domain a and pigment nb in domain b (b ≠a). The interdomain electronic coupling VMaNb is given in Eq. (8), while the function FMaNb(t) reads The interdomain electronic coupling VMaNb is given in Eq. (8), while the function FMaNb(t) reads FMaNb(t) = GMa(t) + GNb(t) −2GMaNb(t), (20) (20) where where a. Limit of uncorrelated fluctuation of site energies. In the limit of uncorrelated fluctuations of site energies in different domains, we can set GMaNb(t) = 0 and obtain the well-known result for the rate constant57,63–66 GKc(t) = ∫ ∞ −∞dω{(1 + n(ω))e−iωt + n(ω)eiωt} × ⎧⎪⎪⎨⎪⎪⎩ ∑ mc,nc ∣c(Kc) mc ∣2∣c(Kc) nc ∣2Jtotal mcnc(ω) ⎫⎪⎪⎬⎪⎪⎭ (21) kGF Ma→Nb = 2π ̵h2 ∣VMaNb∣2 ∫ ∞ ∞dωDNb(ω)D′ Ma(ω) (28) (21) (28) J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 153, 215103-6 © Author(s) 2020 J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 153, 215103-6 © Author(s) 2020 J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 153, 215103-6 © Author(s) 2020 © Author(s) 2020 The Journal of Chemical Physics with the Boltzmann factor with the Boltzmann factor with the Boltzmann factor that contains the overlap integral between the normalized absorp- tion lineshape function DNb(ω) of the acceptor and the normalized fluorescence lineshape function D′ Ma(ω) of the donor, which are given as fma = e−̵hωma /kBT ∑na e−̵hωna /kBT (35) (35) DNb(ω) = 1 2π ∫ ∞ −∞dte−i(ω−˜ωNb )teGNb (t)−GNb (0)e−∣t∣/τNb (29) that takes into account fast equilibration of the population of local- ized excited states in FMO monomer a, where ̵hωma is the site energy of pigment ma. The Förster theory rate constant kF ma→nb between localized excited states ∣ma⟩in domain a and ∣nb⟩in domain b reads60,68 that takes into account fast equilibration of the population of local- ized excited states in FMO monomer a, where ̵hωma is the site energy of pigment ma. The Förster theory rate constant kF ma→nb between localized excited states ∣ma⟩in domain a and ∣nb⟩in domain b reads60,68 (29) and and D′ Ma(ω) = 1 2π ∫ ∞ −∞dtei(ω−˜ωMa )teGMa (t)−GMa (0)e−∣t∣/τMa , (30) (30) kF ma→nb = 2π ̵h2 ∣Vmanb∣2 ∫ ∞ ∞dωDnb(ω)D′ ma(ω), (36) (36) respectively. 2. Interdomain transfer: Generalized Förster theory with correlations respectively. where the normalized absorption lineshape function Dnb(ω) of the acceptor is b. Classical limit of nuclear motion. In the following, we discuss a classical description of nuclear motion. In this high- temperature limit, the Bose–Einstein distribution function in Eq. (16) is approximated as n(ω) ≈kBT/̵hω. Neglecting the exciton relaxation-induced dephasing (τ−1 Ma = τ−1 Nb = 0) in Eq. (19) and using a short-time approximation [cos(ωt) ≈1 −ω2t2/2, sin(ωt) ≈ωt] in the function FMaNb(t) −FMaNb(0) in this equation and Eq. (A8) results in Dnb(ω) = 1 2π ∫ ∞ −∞dte−i(ω−˜ωnb )teGnb (t)−Gnb (0)e−∣t∣/τpd (37) (37) and the normalized fluorescence lineshape function of the D′ ma(ω) of the donor is D′ ma(ω) = 1 2π ∫ ∞ −∞dtei(ω−˜ωma )teGma (t)−Gma (0)e−∣t∣/τpd , (38) FMaNb(t) −FMaNb(0) ≈−kBTE(Ma,Nb) λ ̵h2 t2 −iE(Ma,Nb) λ ̵h t (31) (38) (31) where τpd is a pure dephasing time. The time-dependent function Gkc(t) of the local excited state ∣kc⟩is given as where the reorganization energy is E(Ma,Nb) λ = ∫ ∞ 0 dω̵hωJMaNb(ω) (32) (32) Gkc(t) = ∫ ∞ −∞dω{(1 + n(ω))e−iωt + n(ω)eiωt}Jtotal kckc (ω), (39) (39) that contains the spectral density JMaNb(ω) in Eqs. (A9) and (A10). Performing the time integration in Eq. (19) finally results in the semiclassical generalized Förster rate constant, which is formally identical to the rate constant obtained in Marcus theory of non- adiabatic electron transfer,60,67 where Jtotal kckc (ω) describes the modulation of the site energy of this state. The frequency ˜ωkc in Eqs. (37) and (38) is the 0–0 local tran- sition frequency of pigment k in domain c and is related to the local vertical transition frequency ωkc by ˜ωkc = ωkc −E(kc) λ /̵h, (40) kscGF Ma→Nb = ∣VMaNb∣2 ̵h ¿ Á Á À π kBTE(Ma,Nb) λ × exp ⎧⎪⎪⎨⎪⎪⎩ −(̵h˜ωMaNb −E(Ma,Nb) λ )2 4kBTE(Ma,Nb) λ ⎫⎪⎪⎬⎪⎪⎭ . (33) (40) where E(kc) λ is the local reorganization energy of the exciton- vibrational coupling where E(kc) λ is the local reorganization energy of the exciton- vibrational coupling (33) E(kc) λ = ∫ ∞ 0 dω̵hωJtotal kckc (ω). (41) (41) The correlations in site energy fluctuations are contained in the spectral density JMaNb(ω) in Eq. (A10), where the intradomain cor- relations are described by the off-diagonal elements Jmcnc(ω), and the correlations between different domains a and b are given by Jmanb(ω). 2. Interdomain transfer: Generalized Förster theory with correlations For the numerical implementation, we use the following expres- sion for the rate constant [obtained by inserting Eqs. (37)–(39) into Eq. (36)]: For the numerical implementation, we use the following expres- sion for the rate constant [obtained by inserting Eqs. (37)–(39) into Eq. (36)]: Eq. (36)]: kF ma→nb = ∣Vmanb∣2 ̵h2 × ∫ ∞ −∞dtei˜ωmanb teGma (t)+Gnb (t)−Gma (0)−Gnb (0)e−2∣t∣/τpd . (42) c. Limit of localized excitations: Standard Förster theory. In order to evaluate how the intermonomer excitation energy transfer is influenced by the intramonomer exciton delocalization, we con- sider the limit of localized excitations. In this case, in analogy to Eq. (17), we obtain a rate constant (42) An important difference between the above rate constant and the rate constant obtained in the generalized Förster theory in Eq. (28) is given by the prefactor. The excitonic coupling Vmanb between local ka→b = ∑ ma,nb fmakF ma→nb, (34) (34) J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 153, 215103-7 © Author(s) 2020 The Journal of Chemical Physics The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp scitation.org/journal/jcp BChl a pigments were complete in the crystal structure except for BChl 8, where the phytyl chain is replaced by a methyl group.30 All water molecules were removed except for the axial ligand of BChl 2 and the hydrogen bonding partner of the 3-acetyl group of BChl 1. The force field of the BChl a pigments was adopted from the work of Ceccarelli et al.71 for the use in CHARMM except for the atomic par- tial charges (APCs). The latter were obtained from quantum chem- ical (QC) computations employing QChem.72 In QC computations, the geometry of a BChl a with the phytyl chain truncated to a methyl group was optimized in vacuo with the B3LYP exchange-correlation functional and a 6-31G(d,p) basis set. Excited state calculations were performed on this optimized structure employing time-dependent density functional theory in the random phase approximation with the same basis set and the CAM-B3LYP73 exchange-correlation functional, which is superior to B3LYP for excited states.74 APCs of the ground and first excited state of BChl a were obtained by fit- ting the electrostatic potential (ESP)75,76 of the CAM-B3LYP-derived charge density of the two electronic states. The numerical values of these charges are given in the supplementary material. 3. Exciton state lifetime The lifetime TMd of an exciton state ∣Md⟩is determined by the rate constants of transfer from this state to any other state ∣Nd⟩in the same domain and to any state ∣Na⟩in the other domains a≠d. We define the frequency-resolved average inverse lifetime T(ω)−1 as 1 T(ω) = ⟨∑d,Md(∑Nd≠Md Nd kMd→Nd + ∑a≠d a,Na kMd→Na)δ(ω −ωMd)⟩dis DoE(ω) , (43) (43) where ⟨⋯⟩dis denotes an average over static disorder in site energies and DoE(ω) = ∑ d,Md ⟨δ(ω −ωMd)⟩dis (44) (44) is the density of exciton states. 24 October 2024 05:45:55 For analysis purposes, we also introduce functions Tintra(ω) and Tinter(ω) in which only the intramonomer rate constants kMd→Nd or only the intermonomer rate constants kMd→Na (a ≠d), respectively, are taken into account on the rhs in Eq. (43). q A variant of the above quantities denoted as T′(ω) and DoE′(ω) is used at cryogenic temperatures, where uphill energy transfer is frozen out leading to an infinite lifetime of the lowest exciton state. Therefore, the lowest exciton state of every monomer, in the case of T′ intra(ω), or of the FMO trimer, in the case of T′ inter(ω), is excluded from the sum over Md on the rhs of Eqs. (43) and (44). In another variant, termed DoE′r(ω) and T′r(ω), in addition to leaving out the lowest exciton state(s), instead of the bare exciton frequencies ωMd, the renormalized frequencies ˜ωMd [Eq. (25)], which contain a shift by the exciton-vibrational coupling, are used in the rhs of Eqs. (43) and (44). The geometry of the FMO trimer was optimized with CHARMM using the adopted basis Newton–Raphson (ABNR)79 method. CHARMM was also used for the NMA, resulting in 59 373 eigenfrequencies and normal mode vectors. The first six frequen- cies, corresponding to translation and rotation of the whole FMO trimer, are practically zero (<10−3 cm−1). All other eigenfrequencies ωξ are positive, as expected for a stable minimum. The eigenvectors and eigenfrequencies were used in conjunction with the optimized (equilibrium) coordinates of atoms (nuclei), the ground and excited state APCs of BChl a, and the ground state APCs of the protein from the CHARMM22 force field to calculate the coupling con- stants g(m) ξ [Eq. (13)] entering the intermolecular spectral density Jmk(ω) [Eq. (10)] together with the eigenfrequencies ωξ of the normal modes. III. COMPUTATIONAL DETAILS Computations are based on the crystal structure of the FMO protein from P. aestuarii by Tronrund et al.30 at a resolution of 1.3 Å that includes all eight BChl pigments per monomer (holo form). A starting structure for the geometry optimization was constructed from protein data bank (PDB) file 3EOJ. The protein was included from Thr 8, whose N-terminus was patched with an acetyl group, to the negatively charged C-terminal Gln 366. Hydrogen atoms and missing heavy atoms of the protein (see PDB file 3EOJ) were added by using CHARMM35b369 and the CHARMM22 force field.70 All histidine side chains were modeled as neutral except for His 12, which was positively charged. The remaining titratable residues were calculated to be in their standard protonation state (see Appendix B) and modeled accordingly. 2. Interdomain transfer: Generalized Förster theory with correlations The ground state APCs were used in all CHARMM computations supplement- ing the force field by Ceccarelli et al.71 The ground and excited state APCs were tested in a calculation of site energies employ- ing our Poisson Boltzmann/Quantum chemical (PBQC) method,4 revealing a good correlation with the reference values7 that describe the optical spectra (Appendix B). Additionally, the ground state APCs are very similar in magnitude to those obtained in a recent study77 by using the CHARMM-compatible CGenFF tool78 and, thus, are expected to provide a good description of a polarized BChl a molecule in a condensed matter environment according to the CHARMM philosophy. excited states in Förster theory is replaced by that between delocal- ized excited states VMaNb in the generalized Förster theory. Accord- ing to Eq. (8), VMaNb is related to the Vmanb by the coefficients of the intermonomer exciton states. Since the latter can have posi- tive as well as negative signs, exciton delocalization can lead to an enhancement as well as a decrease in the coupling corresponding to constructive and destructive interference effects, respectively. A. Spectral density The Huang–Rhys factor S3 = 0.31 of the low-energy site BChl 3, which dominates the lowest exciton state of the FMO monomers, agrees well with the experimen- tal estimates S = 0.3 of the intermolecular Huang–Rhys factor from fluorescence line narrowing spectra.62 The intermolecular spectral density J33(ω) obtained for BChl 3 is similar to the average of the diagonal parts of the spectral densities of all pigments in the FMO trimer, FIG. 3. Diagonal elements Jmm(ω) of the intermolecular spectral density [Eq. (10)] for the eight BChl a pigments of the monomeric subunits of the FMO protein and respective Huang–Rhys factors Sm [Eq. (45)]. The spectral densities as well as the Huang–Rhys factors have been averaged over the three equivalent sites in the trimer. JNMA, av(ω) = 1 24 24 ∑ m=1 Jmm(ω). (46) (46) Sm = ∫ ∞ 0 dωJmm(ω), (45) FIG. 5. Huang–Rhys factors Sm [Eq. (45)] of the intermolecular spectral density for the BChl a pigments (m = 1, . . ., 8) in dependence on cutoff radius Rc. Only those amino acids (and other structural elements) with a distance smaller than Rc from the respective pigment m are included in the calculation of the Huang–Rhys factor. Sm was averaged over the three equivalent binding sites in the FMO trimer. (45) varies between S5 = 0.15 and S2 = 0.60. varies between S5 = 0.15 and S2 = 0.60. These Huang–Rhys factors were averaged over the three equiv- alent sites in the FMO trimer. The individual Huang–Rhys factors of the monomers exhibit a similar trend as the averaged values with some deviations between the equivalent sites in the three FMO- monomers (Fig. 4). Obviously, the geometry optimization of the FMO protein leads to some deviations from the perfect C3 symmetry of the crystal structure. y The Huang–Rhys factors are mostly influenced by the direct protein environments of the pigments, as shown in Fig. 5, where the dependence of Sm on a cutoff distance Rc is shown. For a given value of Rc, only those amino acids (and other components of the com- plex) were included in the calculation of Sm that have at least one atom with a smaller distance than Rc to at least one atom of BChl m. A. Spectral density The largest single amino acid con- tribution of 0.2 to the initial rise of S6 up to 0.55 is due to the polar Gln 143. S2 increases steadily up to Rc ≈40 Å and afterward slightly decreases until convergence at Rc ≥60 Å. The Huang–Rhys factor S3 = 0.31 of the low-energy site BChl 3, which dominates the lowest exciton state of the FMO monomers, agrees well with the experimen- tal estimates S = 0.3 of the intermolecular Huang–Rhys factor from fluorescence line narrowing spectra.62 The intermolecular spectral density J33(ω) obtained for BChl 3 is similar to the average of the diagonal parts of the spectral densities of all pigments in the FMO trimer, JNMA av(ω) = 1 24 ∑Jmm(ω). (46) FIG. 3. Diagonal elements Jmm(ω) of the intermolecular spectral density [Eq. (10)] for the eight BChl a pigments of the monomeric subunits of the FMO protein and respective Huang–Rhys factors Sm [Eq. (45)]. The spectral densities as well as the Huang–Rhys factors have been averaged over the three equivalent sites in the trimer FIG. 4. Huang–Rhys factors Sm [Eq. (45)] of the intermolecular spectral density for the BChl a pigments (m = 1, . . ., 8) in the three monomeric subunits of the FMO protein. FIG. 4. Huang–Rhys factors Sm [Eq. (45)] of the intermolecular spectral density for the BChl a pigments (m = 1, . . ., 8) in the three monomeric subunits of the FMO protein. sites m = 2 and 6 with the largest Sm, the latter is influenced by long- range interactions that extend beyond a FMO monomer. S6 exhibits a sharp initial rise, a decrease for intermediate Rc, and a slight but steady increase for large Rc > 20 Å. The largest single amino acid con- tribution of 0.2 to the initial rise of S6 up to 0.55 is due to the polar Gln 143. S2 increases steadily up to Rc ≈40 Å and afterward slightly decreases until convergence at Rc ≥60 Å. A. Spectral density The diagonal part Jmm of the intermolecular spectral density of the eight pigments in the monomeric subunit of the FMO protein, averaged over the three equivalent sites of the trimer, is shown in Fig. 3. It exhibits a strongly asymmetric shape with a sharp rise at small frequencies toward a maximum between 10 cm−1 and 20 cm−1 and a slower decay for larger frequencies reaching values close to zero at a vibrational frequency of roughly 100 cm−1. The integral over the diagonal part Jmm of the spectral density, the Huang–Rhys factor J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 153, 215103-8 153, 215103-8 © Author(s) 2020 The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp FIG. 4. Huang–Rhys factors Sm [Eq. (45)] of the intermolecular spectral density for the BChl a pigments (m = 1, . . ., 8) in the three monomeric subunits of the FMO protein. The Journal of Chemical Physics scitation.org/journal/jcp FIG. 3. Diagonal elements Jmm(ω) of the intermolecular spectral density [Eq. (10)] for the eight BChl a pigments of the monomeric subunits of the FMO protein and respective Huang–Rhys factors Sm [Eq. (45)]. The spectral densities as well as the Huang–Rhys factors have been averaged over the three equivalent sites in the trimer. FIG. 4. Huang–Rhys factors Sm [Eq. (45)] of the intermolecular spectral density for the BChl a pigments (m = 1, . . ., 8) in the three monomeric subunits of the FMO protein. FIG. 3. Diagonal elements Jmm(ω) of the intermolecular spectral density [Eq. (10)] for the eight BChl a pigments of the monomeric subunits of the FMO protein and respective Huang–Rhys factors Sm [Eq. (45)]. The spectral densities as well as the Huang–Rhys factors have been averaged over the three equivalent sites in the trimer. FIG. 4. Huang–Rhys factors Sm [Eq. (45)] of the intermolecular spectral density for the BChl a pigments (m = 1, . . ., 8) in the three monomeric subunits of the FMO protein. sites m = 2 and 6 with the largest Sm, the latter is influenced by long- range interactions that extend beyond a FMO monomer. S6 exhibits a sharp initial rise, a decrease for intermediate Rc, and a slight but steady increase for large Rc > 20 Å. A. Spectral density For most of the sites m, the Huang–Rhys factor Sm is converged for Rc ≥10 Å–15 Å, demonstrating that electrostatic interactions with the local protein environment are dominant. In contrast, for the two FIG. 5. Huang–Rhys factors Sm [Eq. (45)] of the intermolecular spectral density for the BChl a pigments (m = 1, . . ., 8) in dependence on cutoff radius Rc. Only those amino acids (and other structural elements) with a distance smaller than Rc from the respective pigment m are included in the calculation of the Huang–Rhys factor. Sm was averaged over the three equivalent binding sites in the FMO trimer. FIG. 5. Huang–Rhys factors Sm [Eq. (45)] of the intermolecular spectral density for the BChl a pigments (m = 1, . . ., 8) in dependence on cutoff radius Rc. Only those amino acids (and other structural elements) with a distance smaller than Rc from the respective pigment m are included in the calculation of the Huang–Rhys factor. Sm was averaged over the three equivalent binding sites in the FMO trimer. J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 153, 215103-9 The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp A comparison between J33(ω) and JNMA,av(ω) as well as with the experimental spectral density JFLN(ω) extracted from fluorescence line narrowing experiments62 is shown in Fig. 2. JFLN(ω) is smaller at frequencies ω < 25 cm−1 and larger at ω >25 cm−1. The lat- ter deviations are due to the fact that the experimental spec- tral density also includes intramolecular contributions that are due to vibrations of the pigments. Huang–Rhys factors of these intrapigment modes have been estimated from experimental ΔFLN data35 and are shown as green bars in Fig. 2. The large ampli- tude of the calculated NMA spectral density at low frequencies could be due to the harmonic approximation that cannot describe soft and anharmonic motion expected at very low frequencies. As discussed later in more detail, the local reorganization energies of the intermolecular spectral density obtained from the present NMA/CDC approach qualitatively agree with those obtained from combined molecular dynamics/CDC calculations,10 provid- ing some evidence that, overall, the anharmonic effects are not critical. These correlations are contained in the off-diagonal parts of Jmk(ω) shown in Fig. 6 for selected pigments located in the same (left half of this figure) and in different (right half) FMO monomers. scitation.org/journal/jcp FIG. 7. Dependence of generalized Huang–Rhys factors \|S\|mk [red squares, Eq. (48)] and Smk [black circles, Eq. (47)] characterizing the maximum and mini- mum correlation in site energy fluctuations, respectively, of different sites m and k on the distance Rmk between the sites. TABLE I. Summary of different types of spectral densities used in the calculation of energy transfer. Spectral density Content JNMA Jmk(ω), Eq. (10) JNMA (no corr.) Jmk(ω) = δm,kJmm(ω) JNMA (pos. corr.) Jmk(ω) →\|Jmk(ω)\| JNMA,av Equation (46) Jintra Eq. (14), Fig. 2 JFLN Extracted from FLN spectra,62 Fig. 2 TABLE I. Summary of different types of spectral densities used in the calculation of energy transfer. FIG. 7. Dependence of generalized Huang–Rhys factors \|S\|mk [red squares, Eq. (48)] and Smk [black circles, Eq. (47)] characterizing the maximum and mini- mum correlation in site energy fluctuations, respectively, of different sites m and k on the distance Rmk between the sites. disorder, generated from the above distribution functions, rate con- stants of energy transfer are calculated and the distribution and the average values determined. For room temperature calculations, an ensemble of 10 000 sets of site energies is sufficient to obtain con- vergent results, whereas a 100 times larger ensemble had to be used for cryogenic temperatures. In the calculations of rate constants, we use different spectral densities of the exciton-vibrational coupling, as explained in the following and summarized in Table I. The spec- tral density JNMA corresponds to the intermolecular spectral density Jmk(ω) obtained from the NMA [Eq. (10)]. In JNMA (no corr.), only the diagonal part Jmm(ω) of the NMA spectral density is taken into account, and the correlations contained in the off-diagonal parts are neglected. In JNMA (pos. corr.), all anticorrelations are turned into correlations of the same amplitude. JNMA,av describes the aver- age diagonal part of the NMA spectral density [Eq. (46)], Jintra is the intrapigment contribution to the spectral density [Eq. (14), green bars in Fig. 2], and JFLN is the experimental spectral den- sity extracted from FLN spectra of the FMO protein containing intrapigment as well as intermolecular contributions (black line in Fig. 2). Smk = ∫ ∞ 0 dωJmk(ω) (47) (47) and a generalized absolute Huang–Rhys factor and a generalized absolute Huang–Rhys factor ∣S∣mk = ∫ ∞ 0 dω∣Jmk(ω)∣ (48) (48) 24 October 2024 05:45:55 representing a hypothetical system, where all anticorrelations were transformed into correlations. As seen in Fig. scitation.org/journal/jcp 7, the latter Huang– Rhys factor shows no dependence on the interpigment distance Rmk, reflecting the fact of equal amplitudes of intra- and inter- monomer correlations (Fig. 6). In contrast, the amplitude of Smk decreases with the increasing interpigment distance (Fig. 7). Obvi- ously, the compensations between correlations and anticorrelations in the site energy fluctuations due to different normal modes are more complete for pigments at large distance. 2. Physiological temperatures We start with a description of intramonomer exciton relaxation and intermonomer transfer at room temperature (300 K). The den- sity of exciton states DoE(ω) [Eq. (44)] is shown in Fig. 8 together with the frequency-resolved lifetime T(ω) [Eq. (43)] calculated with the different spectral densities described above. Including only the intermolecular part of the spectral density results in lifetimes around 200 fs in the central part of the spectrum that increase to more than a ps at the low and high-energy wings. The inclusions of correlations practically has no influence, and also neglecting the site dependence of the spectral density (using JNMA,av) leads to very similar results. However, including the high-frequency intrapigment spectral den- sity Jintra(ω) decreased the lifetimes to less than 100 fs in the center of the spectrum and to less than 1 ps at the low and high-energy wings. The lifetimes in Fig. 8 are dominated by intramonomer exci- ton relaxation, that is, excluding the intermonomer transfer does not change the lifetimes of exciton states. A. Spectral density In con- trast to the positive diagonal parts Jmm(ω) (Fig. 3), the off-diagonal elements Jmk(ω) can also become negative at certain frequencies, corresponding to a situation where the normal modes at these fre- quencies lead to an anticorrelated fluctuation of site energies. Due to the broken C3 symmetry, discussed already above, the correla- tions are different in the three FMO monomers and also between the monomers. Interestingly, in Fig. 6, the amplitudes of the intra- monomer correlations are comparable to those of the intermonomer correlations despite the much larger interpigment distances in the latter case (see Fig. 1). These amplitudes are somewhat smaller but still in the same order of magnitude than those of the diagonal parts Jmm(ω) in Fig. 3. A difference, however, is that the correla- tions Jmk(ω) with increasing ω decrease steeper to zero than the diagonal parts Jmm(ω). Whereas the former are practically zero for ω > 40 cm−1, the latter still exhibit non-zero values up to a frequency of 100 cm−1. In order to have a global measure of the correlations, we introduce a generalized Huang–Rhys factor An advantage of a microscopic calculation of the spectral den- sity is that correlations in site energy fluctuations can be revealed. FIG. 6. Off-diagonal elements of the intermolecular spectral density Jmk(ω), representing correlations in site energy fluctuations, for selected pigments in the same monomer (left half) and in different monomers (right half). Black, green, and red bars correspond to correlations for equivalent pigment pairs in different monomers (left) and for equivalent pigment pairs across different monomers (right). 24 October 2024 05:45:55 FIG. 6. Off-diagonal elements of the intermolecular spectral density Jmk(ω), representing correlations in site energy fluctuations, for selected pigments in the same monomer (left half) and in different monomers (right half). Black, green, and red bars correspond to correlations for equivalent pigment pairs in different monomers (left) and for equivalent pigment pairs across different monomers (right). J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 153, 215103-10 The Journal of Chemical Physics ARTICLE 1. Parameters In our calculations of energy transfer, presented below, we use the site energies of the FMO protein determined earlier from CDC calculations and a refinement fit of optical spectra.7 The exci- tonic couplings were obtained by using a point-dipole approxima- tion (PDA) and an effective transition dipole moment of 29.8 D2, as determined earlier5 from electrostatic calculations. Note that the validity of the PDA for the FMO protein has been demonstrated.80 The transition dipole of BChl a is assumed to be oriented in the direction of a line connecting the NB and ND atoms (in PDB nomen- clature) and placed in the center between these two atoms. The atomic coordinates were taken from the crystal structure of the FMO trimer.30 The numerical values of the excitonic couplings are given together with that of the site energies in Appendix C. The distribution of intermonomer transfer times (ka→b)−1, taking into account the fast intramonomer equilibration [Eq. (17)], are shown in Fig. 9, calculated for different spectral densities. Sim- ilar distribution functions are obtained with and without including the intrapigment (high-frequency) part of the spectral density. The Static disorder in site energies is taken into account by assign- ing a Gaussian distribution function with a full width at half maxi- mum (FWHF) of 100 cm−1 to every site,3 assuming that there is no correlation between different sites. For every random realization of J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 153, 215103-11 153, 215103-11 The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp FIG. 8. Upper part: Density of exciton states DoE(ω) [Eq. (44)]. Lower part: Frequency-resolved averaged lifetimes of exciton states T(ω) [Eq. (43)] at room temperature (300 K) obtained with different spectral densities, explained in the main text and in Table I, as indicated in the legend. the spectral density leads to a somewhat sharper distribution func- tion but does not change the average dynamics. Hence, the inter- monomer transfer is dominated by the low-frequency intermolec- ular spectral density. Interestingly, practically the same distribution function is obtained with and without including the correlations in site energy fluctuations contained in the off-diagonal elements of the spectral density Jmk(ω), discussed above. 3. Cryogenic temperatures At cryogenic temperatures, uphill energy transfer between the exciton states in the FMO monomers is frozen out and the lifetime of the lowest exciton state is determined by the intermonomer transfer, which, however, occurs on a much longer time scale than intra- monomer exciton relaxation. We, therefore, analyze the lifetimes of the lowest exciton state in the FMO monomers separately. We start with the intramonomer lifetimes of the exciton states, excluding the lowest state in each monomer. The density of these exciton states together with their lifetime T′ intra(ω) at 77 K is shown in Fig. 10. Similar to the room temperature results (Fig. 8), the high-frequency intrapigment contributions to the spectral density are important for a fast sub-picosecond exciton equilibration. The correlations in the intermolecular part of the spectral density have practically no effect on the lifetimes. Compared to the room temperature lifetimes (Fig. 8), those at 77 K are somewhat larger, in particular, those of the low-energy exciton states. FIG. 8. Upper part: Density of exciton states DoE(ω) [Eq. (44)]. Lower part: Frequency-resolved averaged lifetimes of exciton states T(ω) [Eq. (43)] at room temperature (300 K) obtained with different spectral densities, explained in the main text and in Table I, as indicated in the legend. 24 October 2024 05:45:55 maxima of these distribution functions all occur around an inter- monomer transfer time of 10 ps that is about two orders of magni- tude larger than the typical intramonomer exciton relaxation times (Fig. 8). Including the high-frequency intrapigment contribution to FIG. 9. Distribution of inverse intermonomer rate constants at room temperature, calculated assuming fast intramonomer exciton equilibration [Eq. (17)], using dif- ferent spectral densities, explained in the main text and in Table I, as shown in the legend. In the calculations labeled as JNMA(cl), the intermolecular spectral density Jmk(ω) was used, and the rate constants were calculated with the semiclassical expression in Eq. (33). The lifetime of the lowest exciton state of the FMO trimer is not limited by energy transfer since the latter would be uphill in energy and is frozen out at 77 K. However, the second and third lowest exciton states of the FMO trimer, which are the lowest exci- ton states in two FMO monomers, can transfer downhill in energy to the lowest exciton state in the trimer. In Fig. 1. Parameters In order to see whether the compensation between correlations Jmk(ω) > 0 and anticorrelations Jmk(ω) < 0 can be responsible for this result, we recalculated the dis- tribution function for a hypothetical spectral density, where all anti- correlations were transformed into correlations. For this JNMA (pos. corr.), we again find very little deviations from the result obtained for the original spectral density JNMA. Therefore, another factor must be responsible for the small effect of the site energy correla- tions on the transfer times. In addition, the averaged spectral density JNMA,av gives very similar results. Even a classical approximation of nuclear motion in the calculation of the intermonomer rate con- stants kscGF Ma→Nb [Eq. (33)] provides a qualitatively correct description of the intermonomer transfer, with a distribution function that is slightly shifted toward shorter transfer times (the blue solid line in Fig. 9). 3. Cryogenic temperatures The left column contains the distributions of downhill inverse intermonomer rate constants, and the right column contains the resulting frequency-resolved lifetime of exciton states. Note that the blue and the black line in the left bottom figure are on top of each other. FIG. 10. Intramonomer exciton transfer at 77 K. Upper part: Density of exci- ton states without the lowest exciton state in each FMO monomer. Lower part: Intramonomer frequency-resolved lifetime of exciton states (excluding the lowest), calculated with different spectral densities, explained in the main text and in Table I, indicated in the legend. The black solid circles represent the lifetime estimated from 2D experiments.44 FIG. 10. Intramonomer exciton transfer at 77 K. Upper part: Density of exci- ton states without the lowest exciton state in each FMO monomer. Lower part: Intramonomer frequency-resolved lifetime of exciton states (excluding the lowest), calculated with different spectral densities, explained in the main text and in Table I, indicated in the legend. The black solid circles represent the lifetime estimated from 2D experiments.44 24 October 2024 05:45:55 FIG. 11. Intermonomer exciton transfer at 4 K. Upper parts: Frequency-resolved lifetime of exciton states in the low-energy exciton band, calculated using the inter- molecular spectral density JNMA obtained from a NMA (left half) and the spectral density JFLN extracted from fluorescence line narrowing experiments62 (right half). The lowest exciton state in the trimer was excluded from the calculations since its lifetime is not limited by energy transfer. The solid black circles represent life- times extracted from hole-burning experiments.47 Lower parts: Density of exciton states of the three lowest (black lines), the second and third lowest (red lines), and the third lowest (blue lines) exciton states of FMO trimers, taking into account the renormalization of exciton energies by the exciton-vibrational coupling, described by two different spectral densities as in the upper parts of this figure. FIG. 11. Intermonomer exciton transfer at 4 K. Upper parts: Frequency-resolved lifetime of exciton states in the low-energy exciton band, calculated using the inter- molecular spectral density JNMA obtained from a NMA (left half) and the spectral density JFLN extracted from fluorescence line narrowing experiments62 (right half). The lowest exciton state in the trimer was excluded from the calculations since its lifetime is not limited by energy transfer. 3. Cryogenic temperatures Upper parts: Frequency-resolved lifetime of exciton states in the low-energy exciton band, calculated using the inter- molecular spectral density JNMA obtained from a NMA (left half) and the spectral density JFLN extracted from fluorescence line narrowing experiments62 (right half). The lowest exciton state in the trimer was excluded from the calculations since its lifetime is not limited by energy transfer. The solid black circles represent life- times extracted from hole-burning experiments.47 Lower parts: Density of exciton states of the three lowest (black lines), the second and third lowest (red lines), and the third lowest (blue lines) exciton states of FMO trimers, taking into account the renormalization of exciton energies by the exciton-vibrational coupling, described by two different spectral densities as in the upper parts of this figure. FIG. 12. Analysis of intermonomer transfer at 4 K in three different models. The upper row corresponds to the original model (investigated in the left row of Fig. 11). In the middle row, the site energy difference between the low-energy pigment BChl 3 and the remaining pigments was increased by 300 cm−1 (up-shifting the site energies of the latter) in order to study the effect of an increased localiza- tion of the lowest exciton state. In the lower row, we consider the Förster limit of transfer between local excited states [Eqs. (34), (35), and (42), using a pure dephasing time τpd = 1 ps]. The left column contains the distributions of downhill inverse intermonomer rate constants, and the right column contains the resulting frequency-resolved lifetime of exciton states. Note that the blue and the black line in the left bottom figure are on top of each other. FIG. 12. Analysis of intermonomer transfer at 4 K in three different models. The upper row corresponds to the original model (investigated in the left row of Fig. 11). In the middle row, the site energy difference between the low-energy pigment BChl 3 and the remaining pigments was increased by 300 cm−1 (up-shifting the site energies of the latter) in order to study the effect of an increased localiza- tion of the lowest exciton state. In the lower row, we consider the Förster limit of transfer between local excited states [Eqs. (34), (35), and (42), using a pure dephasing time τpd = 1 ps]. 3. Cryogenic temperatures The left column contains the distributions of downhill inverse intermonomer rate constants, and the right column contains the resulting frequency-resolved lifetime of exciton states. Note that the blue and the black line in the left bottom figure are on top of each other FIG. 10. Intramonomer exciton transfer at 77 K. Upper part: Density of exci- ton states without the lowest exciton state in each FMO monomer. Lower part: Intramonomer frequency-resolved lifetime of exciton states (excluding the lowest), calculated with different spectral densities, explained in the main text and in Table I, indicated in the legend. The black solid circles represent the lifetime estimated from 2D experiments.44 an increase was indeed measured in hole burning spectroscopy,47 as discussed above and denoted by two black circles in the upper parts of Fig. 11. An explanation of this behavior is given in terms of the distribution of downhill intermonomer transfer times between the different low-energy exciton states in Fig. 12. The distribution func- tions of the transfer between the third and the second exciton state (3 →2) have a larger amplitude at short transfer times than those of the 3 →1 and 2 →1 transfers, with average transfer times of 21 ps, 141 ps, and 45 ps, respectively (left upper part of Fig. 12). Therefore, the lifetime of the third exciton state is determined by the fast trans- fer to the second exciton state, and that of the second exciton state is determined by the slow transfer to the lowest exciton state. When going to lower energies in the lowest exciton band, it becomes less and less likely to find a FMO trimer that has two more exciton states at lower energies, and hence, the fast transfer 3 →2 will less and less contribute to the lifetime at lower energies, explaining the increase in the lifetime. This effect is lost if the lowest exciton state gets FIG. 10. Intramonomer exciton transfer at 77 K. Upper part: Density of exci- ton states without the lowest exciton state in each FMO monomer. Lower part: Intramonomer frequency-resolved lifetime of exciton states (excluding the lowest), calculated with different spectral densities, explained in the main text and in Table I, indicated in the legend. The black solid circles represent the lifetime estimated from 2D experiments.44 FIG. 11. Intermonomer exciton transfer at 4 K. 3. Cryogenic temperatures 11, we have ana- lyzed the respective lifetimes at 4 K and also provide the density of exciton states of the three lowest exciton states, the second and third lowest exciton states, and the third lowest exciton state of the FMO trimer, calculated with two different spectral densities JNMA(ω) and JFLN(ω). We have included the renormalization of the exciton ener- gies by the exciton-vibrational coupling in order to get as close as possible to the excitation energies that enter the absorption line- shape function [Eq. (29)]. Therefore, the density of exciton states depends now also on the spectral density of the exciton-vibrational coupling. The frequency-resolved lifetime T′r(ω) of exciton states is constant at about 15 ps on the high-energy half of the lowest exciton band and linearly increases up to 130 ps toward lower energies. Such FIG. 9. Distribution of inverse intermonomer rate constants at room temperature, calculated assuming fast intramonomer exciton equilibration [Eq. (17)], using dif- ferent spectral densities, explained in the main text and in Table I, as shown in the legend. In the calculations labeled as JNMA(cl), the intermolecular spectral density Jmk(ω) was used, and the rate constants were calculated with the semiclassical expression in Eq. (33). J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 153, 215103-12 © Author(s) 2020 The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp FIG. 10. Intramonomer exciton transfer at 77 K. Upper part: Density of exci- ton states without the lowest exciton state in each FMO monomer. Lower part: Intramonomer frequency-resolved lifetime of exciton states (excluding the lowest), calculated with different spectral densities, explained in the main text and in Table I, indicated in the legend. The black solid circles represent the lifetime estimated from 2D experiments.44 FIG. 11. Intermonomer exciton transfer at 4 K. Upper parts: Frequency-resolved lifetime of exciton states in the low-energy exciton band, calculated using the inter- molecular spectral density JNMA obtained from a NMA (left half) and the spectral density JFLN extracted from fluorescence line narrowing experiments62 (right half). The lowest exciton state in the trimer was excluded from the calculations since its lifetime is not limited by energy transfer. 3. Cryogenic temperatures The solid black circles represent life- times extracted from hole-burning experiments.47 Lower parts: Density of exciton states of the three lowest (black lines), the second and third lowest (red lines), and the third lowest (blue lines) exciton states of FMO trimers, taking into account the renormalization of exciton energies by the exciton-vibrational coupling, described by two different spectral densities as in the upper parts of this figure an increase was indeed measured in hole burning spectroscopy,47 as discussed above and denoted by two black circles in the upper parts of Fig. 11. An explanation of this behavior is given in terms of the distribution of downhill intermonomer transfer times between the different low-energy exciton states in Fig. 12. The distribution func- tions of the transfer between the third and the second exciton state (3 →2) have a larger amplitude at short transfer times than those of the 3 →1 and 2 →1 transfers, with average transfer times of 21 ps, 141 ps, and 45 ps, respectively (left upper part of Fig. 12). Therefore, the lifetime of the third exciton state is determined by the fast trans- fer to the second exciton state, and that of the second exciton state is determined by the slow transfer to the lowest exciton state. When going to lower energies in the lowest exciton band, it becomes less and less likely to find a FMO trimer that has two more exciton states at lower energies, and hence, the fast transfer 3 →2 will less and less contribute to the lifetime at lower energies, explaining the increase in the lifetime. This effect is lost if the lowest exciton state gets FIG. 12. Analysis of intermonomer transfer at 4 K in three different models. The upper row corresponds to the original model (investigated in the left row of Fig. 11). In the middle row, the site energy difference between the low-energy pigment BChl 3 and the remaining pigments was increased by 300 cm−1 (up-shifting the site energies of the latter) in order to study the effect of an increased localiza- tion of the lowest exciton state. In the lower row, we consider the Förster limit of transfer between local excited states [Eqs. (34), (35), and (42), using a pure dephasing time τpd = 1 ps]. 3. Cryogenic temperatures An advantage of a MD simulation is that it includes the anharmonic motion of nuclei, whereas our present NMA is able to resolve correlations in site energy calculations at very low vibrational frequencies that are difficult to sample with MD simulations. Concerning the site- independent Huang–Rhys factors of the intrapigment modes that were extracted from FLN spectra of the FMO protein,35 we can- not exclude that these factors are biased by the protein environ- ment of the low-energy pigment BChl 3, which dominates the lowest exciton state seen in the low-temperature fluorescence spectrum. On the other hand, a QM NMA on an isolated pigment depends on the limits of the QM method used, as, e.g., on the exchange- correlation functional in (TD)DFT calculations.15 Coker and co- workers10,15 found a way to take into account the site dependence of the intrapigment part of the spectral density, avoiding the geometry mismatch problem. They performed a local QM geometry optimiza- tion of the ground state potential energy surface of the pigments along the classical nuclear trajectories obtained from MD simula- tions and performed a QM NMA as a basis for the calculation of intramolecular Huang–Rhys factors of the pigments. In their careful analysis, using different XC functionals, they arrive at the conclusion (based on results presented in Fig. S5 of the supplementary material of Ref. 15) that for XC-(hybrid) functionals with small amounts or no exact exchange, the intramolecular spectral density of the differ- ent BChls is very similar and does not depend critically on the XC functional. However, with increasing amount of exact exchange, the intrapigment spectral density becomes both site-dependent and also depends on the XC-functional. An alternative solution to the geom- etry mismatch problem was provided by Rhee81,82 and co-workers and by Saito and co-workers16,37 using an interpolation between the MM and the QM potential energy surfaces of the BChls. Whereas Saito et al.,16,37 using the CAM-B3LYP XC-functional, find rather strong variations between sites, the spectral densities calculated by Kim et al.82 with the B3LYP XC-functional are characterized by very similar reorganization energies and Huang–Rhys factors. There is hope15 that the optimally tuned range-separated hybrid func- tionals83,84 will help to settle this issue. At present, it is undecided whether the present assumption of a site-independent intrapigment spectral density is justified. FIG. 13. Effect of exciton delocalization on the intermonomer exciton transfer at 4 K. 3. Cryogenic temperatures The solid black circles represent life- times extracted from hole-burning experiments.47 Lower parts: Density of exciton states of the three lowest (black lines), the second and third lowest (red lines), and the third lowest (blue lines) exciton states of FMO trimers, taking into account the renormalization of exciton energies by the exciton-vibrational coupling, described by two different spectral densities as in the upper parts of this figure. FIG. 12. Analysis of intermonomer transfer at 4 K in three different models. The upper row corresponds to the original model (investigated in the left row of Fig. 11). In the middle row, the site energy difference between the low-energy pigment BChl 3 and the remaining pigments was increased by 300 cm−1 (up-shifting the site energies of the latter) in order to study the effect of an increased localiza- tion of the lowest exciton state. In the lower row, we consider the Förster limit of transfer between local excited states [Eqs. (34), (35), and (42), using a pure dephasing time τpd = 1 ps]. The left column contains the distributions of downhill inverse intermonomer rate constants, and the right column contains the resulting frequency-resolved lifetime of exciton states. Note that the blue and the black line in the left bottom figure are on top of each other. FIG. 12. Analysis of intermonomer transfer at 4 K in three different models. The upper row corresponds to the original model (investigated in the left row of Fig. 11). In the middle row, the site energy difference between the low-energy pigment BChl 3 and the remaining pigments was increased by 300 cm−1 (up-shifting the site energies of the latter) in order to study the effect of an increased localiza- tion of the lowest exciton state. In the lower row, we consider the Förster limit of transfer between local excited states [Eqs. (34), (35), and (42), using a pure dephasing time τpd = 1 ps]. The left column contains the distributions of downhill inverse intermonomer rate constants, and the right column contains the resulting frequency-resolved lifetime of exciton states. Note that the blue and the black line in the left bottom figure are on top of each other. J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 153, 215103-13 © Author(s) 2020 The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp FIG. 13. 3. Cryogenic temperatures Effect of exciton delocalization on the intermonomer exciton transfer at 4 K. Comparison of frequency-resolved lifetimes of exciton states in the low-energy exciton band calculated by including only the pigments shown in the figure legend. The cases “all” and “BChl 3′′ correspond to the limiting cases considered already in the upper and lower part, respectively, of Fig. 12. charge density coupling (CDC) method,9 and a site-independent intrapigment part, obtained from FLN experiments,35 is one of the simplest possible ways to avoid the geometry mismatch prob- lem10,15,16,36,37 that one is facing in traditional QM/MM approaches that combine classical molecular dynamics simulations with quan- tum chemical calculations of transition energy shifts of the pig- ments. Since the CDC method5 just determines the intermolecu- lar pigment–protein Coulomb interactions, it does not suffer from slight distortions of the nuclear coordinates. A workaround, related in spirit, was suggested by Shi and co-workers,36 combining the CDC method with a classical MD simulation to obtain the intermolecular part of the spectral density and performing a quantum mechani- cal normal mode analysis on the isolated, geometry-optimized pig- ment to obtain a site-independent intrapigment part. An advantage of a MD simulation is that it includes the anharmonic motion of nuclei, whereas our present NMA is able to resolve correlations in site energy calculations at very low vibrational frequencies that are difficult to sample with MD simulations. Concerning the site- independent Huang–Rhys factors of the intrapigment modes that were extracted from FLN spectra of the FMO protein,35 we can- not exclude that these factors are biased by the protein environ- ment of the low-energy pigment BChl 3, which dominates the lowest exciton state seen in the low-temperature fluorescence spectrum. On the other hand, a QM NMA on an isolated pigment depends on the limits of the QM method used, as, e.g., on the exchange- correlation functional in (TD)DFT calculations.15 Coker and co- workers10,15 found a way to take into account the site dependence of the intrapigment part of the spectral density, avoiding the geometry mismatch problem. They performed a local QM geometry optimiza- tion of the ground state potential energy surface of the pigments along the classical nuclear trajectories obtained from MD simula- tions and performed a QM NMA as a basis for the calculation of intramolecular Huang–Rhys factors of the pigments. 24 October 2024 05:45:55 24 October 2024 05:45:55 g y Finally, we have investigated which pigments essentially con- tribute to the transfer between the low-energy exciton states of FMO monomers. Besides the lowest-energy pigment BChl 3, obvious can- didates are BChls 4, 2, and 7, which are the pigments with the next higher transition energies (see Table II in Appendix C) and relatively large excitonic couplings to pigments in another FMO monomer (Table III in Appendix C). Including just BChls 3 and 4 in the calculation of intermonomer energy transfer lifetimes gives rise to an inverted wavelength dependence as compared to the case, where all pigments are included (blue and black line in Fig. 13, respectively). Taking into account, in addition, BChls 2 and 7 results in a qualitatively correct wavelength dependence (orange line). A more quantitative agreement with the full calculation results, if besides the low-energy BChls 3, 4, 2, and 7 also BChl 5 (which is strongly coupled to BChl 2′ of the neighboring monomer) is included (brown line). i Concerning the intermolecular contributions to the spectral density, Coker and co-workers,10,15 combining the CDC method5 with MD simulations, arrived at similar local reorganization ener- gies E(k) λ for the BChl pigments in the different binding sites k, as seen in Table II (Appendix C). The largest difference is obtained for BChl 8, which is solvent-exposed. Most likely, interaction with 3. Cryogenic temperatures In their careful analysis, using different XC functionals, they arrive at the conclusion (based on results presented in Fig. S5 of the supplementary material of Ref. 15) that for XC-(hybrid) functionals with small amounts or no exact exchange, the intramolecular spectral density of the differ- ent BChls is very similar and does not depend critically on the XC functional. However, with increasing amount of exact exchange, the intrapigment spectral density becomes both site-dependent and also depends on the XC-functional. An alternative solution to the geom- etry mismatch problem was provided by Rhee81,82 and co-workers and by Saito and co-workers16,37 using an interpolation between the MM and the QM potential energy surfaces of the BChls. Whereas Saito et al.,16,37 using the CAM-B3LYP XC-functional, find rather strong variations between sites, the spectral densities calculated by Kim et al.82 with the B3LYP XC-functional are characterized by very similar reorganization energies and Huang–Rhys factors. There is hope15 that the optimally tuned range-separated hybrid func- tionals83,84 will help to settle this issue. At present, it is undecided whether the present assumption of a site-independent intrapigment spectral density is justified. Concerning the intermolecular contributions to the spectral density, Coker and co-workers,10,15 combining the CDC method5 h MD l d l l l charge density coupling (CDC) method,9 and a site-independent intrapigment part, obtained from FLN experiments,35 is one of the simplest possible ways to avoid the geometry mismatch prob- lem10,15,16,36,37 that one is facing in traditional QM/MM approaches charge density coupling (CDC) method, and a site independent intrapigment part, obtained from FLN experiments,35 is one of the simplest possible ways to avoid the geometry mismatch prob- lem10,15,16,36,37 that one is facing in traditional QM/MM approaches that combine classical molecular dynamics simulations with quan- tum chemical calculations of transition energy shifts of the pig- ments. Since the CDC method5 just determines the intermolecu- lar pigment–protein Coulomb interactions, it does not suffer from slight distortions of the nuclear coordinates. A workaround, related in spirit, was suggested by Shi and co-workers,36 combining the CDC method with a classical MD simulation to obtain the intermolecular part of the spectral density and performing a quantum mechani- cal normal mode analysis on the isolated, geometry-optimized pig- ment to obtain a site-independent intrapigment part. 3. Cryogenic temperatures Comparison of frequency-resolved lifetimes of exciton states in the low-energy exciton band calculated by including only the pigments shown in the figure legend. The cases “all” and “BChl 3′′ correspond to the limiting cases considered already in the upper and lower part, respectively, of Fig. 12. more localized. We have increased the energy gap between the low- energy pigment BChl 3 and the remaining pigments by 300 cm−1 and repeated the calculations of the distribution functions of the inverse rate constants and the frequency-resolved exciton state life- time (middle part in Fig. 12). Now, the distribution functions for the 3 →2 and the 2 →1 transfer are almost equal, resulting in very similar average inverse rate constants of 19 ps and 17 ps, respec- tively. This equality gives rise to a much less frequency-dependent lifetime across the whole low-energy exciton band (middle right part of Fig. 12). For a complete localization of excited states, that is, using standard Förster theory to describe the transfer between localized excited states in different monomers, we find the fastest transfer and practically identical distribution functions for the 3 →2 and 2 →1 transfer with an identical average inverse rate constant of 8 ps, resulting in a frequency-independent lifetime. B. Correlations in site energy fluctuations and energy transfer This time constant is more than twice as large as the ≈10 ps trans- fer time obtained for the realistic intermolecular spectral density at room temperature (Fig. 9). Hence, the effect of the hypothetical cor- relations in Eq. (49) on the intermonomer transfer is not as dramatic as on the intramonomer exciton relaxation but still present. In the hypothetical case that intramonomer exciton relaxation is still fast, the disorder averaged intermonomer rate constant ka→b [Eq. (17)], using the kGF Ma→Nb for perfectly correlated site energies [Eq. (50)], cor- responds to a time constant of 5 ps, which is even faster than that of the uncorrelated transfer. However, nature cannot take advantage of this effect because excitons would not relax in the case of perfect correlations. A main purpose of the present work was to quantify the corre- lations in site energy fluctuations in FMO trimers and to investigate their role in intermonomer energy transfer. So far, there are three studies on correlations,9,85,86 which investigated the monomeric sub- unit of the FMO protein. In a QM/MM approach,85 no correlations were found, whereas our earlier NMA in combination with the CDC method revealed correlations at very low vibrational frequencies.9 Probably, the MD simulations did not resolve these correlations because of the finite simulation times or because the intermolecular site energy correlations were overshadowed by artificially enhanced uncorrelated intrapigment site energy fluctuations caused by the geometry mismatch problem. Cross correlations between site energy fluctuations were reported in another QM/MM study,86 despite the geometry mismatch problem and short (40 ps) simulation times. These correlations were found to have practically no influence on intramonomer exciton relaxation, in agreement with our earlier9 and our present simulations (Fig. 10).i 24 October 2024 05:45:55 What still needs to be understood is why the real correlations in Fig. 6 are obviously so far away from the hypothetical case in Eq. (49). One difference is that the hypothetical Jmk(ω) is positive at all frequencies, whereas the real Jmk(ω) can be positive at some frequencies and negative at others. However, inverting the negative values in the latter has practically no influence on the calculated rate constants for the intermonomer transfer (Fig. 9). Hence, the explanation must be related to the different amplitudes and spectral shapes of the diagonal (Fig. 3) and off-diagonal (Fig. 6) parts of the spectral density Jmk(ω). A. Spectral density The present approach to split the spectral density into an inter- molecular part, obtained from a combination of the NMA and the J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 153, 215103-14 153, 215103-14 The Journal of Chemical Physics The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp water molecules, not included in the present NMA, but in the MD simulation, is responsible for the three times larger reorganization energy obtained with the latter. Both methods agree on the large intermolecular reorganization energy of BChl 2, which has a water ligand and a couple of additional water molecules in its neighbor- hood. The analysis of Rivera et al.10 suggests that about half of the enlarged reorganization energy of BChl 2 is due to the water molecules and the remaining half is due to interaction with polar amino acid side chains of the protein. Since in our analysis only one water molecule, the axial ligand of BChl 2, was taken into account (besides a second water molecule that is hydrogen bonded to BChl 1) the good agreement between the two reorganization energies of BChl 2 seems to be somewhat fortuitous. A remarkable feature of BChl 2 in our calculations is the effect of long-range electrostatic couplings over distances as large as 40 Å (Fig. 5) that increase the Huang–Rhys factor (and the related reorganization energy) of this pigment. becomes zero. In addition, it was found that coherences between different exciton states would live very long.9,25 These results led us to conclude that nature does not protect inter-exciton coherences since excitons would not relax. Introducing Eq. (49) into the expression for the generalized Förster rate constant kGF Ma→Nb [Eq. (19)], noting that the dephasing times τMa and τNb are infinite, because of the missing intermonomer exciton relaxation, and the function FMaNb(t) in Eqs. (20)–(22) is zero, we obtain kGF Ma→Nb = 2π ̵h2 ∣VMaNb∣2δ(˜ωMa,Nb). (50) (50) This equation is simply Fermi’s Golden rule, where the only effect of the exciton-vibrational coupling left is the small renormalization of the exciton energies [Eq. (25)]. For the present site energies and excitonic couplings, an inverse average intermonomer rate constant of 24 ps is obtained, where the average was taken with respect to dis- order in site energies and the different intramonomer exciton states. A. Spectral density This time constant is more than twice as large as the ≈10 ps trans- fer time obtained for the realistic intermolecular spectral density at room temperature (Fig. 9). Hence, the effect of the hypothetical cor- relations in Eq. (49) on the intermonomer transfer is not as dramatic as on the intramonomer exciton relaxation but still present. In the hypothetical case that intramonomer exciton relaxation is still fast, the disorder averaged intermonomer rate constant ka→b [Eq. (17)], using the kGF Ma→Nb for perfectly correlated site energies [Eq. (50)], cor- responds to a time constant of 5 ps, which is even faster than that of the uncorrelated transfer. However, nature cannot take advantage of this effect because excitons would not relax in the case of perfect correlations. B. Correlations in site energy fluctuations and energy transfer The off-diagonal parts with increasing fre- quency decrease faster to zero than the diagonal parts, reflecting the fact that correlations require delocalized vibrational normal modes, whereas the diagonal parts Jmm(ω) are influenced strongest by the intermediate environments of the pigments (Fig. 5). Hence, high- frequency normal modes that are localized in the close environment of the pigments have an influence on the diagonal parts Jmm(ω) but do not affect the off-diagonal parts Jmk(ω). Therefore, Eq. (49) can- not be fulfilled, in particular, at large intermolecular frequencies. Since anharmonic vibrational effects, neglected in the present work, are expected to contribute mainly at low frequencies, it is likely that these effects will not work in favor of Eq. (49) either. Nevertheless, it is of general interest to clarify the role of anharmonic nuclear motion in the correlation of site energy fluctuations. Interestingly, we find similar amplitudes of the intramonomer and the intermonomer correlations in site energy fluctuations (Fig. 6), despite the much larger distances between pigments in different monomers (Fig. 1). Obviously, the low-frequency normal modes of the FMO protein are delocalized over the entire trimer, causing the correlations in site energy fluctuations. Do they have any functional relevance? No, these correlations have practically no influence on intermonomer energy transfer (Fig. 9). 9 g An important result of our earlier work9 is that exciton relax- ation were most affected if the correlations in site energy fluctua- tions, which are contained in the off-diagonal parts of the spectral density Jmk(ω), would be related to the respective diagonal parts Jmm and Jkk, describing the site energy fluctuations, in the following way: Jmk(ω) = 1 2(Jmm(ω) + Jkk(ω)). (49) (49) C. Intramonomer exciton relaxation It is, however, difficult to imagine that nature actively worked on this optimization, considering that the transfer from the baseplate into the FMO protein and the transfer from the FMO protein to the reaction center complex occur on orders of magnitudes slower timescales.87 of the lowest exciton state in the FMO monomers. Interestingly, the minimal model to describe this effect includes not just the two low-energy pigments BChls 3 and 4 but also BChls 2, 5, and 7 (Fig. 13). Due to this delocalization, the square of the excitonic coupling entering the rate constant [Eqs. (19) and (8)] between the third and the second exciton state of the FMO trimer on average is about a factor of 2.4 larger than that between the second and the first exci- ton state, giving rise to the different rate constants that explain the increase in lifetime (Fig. 12). Upon localizing the lowest states of the FMO monomers, this effect is lost, and the lifetime becomes frequency-independent (Figs. 12 and 13). Interestingly, the inter- monomer transfer is faster in this hypothetical limit. So, exciton delocalization in the FMO monomers slows down intermonomer transfer, in contrast to the results of Jankowiak and co-workers,49,50 who, however, did not focus on the present effect and used a differ- ent spectral density in their Förster and in their generalized Förster calculations. We note that an opposite effect was found in the LH2 com- plex of purple bacteria, where standard Förster theory gave an about five times too small rate constant for the transfer between the B800 and the B850 states of the antenna.88 Generalized Förster theory tak- ing into account the delocalization of the B850 states explained64–66 the experimental result. Optically dark exciton states of the B850 manifold accept excitation energy from the B800 states, an effect that cannot be described by Förster theory, where only bright states contribute to the transfer. The FMO trimer represents an example, where quantum mechanic delocalization of excited states acts in the opposite direc- tion, slowing down the transfer. Obviously, the light-harvesting apparatus of green sulfur bacteria can tolerate this effect. At room temperature, we find that Förster theory would result in an aver- age intermonomer transfer time of 6 ps, which is roughly half the time we obtain by using generalized Förster theory. C. Intramonomer exciton relaxation In this hypothetical case, using the equality ∑ka c(Ma) ka c(Na) ka = δMa,Na, it is seen that the Redfield relaxation rate constant kRedf Ma→Na in Eq. (15) In this hypothetical case, using the equality ∑ka c(Ma) ka c(Na) ka = δMa,Na, it is seen that the Redfield relaxation rate constant kRedf Ma→Na in Eq. (15) Intramonomer exciton relaxation in the FMO protein occurs on an ultrafast subpicosecond timescale both at room J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 153, 215103-15 © Author(s) 2020 The Journal of Chemical Physics The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp temperature (Fig. 8) and at cryogenic temperatures (Fig. 10). In both cases, the exciton relaxation is accelerated by the dissipation of high-frequency intrapigment vibrational quanta, reflecting the fact that the differences in exciton energies are in the 0 cm−1– 500 cm−1 range.3,9 Good agreement between calculated lifetimes with those estimated in 2D experiments on C. tepidum at 77 K is obtained except for the bottleneck around 810 nm, which is much more pronounced in the experiment (Fig. 10). The devia- tions could be due to the fact that the present calculations were performed with the Hamiltonian of P. aestuarii, whereas the exper- iments were done on C. tepidum. Although both structures are very similar, their optical spectra show some differences3 that still need to be understood. Another explanation for the long experimental lifetime around 810 nm could be that the pigments contributing to the underlying exciton state (BChls 1 and/or 2)3,25 exhibit a weaker intrapigment exciton-vibrational coupling and therefore the respec- tive exciton state a longer lifetime (Fig. 10). Recent QM/MM cal- culations by Saito and co-workers,16,37 which report a particularly strong exciton-vibrational coupling of BChl 2, seem to suggest that a candidate for weak intrapigment coupling could be BChl 1. On the other hand, as discussed above, the QM/MM calculations of Kim et al.82 obtain only very small variations in Huang–Rhys factors of the different pigments. Saito et al.16 demonstrated that their vari- ation in local reorganization energies optimizes exciton relaxation. C. Intramonomer exciton relaxation Any quantitative estimate of the delocalization effect on the overall energy transfer in green sulfur bacteria has to await the molecular structure of FMO- reaction center supercomplexes. From 2D experiments on whole cells of C. tepidum at 77 K, a time constant of 17 ps has been inferred for the transfer between the FMO protein and the reaction center complex.87 At physiological temperatures, the transfer is most likely somewhat faster, but still in the same order of magnitude as the intermonomer transfer occurring on a 10 ps time scale according to the present calculations. Hence, the system might still take advan- tage of the intermonomer transfer in the FMO protein in order to transfer excitation energy efficiently to the reaction center complex. D. Intermonomer energy transfer Intermonomer energy transfer is calculated to occur with a time constant of about 10 ps at room temperature (Fig. 9). As noted earlier, this transfer is hard, if not impossible, to detect experimen- tally since the lifetime of the exciton states including the lowest one is determined by intramonomer exciton transfer. Even the uphill transfer in the FMO monomer is more than an order of magni- tude faster (Fig. 8) than the intermonomer transfer. Most likely, the time constant of 1.4 ps–2 ps suggested in an early room temperature pump–probe study38 to reflect intermonomer transfer has a different origin. According to the present calculations, this transfer is about five times slower. i In contrast to the intramonomer transfer, the intermonomer transfer is governed by the couplings of excitons to low-frequency intermolecular vibrations (Fig. 9), suggesting that the most impor- tant intermonomer channels involve exciton states at close energies in different FMO monomers. Because of the small vibrational fre- quencies involved in the transfer, the classical description of nuclear motion works so well at room temperature (Fig. 9). SUPPLEMENTARY MATERIAL See the supplementary material for the atomic partial charges of the ground and excited state of BChl a. Trvib{(U(0) Ma (t)) † U(0) Nb (t)W(Ma) eq } = eiω′ MaNb teGMaNb (t)−GMaNb (0), (A7) (A7) VI. CONCLUSIONS (A5) where UMc(t) is the time evolution operator of the vibrational degrees of freedom in the PES of exciton state ∣Mc⟩, where UMc(t) is the time evolution operator of the vibrational degrees of freedom in the PES of exciton state ∣Mc⟩, UMc(t) = ⟨Mc∣U(0) c (t)∣Mc⟩, (A6) (A6) and W(g) eq is the equilibrium statistical operator of the vibrational degrees of freedom in the electronic ground state. The trace in the second line of Eq. (A5) is obtained as ACKNOWLEDGMENTS with ω′ MaNb = ω′ Ma −ω′ Nb, where these frequencies are defined in Eq. (4), and the time-dependent function Financial support by the Austrian Science Fund (FWF) (Grant No. P 33155-NBL) and by the Austrian Federal Ministry of Science, Research and Economy and the State of Upper Austria (Grant No. LIT-2017-4-SEE-009) is gratefully acknowledged. FMaNb(t) = ∫ ∞ −∞dωJMaNb(ω){(1 + n(ω))e−iωt + n(ω)eiωt} (A8) (A8) 24 October 2024 05:45:55 that contains the spectral density VI. CONCLUSIONS The main conclusions of the present work are as follows: (i) Intermonomer transfer in the FMO protein occurs with a time con- stant of about 10 ps at room temperature and may well compete with the transfer between the FMO protein and the reaction center com- plex. (ii) The low-frequency normal modes of the FMO protein are delocalized over the whole trimer and cause correlations and anti- correlations in the fluctuations of site energies of different pigments in the same and in different FMO monomers. (iii) The effect of these correlations on intra- as well as intermonomer energy trans- fer is negligible mainly because of the different spectral shapes of At cryogenic temperatures, the lifetimes of two of the three exciton states in the lowest exciton band of the trimer are lim- ited by intermonomer transfer since intramonomer transfer would be uphill in energy and is, therefore, frozen out. In good agree- ment with estimates from hole burning data,47 we obtain a lifetime of 15 ps–25 ps for the high-energy half of the low-energy exciton band and an increase to more than 100 ps at the low-energy edge. The origin of the increase lies in the intramonomer delocalization J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 153, 215103-16 The Journal of Chemical Physics The Journal of Chemical Physics Trvib{⟨Ma∣U† a(t) ˆVUb(t)∣Nb⟩VNbMaW(Ma) eq } ≈Trvib{⟨Ma∣S† a(t)∣Ma⟩W(g) eq } ×Trvib{(U(0) Ma (t)) † U(0) Nb (t)W(Ma) eq } ×Trvib{⟨Nb∣Sb(t)∣Nb⟩W(g) eq }, (A5) the diagonal and the off-diagonal parts of the intermolecular spec- tral density. (iv) Exciton delocalization in the FMO monomer slows down the intermonomer transfer by about a factor of 2 at physiologi- cal temperatures. (v) A direct signature of this exciton delocalization is seen in the wavelength dependence of the lifetime of low-energy exciton states at cryogenic temperatures detected with hole burning spectroscopy and explained in the present work. (vi) Intramonomer exciton relaxation involves the dissipation of intrapigment vibra- tional quanta, while intermonomer transfer is dominated by the cou- pling to low-frequency intermolecular vibrations. Any fine details of the spectral density are not so critical for intramonomer exciton relaxation and intermonomer energy transfer. A classical descrip- tion of nuclear motion works well for the intermonomer transfer at room temperature. APPENDIX A: DERIVATION OF RATE CONSTANT The rate constant kGF Ma→Nb is obtained in second-order pertur- bation theory in the interdomain coupling ˆV, JMaNb(ω) = ∑ ξ (gξ(Ma, Ma) −gξ(Nb, Nb))2δ(ω −ωξ). (A9) (A9) kGF Ma→Nb = 2 ̵h2 R ∫ ∞ 0 dtTrvib{⟨Ma∣U† a(t) ˆVUb(t)∣Nb⟩VNbMaW(Ma) eq }, (A1) With the help of Eq. (6), the above spectral density can be related to the spectral density of the local exciton-vibrational coupling } (A1) where R denotes the real part, Trvib denotes a trace over the vibra- tional degrees of freedom (DOF), W(Ma) eq is the equilibrium statistical operator of the vibrational DOF in the potential energy surface (PES) of exciton state ∣Ma⟩, and Uc(τ) is the time-evolution operator of domain c, JMaNb(ω) = ∑ ma,na ∣c(Ma) ma ∣2∣c(Ma) na ∣2Jtotal mana(ω) + ∑ mb,nb ∣c(Mb) mb ∣2∣c(Nb) nb ∣2Jtotal mbnb(ω) −2 ∑ ma,nb ∣c(Ma) ma ∣2∣c(Nb) mb ∣2Jmanb(ω). (A10) (A10) Uc(t) = e−i ̵h Hct = U(0) c (t)Sc(t) (A2) (A2) with the time evolution operator with the time evolution operator In this way, the function FMaNb(t) in Eq. (A8) can be decomposed into U(0) c (t) = e−i ̵h H(0) c t (A3) (A3) FMaNb(t) = GMa(t) + GNb(t) −2GMaNb(t) (A11) (A11) containing H(0) c in Eq. (3) and the S-operator of domain c, containing H(0) c in Eq. (3) and the S-operator of domain c, with the function GKc(t) in Eq. (39) that contains the site energy fluctuations and their correlations in domain c and the function GMaNb(t), defined in Eq. (22), that contains the correlations in site energy fluctuations of pigments in different domains a and b. The traces in the first and third line of Eq. (A5) are obtained as55 Sc(t) = Te−i ̵h ∫ t 0 dτ ˆVc(τ) (A4) (A4) where ˆVc(τ) = (U(0) c (τ))† ˆVcU(0) c (τ) contains the time-ordering operator T and Vc in Eq. (5). In secular approximation, the trace over the vibrational degrees of freedom in Eq. (A1) can be factorized according to Ref. 55, Trvib{⟨Ma∣S† a(t)∣Ma⟩W(g) eq } = e−τ/τMa eiΔωMa (A12) (A12) J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 153, 215103-17 © Author(s) 2020 The Journal of Chemical Physics The Journal of Chemical Physics ARTICLE scitation.org/journal/jcp 1. Intermolecular reorganization energies with the dephasing time τMc and the frequency shift ΔωMc in Eqs. (23) and (27), respectively. Table II shows reorganization energies of the intermolecular exciton-vibrational coupling of the BChl pigments in units of cm−1. Table III shows site energies (diagonal) and excitonic couplings (off-diagonal) of BChl pigments in the monomeric subunit (exciton domain) of the FMO protein in units of cm-1. APPENDIX C: INTERMOLECULAR REORGANIZATION ENERGIES AND EXCITON MATRIX and Trvib{⟨Nb∣Sb(t)∣Nb⟩W(g) eq } = e−τ/τNb e−iΔωNb (A13) Trvib{⟨Nb∣Sb(t)∣Nb⟩W(g) eq } = e−τ/τNb e−iΔωNb (A13) (A13) 24 October 2024 05:45:55 FIG. 14. Correlation of site energy shifts of the FMO trimer computed with the PBQC method based on the geometry-optimized structure employing APCs from CAM-B3LYP with fitted site energies from Ref. 7 (see also Table III). The straight line has slope 1 and an axis section corresponding to a reference excitation energy of the S1 state of BChl a of E0 = 12 496 ± 22 cm−1. The standard error of computed site energies against the fitted values is 61 cm−1. APPENDIX B: PROTONATION STATES AND SITE ENERGIES TABLE II. Reorganization energies of the intermolecular exciton-vibrational coupling E(k) λ [Eq. (41)] of the different sites k averaged over the three equivalent sites in the FMO trimer are compared with values from the literature,10 in units of cm−1. TABLE II. Reorganization energies of the intermolecular exciton-vibrational coupling E(k) λ [Eq. (41)] of the different sites k averaged over the three equivalent sites in the FMO trimer are compared with values from the literature,10 in units of cm−1. k 1 2 3 4 5 6 7 8 E(k) λ (Present) 21 30 14 12 10 24 14 13 E(k) λ (From Ref. 10) 15 31 17 13 11 14 20 38 Based on the geometry-optimized structure of the FMO trimer (see Sec. III), site energies of the eight BChl a pigments were computed by employing the PBQC method4 to test the APCs derived from CAM-B3LYP. The PBQC method was implemented as described in Ref. 89 using dielectric constants of ϵp = 4.0, ϵsolv = 80.0 for protonation patterns and ˜ϵp = 1.5, ϵsolv = 80.0 for pigment–protein interactions as well as an ionic strength in the outer medium corresponding to a 0.1M NaCl solution with sol- vent radius 1.4 Å and ion radius 2.0 Å. In the Monte Carlo titra- tion of protonation states, it was found that His 12, the only histidine considered as titratable, was largely positively charged in the pH range from 8 to 11 (i.e., 100% at pH 8.0; 60% at pH 11.0). In the same pH range, all other titratable groups were found to be in their respective standard protonation states. Con- sequently, no re-optimization of the FMO trimer (net charge: −6e) was necessary to account for non-standard protonation states, while His 12 was modeled as charged in all CHARMM compu- tations (see part III). The resulting site energy shifts (in cm−1: 1, 59; 2, 62; 3, −255; 4,-184; 5, 101; 6, 41; 7, 31; and 8, 174; averaged over the three monomers) are plotted against fitted site energies (“holo” from Ref. 7) in Fig. 14, revealing a good correlation and con- firming the consistency of our methods employing the CAM-B3LYP functional. k 1 2 3 4 5 6 7 8 E(k) λ (Present) 21 30 14 12 10 24 14 13 E(k) λ (From Ref. 10) 15 31 17 13 11 14 20 38 2. FMO monomer TABLE III. Site energies (diagonal) and excitonic couplings (off-diagonal) of BChl pigments in the monomeric subunit (exciton domain) of the FMO protein in units of cm−1. The site energies were taken from column “holo” of Table I of Ref. 7. The exci- tonic couplings were obtained as described in the main text. The double prime at BChl 8 indicates that this pigment was assigned to monomer 3 in the crystal structure.30 Hence, in the order of the crystal structure, it is the last pigment (number 24). 1 2 3 4 5 6 7 8′′(24) 1 12 505 −94.8 5.5 −5.9 7.1 −15.1 −12.2 39.5 2 12 425 29.8 7.6 1.6 13.1 5.7 7.9 3 12 195 −58.9 −1.2 −9.3 3.4 1.4 4 12 375 −64.1 −17.4 −62.3 −1.6 5 12 600 89.5 −4.6 4.4 6 12 515 35.1 −9.1 7 12 465 −11.1 8′′(24) 12 700 DATA AVAILABILITY 28M. B. Plenio and S. F. Huelga, New J. Phys. 10, 113019 (2008). 29 29P. Rebentrost, M. Mohseni, I. kassal, S. Lloyd, and A. Aspuru-Guzik, New J. Phys. 11, 033003 (2009). The atomic partial charges of the ground and excited state of BChl a, as well as the parameters of the exciton Hamiltonian, the optimized structural coordinates of the FMO trimer, the eigenfre- quencies ωξ of the NMA, and the coupling constants gξ(m) of the exciton-vibrational coupling can be downloaded from the Zenodo repository via https://doi.org/10.5281/zenodo.4110066. 30D. 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Excitonic couplings between BChl pigments in two monomeric subunits of the FMO protein in units of cm−1. The full coupling matrix of the FMO trimer can be constructed from the present table and Table III by using the C3 symmetry of the complex. The numbers in brackets and the primes and double primes correspond to the pigment order in the crystal structure.30 A prime indicates that this pigment in the crystal structure is assigned to monomer 2 and a double prime indicates assignment to monomer 3. 1′(9) 2′(10) 3′(11) 4′(12) 5′(13) 6′(14) 7′(15) 8 (8) 1 1.7 2.4 2.1 0.4 1.0 0.0 0.5 0.2 2 0.7 −0.1 0.6 0.6 1.5 1.0 0.5 0.9 3 −0.8 −3.5 −3.6 0.6 1.7 1.0 −0.8 2.3 4 0.3 −3.3 7.6 3.0 −0.5 2.2 7.2 −2.3 5 4.0 11.3 6.4 −1.2 2.6 −2.4 −2.4 6.6 6 2.4 7.1 2.9 −0.5 −0.0 −2.2 0.0 −3.5 7 1.3 2.8 7.3 2.9 −0.9 2.7 9.6 −8.6 8′′(24) 0.2 1.1 1.2 −1.4 2.0 −1.4 −3.3 5.3 FIG. 14. Correlation of site energy shifts of the FMO trimer computed with the PBQC method based on the geometry-optimized structure employing APCs from CAM-B3LYP with fitted site energies from Ref. 7 (see also Table III). The straight line has slope 1 and an axis section corresponding to a reference excitation energy of the S1 state of BChl a of E0 = 12 496 ± 22 cm−1. The standard error of computed site energies against the fitted values is 61 cm−1. FIG. 14. Correlation of site energy shifts of the FMO trimer computed with the PBQC method based on the geometry-optimized structure employing APCs from CAM-B3LYP with fitted site energies from Ref. 7 (see also Table III). The straight line has slope 1 and an axis section corresponding to a reference excitation energy of the S1 state of BChl a of E0 = 12 496 ± 22 cm−1. The standard error of computed site energies against the fitted values is 61 cm−1. J. Chem. Phys. 153, 215103 (2020); doi: 10.1063/5.0027994 © Author(s) 2020 J. Chem. 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https://openalex.org/W3135059624	https://dspace.mit.edu/bitstream/1721.1/133368/2/elife-65924-v2.pdf	English	null	A mobile genetic element increases bacterial host fitness by manipulating development	eLife	2,021	cc-by	16,519	MIT Open Access Articles The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Terms of use: Creative Commons Attribution 4.0 International license RESEARCH ARTICLE Introduction Cargo genes include those encoding antibiotic resistances, metabolic pathways, and determinants of pathogenesis and symbiosis (Johnson and Grossman, 2015). Transfer of mobile elements between cells contributes to rapid evolution and spread of associated cargo genes and phenotypes (Frost et al., 2005; Treangen and Rocha, 2011). Despite the benefits cargo genes can provide the maintenance and transfer of mobile genetic A mobile genetic element increases bacterial host fitness by manipulating development Joshua M Jones1, Ilana Grinberg2, Avigdor Eldar2, Alan D Grossman1 1Department of Biology, Massachusetts Institute of Technology, Cambridge, United States; 2The Shmunis School of Biomedicine and Cancer Research, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Abstract Horizontal gene transfer is a major force in bacterial evolution. Mobile genetic elements are responsible for much of horizontal gene transfer and also carry beneficial cargo genes. Uncovering strategies used by mobile genetic elements to benefit host cells is crucial for understanding their stability and spread in populations. We describe a benefit that ICEBs1, an integrative and conjugative element of Bacillus subtilis, provides to its host cells. Activation of ICEBs1 conferred a frequency-dependent selective advantage to host cells during two different developmental processes: biofilm formation and sporulation. These benefits were due to inhibition of biofilm-associated gene expression and delayed sporulation by ICEBs1-containing cells, enabling them to exploit their neighbors and grow more prior to development. A single ICEBs1 gene, devI (formerly ydcO), was both necessary and sufficient for inhibition of development. Manipulation of host developmental programs allows ICEBs1 to increase host fitness, thereby increasing propagation of the element. Introduction For correspondence: avigdor@gmail.com (AE); adg@mit.edu (ADG) Conjugative elements and phages are abundant mobile genetic elements in bacteria, capable of transferring DNA between cells (Frost et al., 2005). Integrative and conjugative elements (ICEs) appear to be the most widespread type of conjugative element (Guglielmini et al., 2011). ICEs are found integrated in a host genome. When activated, they excise and produce conjugation machinery that transfers the element DNA from the host cell to recipients (Carraro and Burrus, 2015; Johnson and Grossman, 2015; Wozniak and Waldor, 2010). Competing interests: The authors declare that no competing interests exist. Funding: See page 20 Received: 21 December 2020 Accepted: 01 March 2021 Published: 03 March 2021 Funding: See page 20 Received: 21 December 2020 Accepted: 01 March 2021 Published: 03 March 2021 Funding: See page 20 Received: 21 December 2020 Accepted: 01 March 2021 Published: 03 March 2021 Funding: See page 20 Received: 21 December 2020 Accepted: 01 March 2021 Published: 03 March 2021 , ; , ) ICEs often carry ‘cargo’ genes that are not necessary for transfer but confer a phenotype to host cells. In fact, ICEs (conjugative transposons) were first identified because of the phenotypes con- ferred by cargo genes (Franke and Clewell, 1981). Cargo genes include those encoding antibiotic resistances, metabolic pathways, and determinants of pathogenesis and symbiosis (Johnson and Grossman, 2015). Transfer of mobile elements between cells contributes to rapid evolution and spread of associated cargo genes and phenotypes (Frost et al., 2005; Treangen and Rocha, 2011). Despite the benefits cargo genes can provide, the maintenance and transfer of mobile genetic elements requires host cellular resources and in some cases is lethal (Baltrus, 2013). Maintenance of a mobile genetic element in host cells requires balancing the costs and benefits to the host or a suffi- ciently high transfer frequency. Many mobile elements, especially ICEs, have been identified bioin- formatically (Bi et al., 2012; Guglielmini et al., 2011). Many of these ICEs contain putative cargo genes. However, the phenotypes conferred by these genes cannot be inferred from sequence nor are they easily detected experimentally (Cury et al., 2017). ICEs often carry ‘cargo’ genes that are not necessary for transfer but confer a phenotype to host cells. In fact, ICEs (conjugative transposons) were first identified because of the phenotypes con- ferred by cargo genes (Franke and Clewell, 1981). Reviewing editor: Petra Anne Levin, Washington University in St. Louis, United States subtilis forms biofilms, the presence of the devI gene in ICEBs1 helps the cells to delay the production of the costly mucus that keeps bacteria together, allowing the organisms to ‘cheat’ for a little while and benefit from the tight-knit community without contributing to it. As nutrients become scarce in biofilms, the gene also allows the bacteria to grow for longer before they start to form spores – the dormant bacterial form that can weather difficult conditions. Mobile elements can carry genes that make bacteria resistant to antibiotics, harmful to humans, or able to use new food sources; they could even be used to artificially introduce genes of interest in these cells. The work by Jones et al. helps to understand the way these elements influence the fate of their host, providing insight into how they could be harnessed for the benefit of human health. (Auchtung et al., 2005). Most of the ICEBs1 genes needed for conjugation are grouped together in an operon that is repressed until activating signals are sensed (Figure 1). Two pathways activate ICEBs1, both of which lead to cleavage of the repressor ImmR by the protease and anti-repressor ImmA (Auchtung et al., 2007; Bose et al., 2008; Bose and Grossman, 2011). ICEBs1 contains the cell-cell signaling genes, rapI and phrI, which regulate ICEBs1 activation by sensing population den- sity and the relative abundance of ICEBs1-containing host cells (Auchtung et al., 2005). RapI is pro- duced at high cell density and during the transition to stationary phase and stimulates the proteolytic cleavage of the repressor ImmR by the protease ImmA (Bose and Grossman, 2011). Overproduction of RapI stimulates activation of ICEBs1 in >90% of cells (Auchtung et al., 2005). RapI activity (and therefore ICEBs1 activation) is inhibited by PhrI, a peptide that is secreted by cells that contain ICEBs1. PhrI levels indicate the relative abundance of ICEBs1-containing cells in the population, preventing the activation and possible redundant transfer of ICEBs1 if most nearby cells already contain the element. ICEBs1 is also activated during the RecA-dependent DNA damage response (Auchtung et al., 2005). Biofilms appear to be hotspots of horizontal gene transfer for bacteria growing in natural settings (Madsen et al., 2012; Molin and Tolker-Nielsen, 2003). Undomesticated strains of B. subtilis form complex biofilms on agar plates and at the air-liquid interface in standing cultures (Vlamakis et al., 2013). There is also extensive spore formation in B. Reviewing editor: Petra Anne Levin, Washington University in St. Louis, United States Despite the benefits cargo genes can provide, the maintenance and transfer of mobile genetic elements requires host cellular resources and in some cases is lethal (Baltrus, 2013). Maintenance of a mobile genetic element in host cells requires balancing the costs and benefits to the host or a suffi- ciently high transfer frequency. Many mobile elements, especially ICEs, have been identified bioin- formatically (Bi et al., 2012; Guglielmini et al., 2011). Many of these ICEs contain putative cargo genes. However, the phenotypes conferred by these genes cannot be inferred from sequence nor are they easily detected experimentally (Cury et al., 2017). Copyright Jones et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. ICEBs1, a relatively small (~20 kb) ICE found in most strains of Bacillus subtilis, was identified bio- informatically (Burrus et al., 2002) and experimentally based on its regulation by cell-cell signaling Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 1 of 24 Research article Research article Microbiology and Infectious Disease eLife digest Many bacteria can ‘have sex’ – that is, they can share their genetic information and trade off segments of DNA. While these mobile genetic elements can be parasites that use the resources of their host to make more of themselves, some carry useful genes which, for example, help bacteria to fight off antibiotics. eLife digest Many bacteria can ‘have sex’ – that is, they can share their genetic information and trade off segments of DNA. While these mobile genetic elements can be parasites that use the resources of their host to make more of themselves, some carry useful genes which, for example, help bacteria to fight off antibiotics. Integrative and conjugative elements (or ICEs) are a type of mobile segments that normally stay inside the genetic information of their bacterial host but can sometimes replicate and be pumped out to another cell. ICEBs1 for instance, is an element found in the common soil bacterium Bacillus subtilis. Scientists know that ICEBs1 can rapidly spread in biofilms – the slimly, crowded communities where bacteria live tightly connected – but it is still unclear whether it helps or hinders its hosts. Using genetic manipulations and tracking the survival of different groups of cells, Jones et al. show that carrying ICEBs1 confers an advantage under many conditions. When B. Reviewing editor: Petra Anne Levin, Washington University in St. Louis, United States subtilis biofilms (Branda et al., 2001; Vlamakis et al., 2008). In addition, during growth in a biofilm, ICEBs1 is naturally activated and transfers efficiently, generating on the order of 10 new ICEBs1-containing host cells (transconju- gants) per donor cell under appropriate conditions (Le´cuyer et al., 2018). B. subtilis biofilms are held together by a matrix composed of secreted exopolysaccharides, protein fibers, and DNA (Vlamakis et al., 2013). This matrix reinforces cell-cell contacts, likely promoting rapid spread of ICEBs1 by conjugation. Additionally, the conditions that promote biofilm formation (high cell den- sity) also promote activation and transfer of ICEBs1 and sporulation (Auchtung et al., 2005; Grossman and Losick, 1988). Although biofilm growth is clearly beneficial to conjugation, it is unknown how ICEBs1 impacts its host cells under these conditions. In this study, we describe a selective advantage provided by ICEBs1 to its host cells during growth in biofilms. This fitness benefit was due to inhibition of host biofilm and spore development. We identified the ICEBs1 gene devI (formerly ydcO) as necessary and sufficient to inhibit host devel- opment and provide a selective advantage to ICEBs1-containing cells. We also provide evidence Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 2 of 24 PphrI ImmR ImmA RecA DNA damage RapI PhrI High total cell density High frequency ICEBs1-containing cells immR immA rapI phrI attR attL PimmR Pxis PrapI devI 1 kb Conjugative Transfer DevI Spo0A Biofilm matrix, Sporulation ? Fi 1 G i d l h f ICEB 1 G d b h i l bl k i di i h di i f i i Research article Microbiology and Infectious Disease Research article Microbiology and Infectious Disease PphrI ImmR ImmA RecA* DNA damage RapI PhrI High total cell density High frequency ICEBs1-containing cells immR immA rapI phrI attR attL PimmR Pxis PrapI devI 1 kb Conjugative Transfer DevI Spo0A Biofilm matrix, Sporulation ? 1 kb PphrI ImmR ImmA RecA* DNA damage RapI PhrI High total cell density High frequency ICEBs1-containing cells immR immA rapI phrI attR attL PimmR Pxis PrapI devI DevI Spo0A Biofilm matrix, Sporulation ? DNA damage High total cell density High total cell density PphrI PrapI immA Figure 1. Genetic map and regulatory pathways of ICEBs1. Genes are represented by horizontal block arrows indicating the direction of transcription. Vertical right-angle arrows mark the positions of promoters, and the arrowhead indicates the direction of transcription. Reviewing editor: Petra Anne Levin, Washington University in St. Louis, United States Genes known to be involved in the conjugative life cycle of ICEBs1 are shaded in gray. The 60 bp direct repeats that mark the ends of ICEBs1 are shown as black rectangles. (Inset) A partial genetic map that highlights factors involved in the regulation of ICEBs1. The major promoter Pxis drives expression of most genes in ICEBs1. Pxis is repressed by the ICE-encoded repressor ImmR. Repression is relieved when ImmR is cleaved by the protease ImmA, and proteolytic cleavage is stimulated by activated RecA (RecA) in response to DNA damage, or, independently by the cell signaling regulator RapI. RapI is made when cells are crowded by potential recipients, but repressed by the ICE-encoded secreted peptide PhrI if the neighboring cells already contain a copy of ICEBs1. devI (formerly ydcO) is the third open-reading frame downstream of Pxis. DevI inhibits sporulation and expression of biofilm matrix genes, likely by inhibiting Spo0A (directly or indirectly). In the genetic pathways, black arrows indicate activation and red T-bars indicate inhibition. indicating that devI likely inhibits the key developmental transcription factor Spo0A, reducing its ability to stimulate biofilm and sporulation gene expression. devI (ydcO) is conserved in other ICEBs1-like elements, indicating that manipulation of host development may be a conserved strat- egy among this family of mobile genetic elements. We postulate that manipulation of host pathways may be a common function of many of the as yet uncharacterized cargo genes in ICEs. Results ICEBs1 spreads efficiently in biofilms by conjugation Biofilm formation is characteristic of many bacteria growing in natural settings, including B. subtilis. We used biofilm growth to determine if ICEBs1 affected the fitness of its host cells under conditions 3 of 24 3 of 24 Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 Research article Research article Research article Microbiology and Infectious Disease that naturally promote its spread. We performed competition experiments in biofilms using strains of undomesticated B. subtilis (NCIB3610 plasmid-free) with or without ICEBs1. We observed highly efficient spread of ICEBs1 at low donor to recipient ratios (Table 1) during growth in biofilms, similar to results reported previously (Le´cuyer et al., 2018). To measure mating, we mixed ICEBs1-containing cells (potential donors) with cells that did not contain ICEBs1 (ICEBs10, potential recipients) and co-cultured the mix on standard biofilm-stimulating growth medium (MSgg agar) (Branda et al., 2001). Since ICEBs1 induction is regulated by cell-cell signaling, we varied the initial frequency of ICEBs1+ cells between approximately 0.01 and 0.9. We inserted unique select- able markers (antibiotic resistances) in the chromosomes of the donors and recipients as well as within ICEBs1. After 4 days of growth at 30˚C (approximately 17 net doublings of the initial popula- tion), biofilms were disrupted and the number of transconjugants was determined by selective plating. We found that after four days of biofilm growth, the frequency of transconjugants ranged from ~0.4 to 0.6 of total cells in the biofilm for starting donor frequencies of ~0.01–0.5 (Table 1). The highest frequency of transconjugants was observed when the starting frequency of ICEBs1-con- taining cells was ~0.1. Enhanced conjugation at low donor to recipient ratios is likely due to regula- tion of ICEBs1 by cell-cell signaling (induction is inhibited by the presence of other potential donors) and the higher likelihood of contacting potential recipients at low frequencies of donors. The high levels of ICEBs1 conjugation during growth in biofilms presented a challenge for quanti- fying the fitness of ICEBs1-containing host cells relative to ICEBs10 cells. Mating converts a large fraction of ICEBs10 cells to transconjugants (ICEBs1-containing), reducing the ICEBs10 proportion of the population in a manner unrelated to host fitness. To measure the effect of ICEBs1 on host fit- ness, we blocked conjugative DNA transfer using the conEK476E mutation (Berkmen et al., 2010). We then compared the proportion of ICEBs1-containing hosts to ICEBs10 cells without the con- founding influence of conjugation. Results ICEBs1 provides a frequency-dependent selective advantage in biofilms We found that cells containing ICEBs1 that is incapable of conjugation {ICEBs1(conEK476E)} had a fitness advantage over cells lacking ICEBs1 during biofilm growth when they were initially present as a minority of the population. As before, we varied the initial frequency of ICEBs1-containing host cells in the inoculum between approximately 0.01 and 0.9. To measure fitness we determined the frequency of ICEBs1-containing cells (fICE) and ICEBs10 (fNULL) cells in the initial inoculum and in mature biofilms (4 days of growth at 30˚C) by selective plating. The relative fitness of the ICEBs1- containing cells was calculated as the fold change in the ratio of fICE / fNULL over the course of the competition. p We found that the fitness of ICEBs1-containing cells was dependent on their initial frequency in the population (Figure 2A). The frequency-dependence was most likely due to regulation of ICEBs1 gene expression by the cell-cell signaling genes rapI-phrI or some other function of rapI. Cells with ICEBs1 had a selective advantage at low frequencies (0.01 or 0.1) when the element is most strongly activated. At high frequencies in the population (0.5 or 0.9), when there is little or no activation, Table 1. Frequency of transconjugants generated in biofilm matings. Initial frequency ICEBs1 donors Final frequency transconjugants† 0.008 ± 0.002 0.42 ± 0.13 0.10 ± 0.03 0.64 ± 0.15 0.47 ± 0.05 0.44 ± 0.07 0.89 ± 0.03 0.063 ± 0.008 ICEBs1-containing cells (JMJ592) were mixed with ICEBs1-cured cells (JMJ550). The initial frequencies reported are the average ± standard deviation from three independent experiments. †The final frequencies of transconjugants reported are the average ± standard deviation from a total of nine biofilms from three independent experiments. The online version of this article includes the following source data for Table 1: Source data 1. Mating in biofilms.Counts of donors, recipients, and transconjugants for ICEBs1 mating in biofilms. DOI: https://doi.org/10.7554/eLife.65924 4 of 24 Table 1. Frequency of transconjugants generated in biofilm matings. Initial frequency ICEBs1 donors Final frequency transconjugants† 0.008 ± 0.002 0.42 ± 0.13 0.10 ± 0.03 0.64 ± 0.15 0.47 ± 0.05 0.44 ± 0.07 0.89 ± 0.03 0.063 ± 0.008 ICEBs1-containing cells (JMJ592) were mixed with ICEBs1-cured cells (JMJ550). The initial frequencies reported the average ± standard deviation from three independent experiments. The online version of this article includes the following source data for Table 1: Source data 1. Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 Research article Results Mating in biofilms.Counts of donors, recipients, and transconjugants for ICEBs1 mating in biofilm Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 Research article Microbiology and Infectious Disease Microbiology and Infectious Disease 0.01 0.1 0.5 0.9 0.1 1 10 Initial frequency ICEBs1 Relative fitness ns 0.01 0.1 0.5 0.9 0.1 1 10 Initial frequency ICEBs1 Relative fitness A. B. 0.01 0.1 0.5 0.9 108 109 1010 Initial frequency ICEBs1 Total CFU yield ns ns ns 0.01 0.1 0.5 0.9 108 109 1010 Initial frequency ICEBs1 Total CFU yield ns ns ns C. D. Wild-type spo0APs 0.1 1 10 Strain background Relative fitness Wild-type spo0APs 0.1 1 10 Strain background Relative fitness E. F. Figure 2. Fitness of ICEBs1-containing cells relative to ICEBs1-cured cells during development. The fitness of ICEBs1-containing cells (JMJ593) relative to ICEBs1-cured cells (JMJ550) was measured by competition during biofilm growth (A) or growth on sporulation medium (B). Total growth yields in biofilms (C) and on sporulation medium (D) were determined by counting CFUs derived from both cells and spores. The fitness of ICEBs1-containing cells relative to ICEBs1-cured cells was compared between the wild-type strain background (JMJ593 vs. JMJ550) and in the sporulation-deficient spo0ADPs background (JMJ788 vs. JMJ786) by competition during biofilm growth (E) or growth on sporulation medium (F). ICEBs1-containing cells were inoculated at an initial frequency of 0.01. Data shown are pooled from three independent experiments. A total of nine populations (biological replicates) were analyzed per condition. Boxes extend from the lower to upper quartiles of the data, and the middle line indicates the median fitness. Figure 2 continued on next page Research article Microbiology and Infectious Diseas 0.01 0.1 0.5 0.9 0.1 1 10 Initial frequency ICEBs1 Relative fitness B. 0.01 0.1 0.5 0.9 0.1 1 10 Initial frequency ICEBs1 Relative fitness ns A. B. A. Initial frequency ICEBs1 0.01 0.1 0.5 0.9 108 109 1010 Initial frequency ICEBs1 Total CFU yield ns ns ns C. 1 D. 0.01 0.1 0.5 0.9 108 109 1010 Total CFU yield ns ns ns D. 0.01 0.1 0.5 0.9 Initial frequency ICEBs1 Wild-type spo0APs 0.1 1 10 Strain background Relative fitness F. Initial frequency ICEBs1 Wild-type spo0APs 0.1 1 10 Strain background Relative fitness E. E. F. Figure 2. Fitness of ICEBs1-containing cells relative to ICEBs1-cured cells during development. Research article Research article Microbiology and Infectious Disease Microbiology and Infectious Disease Microbiology and Infectious Disease Figure 2 continued Whiskers indicate the range of the fitness measurements. Asterisks indicate a p-value<0.05 (two-tailed T-test, unequal variance). Exact p-values: (A) 3.7 105, 8.8 103, 6.0 101; (B) 4.9 1011, 1.6 104, 7.1 103; (C) 3.7 101, 4.2 101, 2.2 102; (D) 8.9 101, 1.9 101, 1.7 101; (E) 5.5 106; (F) 2.4 105. Figure 2 continued Whiskers indicate the range of the fitness measurements. Asterisks indicate a p-value<0.05 (two-tailed T-test, unequal variance). Exact p-values: (A) 3.7 105, 8.8 103, 6.0 101; (B) 4.9 1011, 1.6 104, 7.1 103; (C) 3.7 101, 4.2 101, 2.2 102; (D) 8.9 101, 1.9 101, 1.7 101; (E) 5.5 106; (F) 2.4 105. g Whiskers indicate the range of the fitness measurements. Asterisks indicate a p-value<0.05 (two-tailed T-test, unequal variance). Exact p-values: (A) 3.7 105, 8.8 103, 6.0 101; (B) 4.9 1011, 1.6 104, 7.1 103; (C) 3.7 101, 4.2 101, 2.2 102; (D) 8.9 101, 1.9 101, 1.7 101; (E) 5.5 106; (F) 2.4 105. Whiskers indicate the range of the fitness measurements. Asterisks indicate a p-value<0.05 (two-tailed T-test, unequal variance). Exact p-values: (A) 3.7 105, 8.8 103, 6.0 101; (B) 4.9 1011, 1.6 104, 7.1 103; (C) 3.7 101, 4.2 101, 2.2 102; (D) 8.9 101, 1.9 101, 1.7 101; (E) 5 5 106; (F) 2 4 105 The online version of this article includes the following source data for figure 2: Source data 1. Frequency-dependent fitness. Source data 2. Fitness dependence on sporulation. fitness of ICEBs1-containing cells was approximately neutral (Figure 2A). The final growth yields of the populations were similar regardless of the frequency of ICEBs1-containing cells (Figure 2C). We performed control competitions of two differentially marked ICEBs10 strains to verify that the enhanced fitness we observed was due to the presence of ICEBs1 rather than an inherent fitness dif- ference associated with antibiotic resistances (see Materials and methods, Source data 1). ICEBs1 confers a selective advantage in biofilms without sporulation We blocked sporulation using a mutation that causes a reduction in the amount of the transcription factor Spo0A that is required for spore formation. The spo0ADPs mutation is a deletion of the sigma-H-dependent promoter upstream of spo0A (Siranosian and Grossman, 1994). This mutation reduces production of Spo0A, and cells do not achieve the threshold concentration required to initi- ate sporulation (Chung et al., 1994). spo0ADPs mutant cells formed biofilms that were morphologi- cally similar to those formed by wild-type cells. In biofilms without sporulation, spo0ADPs mutant cells containing ICEBs1 (JMJ788) had a selec- tive advantage compared to spo0ADPs mutant cells without ICEBs1 (JMJ786) (Figure 2E). Notably, the median fitness for spo0ADPs mutant cells containing ICEBs1 at a low frequency in the population was approximately six. Thus, sporulation was not required for a fitness benefit to ICEBs1-containing cells in biofilms. Fitness of cells in biofilms can be affected by production of the biofilm matrix. For example, cells that ‘cheat’ by contributing less to biofilm matrix production reap the benefits of growing with other cells that bear the cost of matrix gene expression (Dragosˇ et al., 2018). We showed that cells con- taining ICEBs1 ‘cheat’ by decreasing expression of biofilm matrix genes compared to cells without ICEBs1 (see below). Research article There was a small cost associated with the marker used to select cells containing ICEBs1 (median relative fitness 0.7 ± 0.09), leading to a slight underestimate of the selective advantage to these cells. There is a large amount of sporulation in B. subtilis biofilms (Branda et al., 2001; Vlamakis et al., 2008). Consistent with this, we found that approximately 80% of viable colony-forming units (CFUs) in a mature biofilm after 3 days were from spores. The selective advantage to cells containing ICEBs1 growing in biofilms could be due to sporulation and/or biofilm development. Results The fitness of ICEBs1-containing cells (JMJ593) relative to ICEBs1-cured cells (JMJ550) was measured by competition during biofilm growth (A) or growth on sporulation medium (B). Total growth yields in biofilms (C) and on sporulation medium (D) were determined by counting CFUs derived from both cells and spores. The fitness of ICEBs1-containing cells relative to ICEBs1-cured cells was compared between the wild-type strain background (JMJ593 vs. JMJ550) and in the sporulation-deficient spo0ADPs background (JMJ788 vs. JMJ786) by competition during biofilm growth (E) or growth on sporulation medium (F). ICEBs1-containing cells were inoculated at an initial frequency of 0.01. Data shown are pooled from three independent experiments. A total of nine populations (biological replicates) were analyzed per condition. Boxes extend from the lower to upper quartiles of the data, and the middle line indicates the median fitness. Figure 2 continued on next page 5 of 24 Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 ICEBs1 confers a selective advantage during sporulation, in the absence of biofilms We found that cells containing ICEBs1 also had a frequency-dependent selective advantage during sporulation, in the absence of biofilms. We prepared mixtures of cells with and without ICEBs1 as described above. These mixtures were spotted onto a medium (DSM agar) that promotes high levels of sporulation. During growth on this medium, there are no complex colony features found in bio- films. As in the biofilm competitions, cells containing ICEBs1 had a frequency-dependent selective advantage during sporulation (Figure 2B). At an initial frequency of approximately 0.01, the median relative fitness of the ICEBs1-containing cells was approximately 14 (14.5 ± 4.3). As in biofilms, the total growth yields of the populations were similar regardless of ICEBs1 host frequency (Figure 2D). These results demonstrate that ICEBs1 confers a selective advantage to cells growing on DSM agar, outside the context of biofilms. This could be due to sporulation or growth under these specific conditions. We found that sporulation was required for the strong selective advantage during growth on sporulation medium (DSM agar). The fitness benefit associated with the ICEBs1-containing cells at a low frequency in the population was greatly reduced in the spo0ADPs mutant (no sporulation) Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 6 of 24 Research article Research article Research article Microbiology and Infectious Disease (Figure 2F). The sporulation mutant with ICEBs1 had a median fitness of approximately 1.5 com- pared to approximately nine for wild-type. Based on these results, we conclude that the presence of ICEBs1 confers a frequency-dependent selective advantage during sporulation. q y p g g p Together, our results demonstrate that cells containing ICEBs1 have a frequency-dependent selective advantage in biofilms and during sporulation. This selective advantage is independent of the ability of ICEBs1 to actually transfer from one cell to another. Biofilm formation (Hamon and Laz- azzera, 2001) and sporulation (Hoch, 1993; Sonenshein, 2000) are both regulated by the transcrip- tion factor Spo0A. Our results indicate that the presence of ICEBs1 could somehow be inhibiting the activity or activation of Spo0A. porulation is delayed in ICEBs1 host cells during biofilm formation Sporulation is delayed in ICEBs1 host cells during biofilm formation We found that sporulation of ICEBs1-containing cells was delayed in a frequency-dependent manner during growth in biofilms (Figure 3A and B). When ICEBs1-containing cells were started at a fre- quency of approximately 0.01, they reached their maximum sporulation frequency (>80% spores) roughly 17 hr later than the cells without ICEBs1 (Figure 3A). After 3 days of biofilm growth, the sporulation frequencies of ICEBs1-containing and ICEBs1-cured cells were indistinguishable. Over this period of time the total frequency of ICEBs1-containing cells in the population typically rose from ~0.01 to ~0.03, giving a relative fitness (~3) consistent with results above (Figure 2A). When the ICEBs1-containing cells were the majority in the population (initial frequency ~0.9) the timing and sporulation frequencies of the ICEBs1-containing and ICEBs1-cured cells were indistinguishable (Figure 3B). Sporulation is delayed in ICEBs1 host cells during sporulation in the absence of biofilms ICEBs1-containing cells have a frequency-dependent delay in sporulation p We hypothesized that some ICEBs1-encoded gene(s) inhibit host cell development. This inhibition could delay development and enable cells to continue growth for a small number of generations. This model derives from analogous phenotypes of mutants that do not enter the sporulation path- way (Dawes and Mandelstam, 1970). Mutants that delay the start of sporulation have a growth advantage as they are able to divide one or a few more times while other cells in the population stop growing and start to sporulate. We found that in mixed populations, sporulation was delayed in cells containing ICEBs1 com- pared to cells without ICEBs1, in a frequency-dependent manner. As above, we used an ICEBs1 mutant that is incapable of conjugation {ICEBs1(conEK476E)}. We started several replicate popula- tions, each of which we sampled once at different times to create a time-course. (This was done because sampling to quantify CFUs [spores and cells] disrupts and prevents monitoring a single pop- ulation over time.) Spore frequency was determined by measuring heat-resistant CFUs as a fraction of total CFUs for ICEBs1-containing and ICEBs1-cured strains that contained different antibiotic resistance markers to distinguish the strains. Sporulation is delayed in ICEBs1 host cells during biofilm formation Sporulation is delayed in ICEBs1 host cells during sporulation in the absence of biofilms We also found that sporulation of ICEBs1-containing cells was delayed in a frequency-dependent manner during sporulation in the absence of biofilm formation (Figure 3C and D). When the ICEBs1 containing cells were inoculated at a low frequency (approximately 0.01), the delay in sporulation was qualitatively similar to that observed in biofilms (Figure 3C). However, the increase in the total frequency of ICEBs1-containing cells in the population was approximately 10-fold, giving a relative fitness of approximately 10, consistent with results described above (Figure 2B). This increase was much greater than the approximately threefold increase during biofilm formation. We suspect that the stronger selective advantage of ICEBs1-containing cells on sporulation medium is due to the earlier onset of sporulation. By 16 hr of growth on sporulation medium, spores made up about 40% of the total CFUs. By the same time in biofilms, spores were undetectable (limit of detection ~0.03% spores). Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 7 of 24 Research article Microbiology and Infectious Disease 0 20 40 60 80 100 % Spores 0 20 40 60 0.01 0.1 Hours ICEBs1 freq. 0 20 40 60 0.01 0.1 Hours ICEBs1 freq. 0 20 40 60 80 100 % Spores 0 20 40 60 80 100 % Spores 0 20 40 60 80 100 % Spores 0 20 40 60 0.5 1 Hours ICEBs1 freq. 0 20 40 60 0.5 1 Hours ICEBs1 freq. A. B. C. D. Figure 3. ICEBs1-containing cells delay sporulation in a frequency-dependent manner. ICEBs1-containing cells (JMJ593, black circles) were mixed with CEBs1-cured cells (JMJ550, open squares) at an initial frequency of 0.01 and spotted onto biofilm growth medium (A) or sporulation medium (C). Mixtures with ICEBs1-containing cells at an initial frequency of 0.9 were also spotted onto biofilm medium (B) and sporulation medium (D). Biofilms and colonies were harvested at the indicated times to determine the fraction of CFUs derived from heat-resistant spores for both the ICEBs1-containing and the ICEBs1-cured cells. Boxes below each graph indicate the frequency of ICEBs1-containing CFUs at each timepoint. Data shown are the average from two populations (biological replicates) per timepoint with error bars indicating the standard deviation. A representative experiment is shown. The online version of this article includes the following source data for figure 3: Source data 1. Sporulation timing during competitions. 0 20 40 60 80 100 % Spores 0 20 40 60 0.5 1 ICEBs1 freq. B. rapI-phrI are necessary but not sufficient for enhanced fitness rapI-phrI are necessary but not sufficient for enhanced fitness The fitness benefits provided by ICEBs1 were dependent on the relative abundance of ICEBs1-con- taining cells, indicating that the cell-cell signaling genes rapI-phrI in ICEBs1 were likely involved, either directly or indirectly. Other Rap proteins in B. subtilis are known to regulate development by inhibiting phosphorylation (activation) of the transcription factor Spo0A (Sonenshein, 2000). RapI, like other Rap proteins in B. subtilis, can inhibit the pathway needed to phosphorylate (activate) the transcription factor Spo0A, and overexpression of rapI in vivo inhibits sporulation (Even-Tov et al., 2016; Parashar et al., 2013; Singh et al., 2013). However, it was unknown whether RapI regulates development in vivo under physiological conditions. Results described below demonstrate that the rapI-phrI system is required for the fitness advantage of ICEBs1-containing cells, but that this requirement is by virtue of causing induction of ICEBs1 gene expression. Another gene in ICEBs1 is both necessary and sufficient for the selective advantage of ICEBs1-containing cells during development. We deleted rapI-phrI (DrapI-phrI) in ICEBs1 and compared the fitness conferred by this mutant to that conferred by ICEBs1 with rapI-phrI. Because loss of rapI prevents induction of gene expression, excision, and replication of ICEBs1, we used ICEBs1 mutants (‘locked-in’) that are incapable of exci- sion or replication (see Materials and methods), regardless of the presence or absence of rapI. Pre- venting excision and replication of ICEBs1 allowed us to compare the fitness of wild-type ICEBs1 to ICEBs1 DrapI-phrI (and other mutants), which would otherwise have a lower gene copy number due to a lower frequency of induction. We verified that locked-in ICEBs1 still conferred a fitness benefit to host cells. During sporulation in biofilms (MSgg agar), cells containing locked-in ICEBs1 had a relative fitness of approximately 14 when they were started at a low frequency in the population (~0.01) (Figure 4A). This benefit was much greater than that conferred by wild-type ICEBs1 that can excise and replicate. We suspect that replication of ICEBs1 incurs a fitness cost to the host cell that reduces the apparent benefit. The sources of this burden could include use of host resources, the host’s response to single-stranded DNA produced by rolling-circle-replication of ICEBs1, and increases in ICEBs1 gene expression due to increased copy number. We found that rapI-phrI were required for the fitness benefit conferred by ICEBs1. Sporulation is delayed in ICEBs1 host cells during sporulation in the absence of biofilms 0 20 40 60 80 100 % Spores 0 20 40 60 0.01 0.1 ICEBs1 freq. A. B. A. 0 Hours 0 20 40 60 0.01 0.1 Hours ICEBs1 freq. 0 20 40 60 80 100 % Spores C. 0 20 40 60 80 100 % Spores Hours 0 20 40 60 0.5 1 Hours ICEBs1 freq. D. D. C. Figure 3. ICEBs1-containing cells delay sporulation in a frequency-dependent manner. ICEBs1-containing cells (JMJ593, black circles) were mixed with ICEBs1-cured cells (JMJ550, open squares) at an initial frequency of 0.01 and spotted onto biofilm growth medium (A) or sporulation medium (C). Mixtures with ICEBs1-containing cells at an initial frequency of 0.9 were also spotted onto biofilm medium (B) and sporulation medium (D). Biofilms and colonies were harvested at the indicated times to determine the fraction of CFUs derived from heat-resistant spores for both the ICEBs1-containing and the ICEBs1-cured cells. Boxes below each graph indicate the frequency of ICEBs1-containing CFUs at each timepoint. Data shown are the average from two populations (biological replicates) per timepoint with error bars indicating the standard deviation. A representative experiment is shown. The online version of this article includes the following source data for figure 3: Source data 1 Sporulation timing during competitions sporulation in a frequency-dependent manner. ICEBs1-containing cells (JMJ593, black circles) were mixed with es) at an initial frequency of 0.01 and spotted onto biofilm growth medium (A) or sporulation medium (C). Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 8 of 24 Research article Research article Microbiology and Infectious Disease rapI-phrI are necessary but not sufficient for enhanced fitness During sporu- lation in biofilms (MSgg agar), the relative fitness of the D(rapI-phrI) host strain was approximately neutral (Figure 4A), in contrast to the high fitness (median ~14) provided by ICEBs1 containing rapI- phrI when the ICEBs1-containing cells were started at a low frequency (~0.01). The requirement for rapI-phrI could be due to a direct role for one of these, likely RapI, or an indirect role in activating expression of ICE genes. rapI-phrI are not sufficient in the absence of other ICEBs1 genes to provide a fitness benefit rapI-phrI are not sufficient in the absence of other ICEBs1 genes to provide a fitness benefit p p a fitness benefit We found that rapI-phrI alone were not sufficient to provide a fitness benefit during sporulation or during sporulation in biofilms. We cloned rapI-phrI and their native promoters and inserted them in an ectopic locus (bcaP) in a strain that did not contain ICEBs1. When this strain was started at a low frequency (~0.01), fitness of this strain was neutral relative to a control strain without rapI-phrI (Figure 4B). To verify that rapI-phrI were functional, we added back ICEBs1 that was missing rapI- phrI. Adding the rest of ICEBs1 restored the fitness advantage during sporulation and in biofilms, indicating that the ectopic copy of rapI-phrI was functional (Figure 4B). The requirement for rapI- phrI and some other ICEBs1 gene(s) indicated that the selective advantage was likely dependent on induction of ICEBs1 by RapI. Activation of ICEBs1 is required for the fitness benefit We found that expression of one or more ICEBs1 genes controlled by the promoter Pxis was required for the selective advantage in biofilms with sporulation. Pxis drives most of the genes in ICEBs1 and is indirectly activated by RapI in a frequency-dependent manner (Auchtung et al., 2005; Bose and Grossman, 2011). We deleted Pxis in a strain in which ICEBs1 was unable to excise or rep- licate (locked-in-ICEBs1). In this strain, only genes not dependent on Pxis could be expressed, Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 9 of 24 Research article Microbiology and Infectious Disease locked-in ICEBs1 rapI-phrI 0.1 1 10 Relative fitness bcaP::rapI-phrI ICEBs10 bcaP::rapI-phrI ICEBs1rapI-phrI 0.1 1 10 Relative fitness bcaP::rapI-phrI ICEBs10 bcaP::rapI-phrI ICEBs1rapI-phrI Biofilm Biofilm Sporulation A. B. Figure 4. The ICEBs1 cell-cell signaling genes, rapI-phrI, are necessary but not sufficient to confer a selective advantage. (A) The fitness of cells containing locked-in ICEBs1 (JMJ646) or an isogenic rapI-phrI mutant (JMJ686) was measured relative to ICEBs1-cured cells (JMJ550) during biofilm competitions. The ICEBs1-containing cells were started at a frequency of 0.01. (B) The fitness of cells containing rapI-phrI alone (JMJ576) or cells containing both rapI-phrI and locked-in ICEBs1DrapI-phrI (JMJ785) was measured relative to ICEBs1-cured cells (JMJ714) during biofilm and sporulation medium competitions. JMJ576 and JMJ785 were started at a frequency of 0.01. Data shown are pooled from three independent experiments with a total of nine populations (biological replicates) analyzed per strain mixture. Boxes extend from the lower to upper quartiles, and the middle line indicates the median fitness. p p a fitness benefit Whiskers indicate the range of the fitness measurements. Asterisks indicate a p-value<0.05 (two-tailed T-test, unequal variance). Exact p-values: (A) 1.3 1012; (B) 2.1 108, 4.6 102. The online version of this article includes the following source data for figure 4: S d 1 Fi d d I h I bcaP::rapI-phrI ICEBs10 bcaP::rapI-phrI ICEBs1rapI-phrI 0.1 1 10 Relative fitness bcaP::rapI-phrI ICEBs10 bcaP::rapI-phrI ICEBs1rapI-phrI Biofilm Sporulation B. locked-in ICEBs1 rapI-phrI 0.1 1 10 Relative fitness Biofilm A. 0 1 1 10 Relative fitness Biofilm A. B. A. Relative fitness Relative fitness Figure 4. The ICEBs1 cell-cell signaling genes, rapI-phrI, are necessary but not sufficient to confer a selective advantage. (A) The fitness of cells containing locked-in ICEBs1 (JMJ646) or an isogenic rapI-phrI mutant (JMJ686) was measured relative to ICEBs1-cured cells (JMJ550) during biofilm competitions. The ICEBs1-containing cells were started at a frequency of 0.01. (B) The fitness of cells containing rapI-phrI alone (JMJ576) or cells containing both rapI-phrI and locked-in ICEBs1DrapI-phrI (JMJ785) was measured relative to ICEBs1-cured cells (JMJ714) during biofilm and sporulation medium competitions. JMJ576 and JMJ785 were started at a frequency of 0.01. Data shown are pooled from three independent experiments with a total of nine populations (biological replicates) analyzed per strain mixture. Boxes extend from the lower to upper quartiles, and the middle line indicates the median fitness. Whiskers indicate the range of the fitness measurements. Asterisks indicate a p-value<0.05 (two-tailed T-test, unequal variance). Exact p-values: (A) 1.3 1012; (B) 2.1 108, 4.6 102. The online version of this article includes the following source data for figure 4: Source data 1. Fitness dependence on rapI-phrI. including rapI-phrI. Fitness of this strain was approximately neutral during sporulation in biofilms (Figure 5B). This indicated that expression of one or more genes controlled by Pxis, either alone or in combination with rapI, was required for the fitness benefit conferred by ICEBs1. Since most of the genes controlled by Pxis have known roles in the conjugative life cycle, we focused our search on genes without a known function, starting with the genes near Pxis. Of these, we found that a deletion of devI (ydcO) reduced fitness. Results described below demonstrate that a single ICEBs1 gene, devI (ydcO), is both necessary and sufficient to inhibit development and provide a selective advantage. p p a fitness benefit The primary role of rapI in the fitness benefit appears to be the induction of devI expression. devI is necessary for the fitness benefit conferred by ICEBs1 Effects of deletions in ICEBs1 on host fitness. (A) Abbreviated genetic map of locked-in-ICEBs1 showing genes as open block arrows, promoters as thin right-angle arrows, and the left attachment site (attL) as a black bar. (B to E) Brackets under the map of ICEBs1 indicate regions contained in isogenic derivatives of locked-in-ICEBs1. Open spaces represent regions deleted. The fitness of strains containing locked-in-ICEBs1 (JMJ646) and its derivatives (DPxis, JMJ662; DydzL, JMJ704; DdevI, JMJ703; DsncO, JMJ688) is indicated at the right. Fitness was measured relative to ICEBs1-cured cells (JMJ550) during competitions in biofilms. ICEBs1-containing cells were started at a frequency of 0.01. Data shown are the median ± standard deviation from at least three independent experiments (at least nine total biological replicates per strain mixture), with the exception of JMJ688 which was measured only in one experiment (three total biological replicates). Asterisks indicate a statistically significant difference in fitness compared to JMJ646 (p-value<0.05, two-tailed T-test, unequal variance). Exact p-values: (B) 2.3 1016; (C) 4.5 101; (D) 6.3 1012; (E) 6.2 102. The online version of this article includes the following source data for figure 5: Source data 1. Effects of gene deletions on ICEBs1 fitness. Source data 1. Effects of gene deletions on ICEBs1 fitness. devI is sufficient to inhibit sporulation and provide a fitness benefit We found that when expressed constitutively, devI alone, in the absence of all other ICEBs1 genes, was sufficient to inhibit sporulation and provide a selective advantage. We cloned devI under the control of Pxis at an ectopic locus (lacA) in a strain without ICEBs1. In the absence of ICEBs1 (and its repressor ImmR), Pxis is constitutively active (Auchtung et al., 2007). Fitness was measured relative to a control strain that had Pxis with no gene downstream. Sporulation of the Pxis-devI strain was strongly inhibited under conditions that normally support robust sporulation, including in biofilms (Figure 6). During sporulation either with (Figure 6A) or without biofilm formation (Figure 6B), the frequency of the Pxis-devI strain in the population rose from ~0.01 to ~0.05, giving a relative fitness of ~5. This is greater than the typical fitness conferred by ICEBs1 in biofilms, but less than that observed during sporulation without biofilms. devI is necessary for the fitness benefit conferred by ICEBs1 y y C We found that an ICEBs1 gene of unknown function, devI (ydcO), was necessary for the fitness advantage of ICEBs1 host cells. We constructed a deletion of devI in the locked-in-ICEBs1 strain. When started at a low frequency in the population (~0.01) the relative fitness of the devI mutant was approximately 3.5 (Figure 5D), much less than that of the isogenic devI+ cells (median fitness ~14) in biofilms with sporulation (Figure 5A). Interestingly, the deletion of devI did not reduce fitness fully to neutral, indicating a possible role for other ICEBs1 genes. devI (ydcO) is predicted to encode an 86 amino acid protein. A search for conserved motifs and structural similarity between DevI (YdcO) and other proteins did not significantly inform our understanding of DevI function. However, devI (ydcO) homologs are found in other Bacillus species (see below). 10 of 24 Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 Research article Microbiology and Infectious Disease immA attL PimmR Pxis PsncO int immR xisydzL devI helP sncO A. B. C. D. E. ∆Pxis ∆ydzL ∆devI ∆sncO ∆attR::tet Relative fitness 13.8 ± 3.4 0.6 ± 0.2 11.6 ± 1.8 3.5 ± 0.7 9.3 ± 1.3 ✱ ✱ ns ns Figure 5. Effects of deletions in ICEBs1 on host fitness. (A) Abbreviated genetic map of locked-in-ICEBs1 showing genes as open block arrows, promoters as thin right-angle arrows, and the left attachment site (attL) as a black bar. (B to E) Brackets under the map of ICEBs1 indicate regions contained in isogenic derivatives of locked-in-ICEBs1. Open spaces represent regions deleted. The fitness of strains containing locked-in-ICEBs1 (JMJ646) and its derivatives (DPxis, JMJ662; DydzL, JMJ704; DdevI, JMJ703; DsncO, JMJ688) is indicated at the right. Fitness was measured relative to ICEBs1-cured cells (JMJ550) during competitions in biofilms. ICEBs1-containing cells were started at a frequency of 0.01. Data shown are the median ± standard deviation from at least three independent experiments (at least nine total biological replicates per strain mixture), with the exception of JMJ688 which was measured only in one experiment (three total biological replicates). Asterisks indicate a statistically significant difference in fitness compared to JMJ646 (p-value<0.05, two-tailed T-test, unequal variance). Exact p-values: (B) 2.3 1016; (C) 4.5 101; (D) 6.3 1012; (E) 6.2 102. The online version of this article includes the following source data for figure 5: Source data 1. Effects of gene deletions on ICEBs1 fitness. Figure 5. devI is necessary for the fitness benefit conferred by ICEBs1 We suspect these differences are due to constitutive expression of devI in the absence of ICEBs1’s regulatory systems and the earlier onset of starvation on DSM agar compared to MSgg agar; cells that are unable to sporulate eventually die. 11 of 24 Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 Research article Microbiology and Infectious Disease 0 20 40 60 80 100 % Spores 0 20 40 60 80 100 % Spores 0 20 40 60 80 0.01 0.1 Hours devI freq. 0 20 40 60 80 0.01 0.1 Hours devI freq. A. B. Figure 6. devI alone is sufficient to inhibit sporulation and provide a selective advantage. ICEBs1-cured cells that constitutively express devI (JMJ725, black circles) were mixed at an initial frequency of 0.01 with ICEBs1-cured cells containing an empty expression construct (JMJ727, open squares). The mixture was spotted onto biofilm growth medium (A) or sporulation medium (B). Biofilms and colonies were harvested at the indicated times to determine the fraction of CFUs derived from heat-resistant spores for both the devI-containing and control cells. Boxes below each graph indicate the frequency of devI-containing CFUs at each timepoint. Data shown are the average from two populations (biological replicates) per timepoint with error bars indicating the standard deviation. The online version of this article includes the following source data for figure 6: Source data 1. DevI inhibits sporulation and provides benefit. 0 20 40 60 80 100 % Spores 0 20 40 60 80 0.01 0.1 Hours devI freq. A. 0 20 40 60 80 100 % Spores 0 20 40 60 80 0.01 0.1 Hours devI freq. B. B. A. Figure 6. devI alone is sufficient to inhibit sporulation and provide a selective advantage. ICEBs1-cured cells that constitutively express devI (JMJ725, black circles) were mixed at an initial frequency of 0.01 with ICEBs1-cured cells containing an empty expression construct (JMJ727, open squares). The mixture was spotted onto biofilm growth medium (A) or sporulation medium (B). Biofilms and colonies were harvested at the indicated times to determine the fraction of CFUs derived from heat-resistant spores for both the devI-containing and control cells. Boxes below each graph indicate the frequency of devI-containing CFUs at each timepoint. Data shown are the average from two populations (biological replicates) per timepoint with error bars indicating the standard deviation. The online version of this article includes the following source data for figure 6: Source data 1. devI is necessary for the fitness benefit conferred by ICEBs1 DevI inhibits sporulation and provides benefit. DevI likely targets the developmental transcription factor Spo0A Results described above demonstrated that devI is a robust inhibitor of sporulation. Sporulation is controlled by the transcription factor Spo0A (reviewed in Hoch, 1993; Sonenshein, 2000}) which both directly and indirectly regulates the expression of many genes needed for development, includ- ing biofilm formation (Hamon and Lazazzera, 2001). The results described below indicate that DevI most likely targets Spo0A, either directly or indirectly. devI inhibits early sporulation gene expression We found that devI inhibits expression of genes normally activated early during sporulation. Sporula- tion is initiated when Spo0A~P directly stimulates transcription of several genes, including the three sporulation operons, spoIIA, spoIIE, and spoIIG (Sonenshein, 2000). Using lacZ fusions to the pro- moters of each of these operons, we found that Pxis-devI inhibited activity of each promoter com- pared to wild-type during sporulation in liquid sporulation medium (Figure 7A). This indicates that DevI inhibits the initiation of sporulation, perhaps by affecting the activity or accumulation of Spo0A~P. devI inhibits biofilm gene expression We also found that devI inhibits expression of genes needed for extracellular matrix production dur- ing biofilm formation. We measured expression of biofilm matrix genes epsB and tasA (Hahn et al., e also found that devI inhibits expression of genes needed for extracellular matrix production dur- biofilm formation. We measured expression of biofilm matrix genes epsB and tasA (Hahn et al., 12 of 24 Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 Research article Research article Microbiology and Infectious Disease -2 0 2 4 6 0 50 100 150 Hours post-exponential -gal specific activity PspoIIA-lacZ -2 0 2 4 6 0 50 100 150 Hours post-exponential -gal specific activity PspoIIE-lacZ -2 0 2 4 6 0 20 40 60 80 Hours post-exponential -gal specific activity PspoIIG-lacZ A. B. -2 -1 0 1 2 3 0 2 4 6 Hours post-exponential mRNA (arbitrary units) epsB -2 -1 0 1 2 3 0 1 2 3 4 5 Hours post-exponential mRNA (arbitrary units) tasA Figure 7. devI inhibits expression of genes associated with sporulation initiation and biofilm formation. (A) ICEBs1-cured cells that constitutively expre devI (black circles) or contain an empty expression construct (open squares) were grown in liquid sporulation medium. Cells were harvested at the indicated times and b-galactosidase specific activity was measured. devI is necessary for the fitness benefit conferred by ICEBs1 (B) ICEBs1-cured cells that constitutively express devI (JMJ725, black circles) and ICEBs1-cured cells containing an empty expression construct (JMJ727, open squares) were grown in liquid biofilm medium, and cells were harvested at the indicated times. cDNA was synthesized using reverse transcriptas and RT-qPCR was used to measure expression of biofilm-associated genes epsB and tasA. The transcript copy numbers of these genes were measure relative to a housekeeping gene gyrA. The data reported are the average of three technical replicates from one experiment. The relative expression levels are normalized to wild-type at T-1. A representative experiment is shown. The online version of this article includes the following source data for figure 7: Source data 1. Sporulation and biofilm gene expression. B. B. -2 -1 0 1 2 3 0 2 4 6 Hours post-exponential mRNA (arbitrary units) epsB -2 -1 0 1 2 3 0 1 2 3 4 5 Hours post-exponential mRNA (arbitrary units) tasA Figure 7. devI inhibits expression of genes associated with sporulation initiation and biofilm formation. (A) ICEBs1-cured cells that constitutively exp devI (black circles) or contain an empty expression construct (open squares) were grown in liquid sporulation medium. Cells were harvested at the al -2 -1 0 1 2 3 0 1 2 3 4 5 Hours post-exponential mRNA (arbitrary units) tasA -2 -1 0 1 2 3 0 1 2 3 4 5 Hours post-exponential mRNA (arbitrary units) tasA Hours post-exponential Figure 7. devI inhibits expression of genes associated with sporulation initiation and biofilm formation. (A) ICEBs1-cured cells that constitutively express devI (black circles) or contain an empty expression construct (open squares) were grown in liquid sporulation medium. Cells were harvested at the indicated times and b-galactosidase specific activity was measured. Strains: PspoIIA-lacZ Pxis-devI (JMJ732), PspoIIA-lacZ WT (JMJ735), PspoIIE-lacZ Pxis-devI (JMJ731), PspoIIE-lacZ WT (JMJ734), PspoIIG-lacZ Pxis-devI (JMJ733), PspoIIG-lacZ WT (JMJ736). A representative experiment is shown. (B) ICEBs1-cured cells that constitutively express devI (JMJ725, black circles) and ICEBs1-cured cells containing an empty expression construct (JMJ727, open squares) were grown in liquid biofilm medium, and cells were harvested at the indicated times. cDNA was synthesized using reverse transcriptase and RT-qPCR was used to measure expression of biofilm-associated genes epsB and tasA. The transcript copy numbers of these genes were measured relative to a housekeeping gene gyrA. The data reported are the average of three technical replicates from one experiment. devI is necessary for the fitness benefit conferred by ICEBs1 Strains: PspoIIA-lacZ Pxis-devI (JMJ732), PspoIIA-lacZ WT (JMJ735), PspoIIE-lacZ Pxis-devI (JMJ731), PspoIIE-lacZ WT (JMJ734), PspoIIG-lacZ Pxis-devI (JMJ733), PspoIIG-lacZ WT (JMJ736). A representative experiment is shown. (B) ICEBs1-cured cells that constitutively express devI (JMJ725, black circles) and ICEBs1-cured cells containing an empty expression construct (JMJ727, open squares) were grown in liquid biofilm medium, and cells were harvested at the indicated times. cDNA was synthesized using reverse transcriptas and RT-qPCR was used to measure expression of biofilm-associated genes epsB and tasA. The transcript copy numbers of these genes were measure relative to a housekeeping gene gyrA. The data reported are the average of three technical replicates from one experiment. The relative expression levels are normalized to wild-type at T-1. A representative experiment is shown. The online version of this article includes the following source data for figure 7: -2 0 2 4 6 0 50 100 150 Hours post-exponential -gal specific activity PspoIIA-lacZ -2 0 2 4 6 0 50 100 150 Hours post-exponential -gal specific activity PspoIIE-lacZ -2 0 2 4 6 0 20 40 60 80 Hours post-exponential -gal specific activity PspoIIG-lacZ A. -2 0 2 4 6 0 50 100 150 Hours post-exponentia -gal specific activity PspoIIA-lacZ A. A. -2 0 2 4 6 0 20 40 60 80 -gal specific activity PspoIIG-lacZ Hours post-exponential Hours post-exponential Hours post-exponential B. -2 -1 0 1 2 3 0 2 4 6 Hours post-exponential mRNA (arbitrary units) epsB B. -2 -1 0 1 2 3 0 2 4 6 Hours post-exponential mRNA (arbitrary units) epsB -2 -1 0 1 2 3 0 1 2 3 4 5 Hours post-exponential mRNA (arbitrary units) tasA B. -2 -1 0 1 2 3 0 2 4 6 Hours post-exponential mRNA (arbitrary units) epsB -2 -1 0 1 2 3 0 1 2 3 4 5 Hours post-exponential mRNA (arbitrary units) tasA Figure 7. devI inhibits expression of genes associated with sporulation initiation and biofilm formation. (A) ICEBs1-cured cells that constitutively expre devI (black circles) or contain an empty expression construct (open squares) were grown in liquid sporulation medium. Cells were harvested at the indicated times and b-galactosidase specific activity was measured. Strains: PspoIIA-lacZ Pxis-devI (JMJ732), PspoIIA-lacZ WT (JMJ735), PspoIIE-lacZ Pxis-devI (JMJ731), PspoIIE-lacZ WT (JMJ734), PspoIIG-lacZ Pxis-devI (JMJ733), PspoIIG-lacZ WT (JMJ736). A representative experiment is shown. Discussion Our work demonstrates that ICEBs1 confers a selective advantage on its host cells by delaying bio- film and spore development, enabling the host to grow more than cells without ICEBs1. When ICEBs1-containing cells are the minority in a mixed population, ICEBs1 genes are induced. One of these genes, devI, is necessary and sufficient to inhibit biofilm- and sporulation-associated gene expression, likely by inhibiting the key developmental regulator Spo0A, either directly or indirectly. Together with previous findings we conclude that ICEBs1 encodes at least three distinct strategies to benefit its host cells. (1) Inhibition of development (described here) provides a growth advantage in biofilms and during sporulation. (2) Exclusion, mediated by yddJ, blocks the conjugation machin- ery and protects the host cell from lethal excessive transfer (Avello et al., 2019). (3) An abortive infection mechanism, mediated by spbK (yddK) protects populations of ICEBs1 host cells from pre- dation by the lysogenic phage SPb (Johnson et al., 2020). We propose that all three strategies pro- vide a competitive advantage for ICEBs1 and its host cells in different conditions. Expression of devI reduces biofilm matrix expression and delays the initiation of sporulation. Pro- duction of the biofilm matrix is a public good, benefiting the whole community (Dragosˇ et al., 2018). Avoidance of matrix production can therefore be considered an exploitative behavior. Exploi- tation can be detrimental to the population as a whole (Smith and Schuster, 2019), but we did not observe any negative effects on populations under conditions where ICEBs1 host cells had an advan- tage. This is in agreement with the facultative nature of ICEBs1 cheating (Even-Tov et al., 2016; Pollak et al., 2016). Quorum-sensing by rapI-phrI ensures that ICEBs1 cheats only as a minority, where its impact on total public goods levels is negligible. Interestingly, the pBS32 plasmid utilizes direct regulation of biofilm formation by a Rap receptor to its benefit (Omer Bendori et al., 2015; Pollak et al., 2015), while in ICEBs1 this regulation was moved from the Rap receptor to one of its regulated genes. The fitness consequences of sporulation inhibition are complicated (Mutlu et al., 2018). Delaying sporulation too long would result in a loss of viability of the starved cells. Inhibition of sporulation by ICEBs1 appears to be transient; ICEBs1 host cells eventually sporulate and do not lose significant viability as a consequence of delaying sporulation. devI is conserved among ICEs homologous to ICEBs1 We found that devI (ydcO) is conserved among Bacillus species and in many cases is located within what appear to be ICEs similar to ICEBs1. We used NCBI BLAST to search for homologous protein sequences using both pBLAST (protein database) and tBLASTn (translated nucleotide database). Homologs with 100% sequence coverage and greater than 70% identity to YdcO from B. subtilis NCIB3610 were found in dozens of other B. subtilis strains and in closely related species including B. licheniformis, B. atrophaeus, and B. amyloliquefaciens. Excluding Bacillus species from the searches to possibly identify more distantly related proteins with known functions produced no hits. We analyzed the sequence surrounding the devI (ydcO) homologs identified to determine if there is similarity to ICEBs1. All of the devI (ydcO) homologs appear to be within mobile element regions similar to ICEBs1, though some are clearly missing genes present in ICEBs1. Although we cannot infer whether any of these regions are functional mobile elements, we suspect that the ability to inhibit host development may be a conserved strategy among ICEBs1-like elements and possibly other ICEs with cargo genes of unknown function. devI is necessary for the fitness benefit conferred by ICEBs1 The relative expression levels are normalized to wild-type at T-1. A representative experiment is shown. The online version of this article includes the following source data for figure 7: Source data 1. Sporulation and biofilm gene expression. Source data 1. Sporulation and biofilm gene expression. 1995; Hamon et al., 2004; Bai et al., 1993; Kearns et al., 2005) by RT-qPCR with primers internal to each gene. In early stationary phase in liquid biofilm medium, transcript levels of epsB and tasA were reduced by about 5-fold and 3-fold, respectively, in the Pxis-devI strain compared to wild-type (Figure 7B). We suspect that inhibition of biofilm matrix genes, in addition to delaying sporulation, is an important mechanism of selection for ICEBs1 host cells during growth in a biofilm. This is con- sistent with the selective advantage of ICEBs1 host cells in biofilms without sporulation described Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 13 of 24 Research article Research article Research article Microbiology and Infectious Disease earlier. Inhibition of biofilm and early sporulation genes is consistent with DevI functioning as an inhibitor of Spo0A or its activation by phosphorylation. Diversity of cargo genes and associated phenotypes Diversity of cargo genes and associated phenotypes Mobile genetic elements, especially ICEs, are widespread in bacteria (Frost et al., 2005; Guglielmini et al., 2011). Many known mobile genetic elements encode cargo genes that confer easily recognizable phenotypes, notably antibiotic resistance. Other cargo genes provide less obvi- ous phenotypes but still fundamentally alter the physiology of the host cell. A large (500 kb) ICE was discovered in Mesorhizobium loti because its horizontal transfer conferred the ability to form nitro- gen-fixing symbiotic rood nodules on Lotus plant species (Sullivan and Ronson, 1998). In many pathogens, cargo genes in mobile elements are largely responsible for virulence. For example, Vibrio cholerae is capable of a pathogenic lifestyle in human hosts due to the toxin-encoding phage CTXF (Waldor and Mekalanos, 1996). In the sporulating pathogen Bacillus anthracis, mobile genetic ele- ments regulate both virulence and host development. Two plasmids, pXO1 and pXO2, provide the genes for toxin synthesis and production of a protective capsule, respectively (Green et al., 1985; Mikesell et al., 1983). pXO1 also contains a regulatory gene, atxA, that regulates virulence factor production and inhibits host cell sporulation (Dale et al., 2018). Co-regulation of virulence factors and sporulation is likely important during infection, as B. anthracis spores are thought to be more susceptible than vegetative cells to eradication by the immune system (Mock and Fouet, 2001). Mobile elements are also known to alter the host’s interaction with other horizontally acquired DNA, which has implications for the fitness and evolvability of the host. For example, the plasmid pBS32 in B. subtilis encodes an inhibitor of the host’s DNA uptake machinery, blocking natural trans- formation (Konkol et al., 2013). Interestingly, genes with roles in defense against foreign DNA, CRISPR-Cas systems, are also identified within mobile elements (Faure et al., 2019; McDonald et al., 2019; Millen et al., 2012). Competition between mobile elements not only shapes the repertoire of cargo genes in a given cell, but it may also protect the host from harmful elements. Many mobile genetic elements have been identified bioinformatically from genome sequences or discovered by means other than the phenotypes they provide (Bi et al., 2012; Guglielmini et al., 2011; Johnson and Grossman, 2015). Many elements lack obvious cargo genes, or at least lack cargo genes that have recognizable functions (Cury et al., 2017). Discussion Regulation of devI expression by the cell-cell sig- naling genes rapI-phrI is likely critical for transient developmental inhibition. Because commitment to sporulation is irreversible, sporulating too early is detrimental if nutrient deprivation is short-lived. B. subtilis cells with activated Spo0A that have not yet committed to sporulate also delay commitment to sporulation by killing sibling cells to liberate nutrients (‘cannibalism’) (Gonza´lez-Pastor et al., 2003). Cannibalism is regulated by Spo0A and the subpopulation of cannibal cells (those doing the killing) overlaps with those producing the biofilm matrix (Lo´pez et al., 2009). Because of this over- lap, it seems unlikely that devI delays sporulation by stimulating cannibalism. Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 14 of 24 Research article Research article Microbiology and Infectious Disease ICEBs1 stability during sporulation We hypothesize that in addition to providing a fitness advantage to its host cell, delaying sporulation may also improve stability of ICEBs1 in the host during development. Sporulation involves the forma- tion of an asymmetric division septum generating the larger mother cell and the smaller forespore (Errington, 2001; Higgins and Dworkin, 2012). Sporulation is induced when cells are at a high pop- ulation density and running out of nutrients, conditions that also activate ICEBs1 (Auchtung et al., 2005; Grossman and Losick, 1988). The plasmid form of ICEBs1 that is generated after excision from the chromosome is not known to have a mechanism for active partitioning and is more likely to remain in the larger mother cell if the cells do enter the sporulation pathway and divide asymmetri- cally. Therefore, the ability of ICEBs1 to delay the initiation of sporulation under conditions where the element is activated could help prevent loss of the element and maintain ICEBs1 in host cells. p p Mobile genetic elements employ various strategies to promote their maintenance during sporula- tion. Rates of curing during sporulation for various plasmids in Bacillus species vary widely and do not necessarily correlate with their stability during normal cell division (Tokuda et al., 1993; Turgeon et al., 2008). Mechanisms encoded by plasmids to promote their stability during growth and sporulation include the production of dynamic cytoskeletal filaments (Becker et al., 2006) and post-segregational killing of plasmid-cured pre-spores with toxin-antitoxin systems (Short et al., 2015). Interestingly, even lytic phage genomes can be incorporated into spores (first described in the 1960 s) by co-opting the host’s chromosomal partitioning system (Meijer et al., 2005). Diversity of cargo genes and associated phenotypes We suspect that many elements with uncharacterized cargo genes provide important traits to their hosts beyond the scope of the phenotypes currently attributed to mobile elements. Although mobile genetic elements can have remarkably broad host ranges, such as the Tn916-Tn1545 group of ICEs (Clewell et al., 1995; Roberts and Mullany, 2009) and the IncP-1 group of plasmids (Popowska and Krawczyk-Balska, 2013), cargo genes and their associated functions could be highly specific to certain hosts. g g g y Characterization of unknown cargo genes is likely to expand the diversity of traits currently attrib- uted to mobile genetic elements. We speculate that many of these genes modulate normal host Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 15 of 24 Microbiology and Infectious Disease functions rather than provide entirely new phenotypes. Understanding cargo gene function is critical for understanding interactions between and co-evolution of mobile elements and their hosts. functions rather than provide entirely new phenotypes. Understanding cargo gene function is critical for understanding interactions between and co-evolution of mobile elements and their hosts. functions rather than provide entirely new phenotypes. Understanding cargo gene function is critical for understanding interactions between and co-evolution of mobile elements and their hosts. Materials and methods Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (Bacillus subtilis NCIB3610) DS2569 Konkol et al., 2013. PMID:23836866 NCIB3610 cured of pBS32 plasmid. Gift of Daniel Kearns to Avigdor Eldar. Continued on next page Diversity of cargo genes and associated phenotypes Strain, strain background (Bacillus subtilis DS2569) JMJ550 This paper ICEBs10 lacA:: {Ppen-mApple2 kan} Strain, strain background (Bacillus subtilis DS2569) JMJ574 This paper ICEBs10 lacA::{Pveg- mTagBFP mls} Strain, strain background (Bacillus subtilis DS2569) JMJ576 This paper ICEBs10 bcaP::{PrapI- rapIphrI kan} lacA::{Pveg-mTagBFP mls} Strain, strain background (Bacillus subtilis DS2569) JMJ592 This paper ICEBs1 yddJ-cat-yddK lacA:: {Pveg-mTagBFP mls} Strain, strain background (Bacillus subtilis DS2569) JMJ593 This paper ICEBs1 conEK476E yddJ-cat-yddK lacA::{Pveg-mTagBFP mls} Strain, strain background (Bacillus subtilis DS2569) JMJ646 This paper ICEBs1 oriT attR::tet lacA: :{Pveg-mTagBFP mls} Strain, strain background (Bacillus subtilis DS2569) JMJ662 This paper ICEBs1 DPxis oriT* attR::tet lacA:: {Pveg-mTagBFP mls} Strain, strain background (Bacillus subtilis DS2569) JMJ686 This paper ICEBs1 oriT* DrapIphrI attR::tet lacA:: {Pveg-mTagBFP mls} Strain, strain background (Bacillus subtilis DS2569) JMJ688 This paper ICEBs1 oriT* DsncO attR::tet lacA:: {Pveg-mTagBFP mls} Strain, strain background (Bacillus subtilis DS2569) JMJ703 This paper ICEBs1 oriT* DdevI attR::tet lacA:: {Pveg-mTagBFP mls} Strain, strain background (Bacillus subtilis DS2569) JMJ704 This paper ICEBs1 oriT* DydzL attR::tet lacA:: {Pveg-mTagBFP mls} Strain, strain background (Bacillus subtilis DS2569) JMJ714 This paper ICEBs10 lacA::spec bcaP::kan Strain, strain background (Bacillus subtilis DS2569) JMJ725 This paper ICEBs10 lacA::{Pxis-devI mls} Strain, strain background (Bacillus subtilis DS2569) JMJ727 This paper ICEBs10 lacA::{Pxis-empty mls} Strain, strain background (Bacillus subtilis DS2569) JMJ731 This paper ICEBs10 lacA::{Pxis-devI mls} amyE::{PspoIIE-lacZ cat} Strain, strain background (Bacillus subtilis DS2569) JMJ732 This paper ICEBs10 lacA::{Pxis-devI mls} amyE::{PspoIIA-lacZ cat} Strain, strain background (Bacillus subtilis DS2569) JMJ733 This paper ICEBs10 lacA::{Pxis-devI mls} amyE::{PspoIIG-lacZ cat} Strain, strain background (Bacillus subtilis DS2569) JMJ734 This paper ICEBs10 lacA::{Pxis-empty mls} amyE::{PspoIIE-lacZ cat} Strain, strain background (Bacillus subtilis DS2569) JMJ735 This paper ICEBs10 lacA::{Pxis-empty mls} amyE::{PspoIIA-lacZ cat} Strain, strain background (Bacillus subtilis DS2569) JMJ736 This paper ICEBs10 lacA::{Pxis-empty mls} amyE::{PspoIIG-lacZ cat} Strain, strain background (Bacillus subtilis DS2569) JMJ785 This paper ICEBs1 oriT* DrapIphrI attR::tet bcaP::{PrapI-rapIphrI kan} lacA:: {Pveg-mTagBFP mls} Continued on next page Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 16 of 24 16 of 24 Research article Microbiology and Infectious Disease Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (Bacillus subtilis DS2569) JMJ786 This paper ICEBs10 spo0ADPs lacA:: {Ppen-mApple2 kan} Strain, strain background (Bacillus subtilis DS2569) JMJ788 This paper ICEBs1 conEK476E yddJ-cat-yddK spo0ADPs lacA::{Pveg-mTagBFP mls} Strain, strain background (Escherichia coli MC1061) AG1111 Ireton et al., 1993 PMID:8436298 E. coli strain for cloning and maintaining plasmids. MC1061 with F’ proAB+ lacIq lacZM15 Tn10. Media and growth conditions Diversity of cargo genes and associated phenotypes The sporulation medium used was DSM (in liquid form or as plates solidified with 1.5% agar) (Harwood and Cutting, 1990). MSgg agar and DSM agar plates were dried for 20–24 hr at 37˚C prior to use. Antibiotics were used at the following concentrations for selection on LB agar plates: chloramphenicol (5 mg/ml), kanamycin (5 mg/ml), spectinomycin (100 mg/ml), tetracycline (12.5 mg/ml), and a combination of erythromycin (0.5 mg/ml) and lincomycin (12.5 mg/ml) to select for macrolide-lincosamide-streptogramin (MLS) resistance. and 1x Spizizen’s salts (2 g/l (NH4)SO4, 14 g/l K2HPO4, 6 g/l KH2PO4, 1 g/l Na3citrate-2H2O, and 0.2 g/l MgSO4-7H2O) (Harwood and Cutting, 1990). Cells were resuspended from light lawns and grown at 37˚C with shaking in S750 defined minimal medium (Jaacks et al., 1989) with 1% w/v glu- cose and 0.1% w/v monopotassium glutamate. Biofilms were grown at 30˚C on MSgg agar plates (as defined in Branda et al., 2001 with the exception of tryptophan and phenylalanine, which we did not include). The sporulation medium used was DSM (in liquid form or as plates solidified with 1.5% agar) (Harwood and Cutting, 1990). MSgg agar and DSM agar plates were dried for 20–24 hr at 37˚C prior to use. Antibiotics were used at the following concentrations for selection on LB agar plates: chloramphenicol (5 mg/ml), kanamycin (5 mg/ml), spectinomycin (100 mg/ml), tetracycline (12.5 mg/ml), and a combination of erythromycin (0.5 mg/ml) and lincomycin (12.5 mg/ml) to select for macrolide-lincosamide-streptogramin (MLS) resistance. Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 1All strains derived from NCIB3610 plasmid-free. Diversity of cargo genes and associated phenotypes Recombinant DNA reagent pJMJ196 (plasmid) This paper For generating oriT* nick; derived from pCAL1422. Recombinant DNA reagent pJMJ430 (plasmid) This paper For generating unmarked rapI-phrI deletion; derived from pCAL1422. Recombinant DNA reagent pJMJ199 (plasmid) This paper For generating unmarked Pxis deletion; derived from pCAL1422. Recombinant DNA reagent pELS5 (plasmid) Other For generating unmarked ydzL deletion; derived from pCAL1422. From Grossman lab collection. Recombinant DNA reagent pELS1 (plasmid) Other For generating unmarked devI deletion; derived from pCAL1422. From Grossman lab collection. Recombinant DNA reagent pELC815 (plasmid) Other For generating unmarked sncO deletion; derived from pCAL1422. From Grossman lab collection. Recombinant DNA reagent pJT245 (plasmid) Other Source of oriT* nicK allele. From Grossman lab collection. Recombinant DNA reagent pCAL1422 (plasmid) Thomas et al., 2013. PMID:23326247 For generating markerless deletions/mutations. Recombinant DNA reagent pMMH253 (plasmid) Other Vector for integration of constructs at bcaP. From Grossman lab collection. Recombinant DNA reagent pJMJ354 (plasmid) This paper Native rapI-phrI expression construct for integration at bcaP; derived from pMMH253 Sequence- based reagent oJJ363 Sigma-Aldrich qPCR primer CGGAACAATATCGCACCATTC Sequence- based reagent oJJ364 Sigma-Aldrich qPCR primer CGCTGCACTGAACGATTTAC Sequence- based reagent oJJ367 Sigma-Aldrich qPCR primer GGATCACTTGCGATCAAAGAAG Sequence- based reagent oJJ368 Sigma-Aldrich qPCR primer CTTCAAACTGGCTGAGGAAATC Sequence- based reagent oMEA128 Sigma-Aldrich qPCR primer TGGAGCATTACCTTGACCATC Sequence- based reagent oMEA129 Sigma-Aldrich qPCR primer AGCTCTCGCTTCTGCTTTAC Commercial assay or kit RNeasy PLUS Qiagen Cat No. 74136 Commercial assay or kit iScript Reverse Transcription Supermix Bio-Rad Cat No. 1708840 Commercial assay or kit SsoAdvanced SYBR master mix Bio-Rad Cat No. 1725270 d d h d Media and growth conditions Prior to competition experiments, cells were grown as light lawns for approximately 20 hr at room temperature on 1.5% agar plates containing 1% w/v glucose, 0.1% w/v monopotassium glutamate, 17 of 24 Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 Research article Research article Research article Microbiology and Infectious Disease and 1x Spizizen’s salts (2 g/l (NH4)SO4, 14 g/l K2HPO4, 6 g/l KH2PO4, 1 g/l Na3citrate-2H2O, and 0.2 g/l MgSO4-7H2O) (Harwood and Cutting, 1990). Cells were resuspended from light lawns and grown at 37˚C with shaking in S750 defined minimal medium (Jaacks et al., 1989) with 1% w/v glu- cose and 0.1% w/v monopotassium glutamate. Biofilms were grown at 30˚C on MSgg agar plates (as defined in Branda et al., 2001 with the exception of tryptophan and phenylalanine, which we did not include). Construction of lacA::Pxis-devI We expressed devI from the Pxis promoter by cloning existing elements from a Pxis gene expression construct marked with mls at lacA and inserting the devI ORF by isothermal assembly. The resulting product was introduced to the chromosome by transformation and selection for MLS resistance. The Pxis-empty control strain contains an identical construct lacking an ORF fused to Pxis. Strains and alleles The B. subtilis strains used are listed in Table 2. The strain background used in all experiments was a derivative of the undomesticated strain NCIB3610 lacking its endogenous plasmid pBS32 (NCIB3610 plasmid-free). ICEBs10 indicates the strain is cured of ICEBs1. Standard techniques were used for cloning and strain construction (Harwood and Cutting, 1990). Some alleles were previously described and are summarized below. Variants of ICEBs1 that were blocked for transfer contained the conEK476E mutation derived from MMB1118 (Berkmen et al., 2010). The spo0ADPs allele was derived from AG1242 (Siranosian and Grossman, 1994). The amyE::PspoIIA-lacZ cat allele was derived from KI938 (Chung et al., 1994). Essentially identical alleles with PspoIIE and PspoIIG were also used. Table 2. B. subtilis strains used. Strain Relevant genotype JMJ550 ICEBs10 lacA::{Ppen-mApple2 kan} JMJ574 ICEBs10 lacA::{Pveg-mTagBFP mls} JMJ576 ICEBs10 bcaP::{PrapI-rapIphrI kan} lacA::{Pveg-mTagBFP mls} JMJ592 ICEBs1 yddJ-cat-yddK lacA::{Pveg-mTagBFP mls} JMJ593 ICEBs1 conEK476E yddJ-cat-yddK lacA::{Pveg-mTagBFP mls} JMJ646 ICEBs1 oriT attR::tet lacA::{Pveg-mTagBFP mls} JMJ662 ICEBs1 DPxis oriT* attR::tet lacA::{Pveg-mTagBFP mls} JMJ686 ICEBs1 oriT* DrapIphrI attR::tet lacA::{Pveg-mTagBFP mls} JMJ688 ICEBs1 oriT* DsncO attR::tet lacA::{Pveg-mTagBFP mls} JMJ703 ICEBs1 oriT* DdevI attR::tet lacA::{Pveg-mTagBFP mls} JMJ704 ICEBs1 oriT* DydzL attR::tet lacA::{Pveg-mTagBFP mls} JMJ714 ICEBs10 lacA::spec bcaP::kan JMJ725 ICEBs10 lacA::{Pxis-devI mls} JMJ727 ICEBs10 lacA::{Pxis-empty mls} JMJ731 ICEBs10 lacA::{Pxis-devI mls} amyE::{PspoIIE-lacZ cat} JMJ732 ICEBs10 lacA::{Pxis-devI mls} amyE::{PspoIIA-lacZ cat} JMJ733 ICEBs10 lacA::{Pxis-devI mls} amyE::{PspoIIG-lacZ cat} JMJ734 ICEBs10 lacA::{Pxis-empty mls} amyE::{PspoIIE-lacZ cat} JMJ735 ICEBs10 lacA::{Pxis-empty mls} amyE::{PspoIIA-lacZ cat} JMJ736 ICEBs10 lacA::{Pxis-empty mls} amyE::{PspoIIG-lacZ cat} JMJ785 ICEBs1 oriT* DrapIphrI attR::tet bcaP::{PrapI-rapIphrI kan} lacA::{Pveg-mTagBFP mls} JMJ786 ICEBs10 spo0ADPs lacA::{Ppen-mApple2 kan} JMJ788 ICEBs1 conEK476E yddJ-cat-yddK spo0ADPs lacA::{Pveg-mTagBFP mls} 1All i d i d f NCIB3610 l id f Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 18 of 24 Research article Research article Microbiology and Infectious Disease Construction of selective markers for mating and competition experiments ICEBs1 was marked with the cat gene (conferring chloramphenicol resistance) between the diver- gently transcribed genes yddJ and spbK (yddK). Markers used to select ICEBs1-containing and ICEBs10 cells were all integrated at lacA and contained spec (spectinomycin resistance), mls (macro- lide-lincosamide-streptogramin resistance), or kan (kanamycin resistance). The mls and kan con- structs also contained constitutively expressed fluorescent proteins BFP and RFP, respectively. The plating efficiency of all markers was verified, and control competitions (described below) were per- formed to measure marker-associated fitness effects. Construction of ICEBs1 mutants Fitness measurements of ICEBs1 mutants were performed in a version of ICEBs1 unable to excise and replicate (locked-in ICEBs1). All mutants were isogenic to JMJ646 (ICEBs1 oriT* DattR::tet), which is unable to excise due to the attR::tet deletion (Lee and Grossman, 2007). The origin of transfer was mutated (oriT) to prevent ICEBs1 replication while integrated, which is detrimental (Lee and Grossman, 2007; Menard and Grossman, 2013). The markerless oriT mutation was con- structed by cloning nicK(oriT) from pJT245 and ~1 kb of upstream sequence into pCAL1422 (a plas- mid that contains E. coli lacZ) by isothermal assembly (Gibson et al., 2009), essentially as previously described (Thomas et al., 2013). The resulting plasmid, pJMJ196, was integrated into the chromo- some by single-crossover recombination. Transformants were screened for loss of lacZ, indicating loss of the integrated plasmid, and PCR was used to identify a clone containing the oriT allele. Mar- kerless deletions of ICEBs1 sequence were also generated using pCAL1422-derived plasmids. The rapI-phrI deletion was generated using pJMJ430 and removes the rapI and phrI ORFs. The Pxis dele- tion was generated using pJMJ199 and removes sequence from 149 bp to 27 bp upstream of the xis ORF. This removes the presumed 35 and 10 of the promoter but does not remove the known regulatory sites at the neighboring immR promoter (Auchtung et al., 2007). The ydzL deletion was generated using pELS5 and fuses the first four and last two codons of ydzL. The devI deletion was generated using pELS1 and fuses the first four and last two codons of devI. The sncO deletion was generated using pELC815 and removes the sncO gene and 44 bp of upstream sequence. Construction of ectopic rapI-phrI construct The rapI-phrI ORFs plus the promoter region (352 bp upstream of rapI) and 112 bp of downstream sequence were cloned into pMMH253 (vector for integration at bcaP). The resulting plasmid (pJMJ354) was linearized and introduced to B. subtilis by transformation and selection for kanamycin resistance. The corresponding empty control construct was generated by transforming linearized pMMH253. Construction of lacA::Pxis-devI Research article Biofilm mating experiments Cultures were started from resuspended light lawns (described above) diluted to an initial OD600 of 0.05 in S750 minimal medium. Cultures were grown to mid-exponential phase (OD600 ~0.5) at 37˚C with shaking. Cells were pelleted, resuspended in 1x Spizizen’s salts, and diluted to an OD600 of 0.01. Donor and recipient strains were mixed at the indicated frequencies and 50 ml of the mixture was spotted onto the center of MSgg agar plates. Spots were allowed to dry at 30˚C before flipping the plates. Plates were incubated at 30˚C for 4 days. At the time of inoculation, the strain mixes were diluted and plated in duplicate on LB agar plates containing the appropriate antibiotics to determine the initial CFU/ml of the donor and recipient strains. After 4 days, the mature biofilms were scraped from the agar surface with sterile wooden sticks and resuspended in 5 ml 1x Spizizen’s salts, followed by mild sonication to disperse the cells. Cells were diluted and selectively plated to determine the final CFU/ml of transconjugants. Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 19 of 24 Research article Research article Microbiology and Infectious Disease Gene expression assays Expression of sporulation genes was measured in cultures grown from single colonies in liquid DSM at 37˚ with shaking. Cells were harvested at the indicated timepoints. For b-galactosidase assays, growth was stopped by the addition of toluene (~1.5% final concentration). b-galactosidase specific activity ([DA420 per minute per ml of culture per OD600] x 1000) was measured as described (Miller, 1972) after pelleting cell debris. Biofilm gene expression was measured in cultures grown from single colonies in liquid MSgg at 37˚ with shaking. For RT-qPCR assays, cells were harvested directly into ice-cold methanol (1:1 meth- anol to culture volume) and pelleted. RNA was isolated using Qiagen RNeasy PLUS kit with 10 mg/ ml lysozyme. iScript Supermix (Bio-Rad) was used for reverse transcriptase reactions to generate cDNA. Control reactions without reverse transcriptase were performed to assess the amount of DNA present in the RNA samples. RNA was degraded by adding 75% vol of 0.1 M NaOH and incu- bating at 70˚C for 10 min, followed by neutralization with an equal volume of 0.1 M HCl. qPCR was done using SSoAdvanced SYBR master mix and CFX96 Touch Real-Time PCR system (Bio-Rad). Pri- mers used to measure epsB were oJJ363 (5’-CGGAACAATATCGCACCATTC-3’) and oJJ364 (5’- CGCTGCACTGAACGATTTAC-3’). Primers used to quantify tasA were oJJ367 (5’-GGATCAC TTGCGATCAAAGAAG-3’) and oJJ368 (5’-CTTCAAACTGGCTGAGGAAATC-3’). Primers used to measure the control locus gyrA were oMEA128 (5’-TGGAGCATTACCTTGACCATC-3’) and oMEA129 (5’-AGCTCTCGCTTCTGCTTTAC-3’). The relative transcript copy numbers (as indicated by the Cp values measured by qPCR) of epsB and tasA were normalized to gyrA after subtracting the signal from control reactions without reverse transcriptase. Competition experiments Cells were prepared for competition experiments as described above for biofilm mating experi- ments. Strain mixtures at the indicated frequencies were spotted onto MSgg agar plates for biofilm competitions and DSM agar plates for sporulation competitions. Plates were incubated at 30˚C for 4 days unless otherwise indicated. Biofilms/colonies were collected, disrupted, and plated as described above. For time-course competitions, two replicate biofilms/colonies were collected at each of the indicated times. Sporulation frequency was determined by selective plating before and after a heat treatment at 85˚C for 20 min to enumerate total CFUs and CFUs derived from heat-resis- tant spores. Relative fitness of ICEBs1-containing cells over ICEBs10 cells was determined as (pf/(1- pf))/(pi/(1-pi)), where pf, pi are the frequencies of ICEBs1-containing cells in the final and initial popu- lations, respectively. Control competitions between ICEBs1-cured cells were performed to deter- mine the fitness associated with the lacA::{Pveg-mTagBFP mls} marker (JMJ574) used in ICEBs1- containing cells relative to the lacA::{Ppen-mApple2 kan} marker used in ICEBs1-null cells (JMJ550). When the mls-marked cells were started at a frequency of 0.01, their relative fitness was 0.7 ± 0.09 (average and standard deviation from three independent experiments and a total of 9 biofilms). Acknowledgements We thank Mary Anderson and Janet Smith for helpful comments on the manuscript. Research reported here is based upon work supported, in part, by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM050895 and R35 GM122538 to ADG. Any opinions, findings, and conclusions or recommendations expressed in this report are those of the authors and do not necessarily reflect the views of the National Institutes of Health. Funding for travel was provided by the MIT International Science and Technology Initiatives (MISTI) MIT-Israel Seed Fund. Additional information Data availability y All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures. Author contributions Joshua M Jones, Conceptualization, Formal analysis, Validation, Investigation, Visualization, Method- ology, Writing - original draft, Writing - review and editing; Ilana Grinberg, Investigation, Methodol- ogy; Avigdor Eldar, Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - review and editing; Alan D Grossman, Conceptualization, Supervision, Funding acquisition, Visualiza- tion, Project administration, Writing - review and editing Author ORCIDs Joshua M Jones http://orcid.org/0000-0002-3327-8899 Avigdor Eldar https://orcid.org/0000-0001-8485-9370 Alan D Grossman https://orcid.org/0000-0002-8235-7227 Decision letter and Author response Decision letter https://doi.org/10.7554/eLife.65924.sa1 Author response https://doi.org/10.7554/eLife.65924.sa2 Author ORCIDs Joshua M Jones http://orcid.org/0000-0002-3327-8899 Avigdor Eldar https://orcid.org/0000-0001-8485-9370 Alan D Grossman https://orcid.org/0000-0002-8235-7227 Decision letter and Author response Decision letter and Author response Decision letter https://doi.org/10.7554/eLife.65924.sa1 Author response https://doi.org/10.7554/eLife.65924.sa2 Additional files . Source data 1. Neutral fitness due to marker effects. Counts of two ICEBs1-cured strains each bearing an antibiotic resistance marker (used to distinguish strains) at the start and end of competitions. Additional information Funding Funder Grant reference number Author National Institute of General Medical Sciences R01 GM050895 Alan D Grossman 20 of 24 Jones et al. eLife 2021;10:e65924. DOI: https://doi.org/10.7554/eLife.65924 Research article Research article Research article Microbiology and Infectious Disease National Institute of General Medical Sciences R35 GM122538 National Institute of General Medical Sciences R35 GM122538 Alan D Grossman The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Microbiology and Infectious Disease References Auchtung JM, Lee CA, Monson RE, Lehman AP, Grossman AD. 2005. 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https://openalex.org/W2767471152	http://norden.diva-portal.org/smash/get/diva2:1155273/FULLTEXT01	English	null	Nordic Foundries	TemaNord	2,017	cc-by	33,670	Nordic Foundries Nordic Foundries Best Available Techniques (BAT) Martin Wänerholm TemaNord 2017:562 Nordic Foundries Best Available Techniques (BAT) Martin Wänerholm ISBN 978-92-893-5226-0 (PRINT) ISBN 978-92-893-5227-7 (PDF) ISBN 978-92-893-5228-4 (EPUB) http://dx.doi.org/10.6027/TN2017-562 TemaNord 2017:562 ISSN 0908-6692 Standard: PDF/UA-1 ISO 14289-1 © Nordic Council of Ministers 2017 Cover photo: Fredrik Lind Print: Rosendahls Printed in Denmark Disclaimer This publication was funded by the Nordic C reflect the Nordic Council of Ministers’ views Nordic Foundries Disclaimer Disclaimer This publication was funded by the Nordic Council of Ministers. However, the content does not necessarily reflect the Nordic Council of Ministers’ views, opinions, attitudes or recommendations. 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The Nordic Council of Ministers Nordens Hus Ved Stranden 18 DK-1061 Copenhagen K, Denmark Tel.: +45 3396 0200 www.norden.org p g , Tel.: +45 3396 0200 www.norden.org Download Nordic publications at www.norden.org/nordpub Contents Summary ................................................................................................................................. 9 Preface .................................................................................................................................... 15 List of abbreviations ................................................................................................................ 17 Introduction ........................................................................................................................... 19 Methodology .................................................................................................................... 19 The objectives .................................................................................................................. 20 1. The Nordic foundries .........................................................................................................21 1.1 Finland ....................................................................................................................21 1.2 Åland Island ............................................................................................................21 1.3 Denmark .................................................................................................................21 1.4 Norway .................................................................................................................. 22 1.5 Sweden .................................................................................................................. 22 1.6 Iceland ................................................................................................................... 22 1.7 Faroe Islands .......................................................................................................... 22 2. Brief regulatory review ......................................................................................................23 2.1 Finland ....................................................................................................................23 2.2 Åland Islands ...........................................................................................................23 2.3 Denmark .................................................................................................................23 2.4 Norway .................................................................................................................. 24 2.5 Sweden .................................................................................................................. 24 2.6 Iceland ................................................................................................................... 24 2.7 Faroe Islands .......................................................................................................... 24 3. Casting ............................................................................................................................. 25 3.1 Introduction ........................................................................................................... 25 3.2 Non-permanent moulds ......................................................................................... 25 3.3 Permanent moulds................................................................................................. 30 3.4 Melting and pouring ................................................................................................ 35 3.5 Shake-out and the last steps ................................................................................... 37 4. Key environmental issues (KEI) and environmental impact of foundry and casting ............ 39 4.1 Air emissions.......................................................................................................... 39 4.2 Noise emissions and vibrations ...............................................................................41 4.3 Emissions to water and water use........................................................................... 42 4.4 Chemicals use and risks .......................................................................................... 42 4.5 Waste/Residues ..................................................................................................... 43 4.6 Energy use ............................................................................................................. 43 4.7 Raw material .......................................................................................................... 44 4.8 Transports ............................................................................................................. 44 5. BAT for foundries .............................................................................................................. 45 5.1 BAT for control of air emissions .............................................................................. 45 5.2 BAT to prevent noise emissions and vibrations ....................................................... 46 5.3 BAT for control of wastewater emission .................................................................. 47 5.4 BAT High pressure die casting ................................................................................ 48 5.5 BAT sand and binders ............................................................................................. 49 5.6 BAT for chemicals handling and substitution .......................................................... 50 5.7 BAT for waste management and minimisation ....................................................... 50 5.8 BAT for construction simulation ............................................................................. 51 5.9 BAT for energy ....................................................................................................... 51 5.10 BAT metal and melting ........................................................................................... 54 5.11 BAT for environmental management ...................................................................... 55 6. BAT Candidates ................................................................................................................ 57 7. BAT Candidates Emissions to Air ....................................................................................... 61 7.1 Bag-house filter ...................................................................................................... 61 7.2 Pressure drop gauge ............................................................................................... 63 7.3 Dust filter measurements ....................................................................................... 64 7.4 Filter heating .......................................................................................................... 65 7.5 Using fluorescent powder to check for problem in bag-house filter......................... 65 7.6 Particle monitor ..................................................................................................... 66 7.7 Acid scrubber to clean amine .................................................................................. 67 7.8 Oil mist filter .......................................................................................................... 68 7.9 Plan for reduction of VOC ....................................................................................... 69 7.10 Lid on coating vessel with alcohol based coating..................................................... 71 7.11 Use coating only when needed ............................................................................... 72 7.12 Coal dust replacement ............................................................................................ 73 7.13 Cleaning VOC and odour with biofilter .................................................................... 74 7.14 Cleaning VOC and odour with RTO ......................................................................... 75 8. BAT Candidates Noise emission ........................................................................................ 77 8.1 Tipping scrap indoors ............................................................................................. Contents 77 8.2 Noise reducing plan ................................................................................................ 78 8.3 Noise simulation ..................................................................................................... 80 8.4 Silencers on ventilation systems ............................................................................. 82 8.5 Additional insulation of fans and ducts.................................................................... 83 9. BAT Candidates Chemicals ................................................................................................ 85 9.1 Chemicals responsibility ......................................................................................... 85 10. BAT Candidates Waste and residues .................................................................................. 87 10.1 Recycling management .......................................................................................... 87 10.2 Equipment for mechanical sand regeneration ......................................................... 88 10.3 Reuse of rest sand outside the foundry ................................................................... 89 10.4 Briquetting ............................................................................................................. 90 11. BAT Candidates Simulation ............................................................................................... 93 11.1 Simulation .............................................................................................................. 93 11.2 Optimized inlet and gating system ......................................................................... 94 12. BAT Candidates Energy ..................................................................................................... 97 12.1 Energy audit ........................................................................................................... 97 12.2 Blasting raw material before melting ...................................................................... 98 12.3 Crushing inlet and gating system ............................................................................ 98 12.4 Insulated ladles ....................................................................................................... 99 12.5 Use of lid on furnace and ladle .............................................................................. 100 12.6 Flameless ladle heating ........................................................................................ 101 13. BAT Candidates Metal and melting .................................................................................. 103 13.1 Scrap handling ...................................................................................................... 103 13.2 Using deoxidiser/degassing ................................................................................... 104 13.3 Flushing ladle with argon ...................................................................................... 105 13.4 Inoculation control ................................................................................................ 106 13.5 Automatized in-stream inoculation ....................................................................... 107 14. BAT Candidates High and Low pressure die casting ......................................................... 109 14.1 Maintenance of die tool ........................................................................................ 109 14.2 Preheat die tool .................................................................................................... 110 14.3 IR camera .............................................................................................................. 111 14.4 Use of vacuum dosing furnace ............................................................................... 113 15. BAT Candidates Management and general ...................................................................... 115 15.1 Circular economy .................................................................................................. 115 15.3 Management system ............................................................................................ 117 15.4 Digitalization ........................................................................................................ 119 15.5 Environmental communication ............................................................................. 120 16. Emerging techniques ....................................................................................................... 123 16.1 New release agent for High pressure die casting .................................................... 123 16.2 Ignition of VOC at the sand mould ......................................................................... 124 16.3 New inorganic sand binder .................................................................................... 125 16.4 Reclaim green sand to core production ................................................................. 125 References and contacts ........................................................................................................ 127 Sammanfattning ................................................................................................................... 129 BAT kandidater utsläpp till luft......................................................................................... 130 BAT kandidater buller ...................................................................................................... 131 BAT kandidater kemikalier............................................................................................... 131 BAT kandidater avfall och restprodukter .......................................................................... 131 BAT kandidater simulering .............................................................................................. 131 BAT kandidater energi ..................................................................................................... 131 BAT kandidater metall och smältning .............................................................................. 132 BAT kandidater pressgjutning .......................................................................................... 132 Kommande tekniker ........................................................................................................ 132 Appendix ............................................................................................................................... 133 Air ............................................................................................................................. 133 Chemicals (process chemicals and auxiliary chemicals) .................................................... 133 Energy ............................................................................................................................. 133 Management ................................................................................................................... 134 Waste ............................................................................................................................. 134 Simulation ....................................................................................................................... 134 Sand ............................................................................................................................. 134 Binder ............................................................................................................................. 134 Water ............................................................................................................................. 135 Die casting....................................................................................................................... 135 Blacking (coating of cores) ............................................................................................... 135 Oven ............................................................................................................................. 135 Other ............................................................................................................................. 135 Summary The BAT Group of the Nordic Council of Ministers has decided to conduct a project on Best Available Techniques (BAT) for the foundries and smitheries in the Nordic countries. No smithieries satisfies the criteria of the Industrial Emissions Directive (IED) and therefore this report focuses only on foundries. The objectives when compiling this report have been to:  Provide an overview of the foundry sector in the Nordic countries.  Provide an overview of the foundry sector in the Nordic countries.  Present currently used and potential environmental techniques in foundries in the Nordic countries.  Present the Key environmental issues with the perspective of foundries in the Nordic countries.  Present and describe techniques that shall be included in the considerations of representing BAT in foundries. The scope of the work has been to include foundries using permanent and non- permanent moulds. The focus has been on processes specific for the foundry industry, from simulation of the casting to cast products, and to a minor extent some general processes of significant relevance to casting. The provided information can be utilised by operators, environmental consultants and environmental authorities. The projects might also be useful as a Nordic input to the Technical Working Group in the revision of the BAT conclusions for the “Smitheries and Foundries Industry” according to the IED. Metal casting has a long tradition and it has previously been a large industry in the Nordic countries but the number of foundries has decreased. Today Sweden has the largest number of foundries in the Nordic countries. Nordic Foundries Summary The key environmental issues (KEI) in the scope of this study are (in non-prioritised order) The key environmental issues (KEI) in the scope of this study are (in non-prioritised order)  Air emissions (dust and VOC)  Noise emission and vibrations  Emissions to water and water use  Chemical use and risks  Waste/residues  Energy use  Raw material  Transports Presented BAT at foundries describes  BAT for control of air emissions  BAT to prevent noise emissions and vibrations  BAT for control of wastewater emission  BAT High pressure die casting  BAT sand and binders  BAT for chemicals handling and substitution  BAT for waste management and minimisation  BAT for construction simulation  BAT for energy  BAT metal and melting  BAT for environmental management Technical applicability, economic cost and environmental benefits from the different BAT varies widely and is strongly case dependent. It is also worth stressing that due to processes, products, current foundry design and if it is an old existing or new foundry the foundries are not always free to choose BAT. Presented BAT candidates are listed below. 10 Nordic Foundries BAT Candidates Emissions to Air BAT Candidates Emissions to Air  Bag-house filter  Pressure drop gauge  Dust filter measurements  Filter heating  Bag-house filter  Pressure drop gauge 10  Using fluorescent powder to check for problem in bag-house filter  Particle monitor  Acid scrubber to clean amine  Oil mist filter  Plan for reduction of VOC  Lid on coating vessel  Use coating only when needed  Coal Dust replacement  Cleaning VOC and odour with biofilter  Cleaning VOC odour with RTO BAT Candidates Noise emission  Tipping scrap indoors  Noise reducing plan  Noise simulation  Silencers on ventilation systems  Additional insulation of fans and ducts BAT Candidates Chemicals Chemicals responsibility BAT Candidates Waste and residues  Recycling management  Equipment for mechanical sand regeneration  Reuse of rest sand outside the foundry  Bricketing BAT Candidates Simulation  Simulation  Optimized inlet and gating system  Using fluorescent powder to check for problem in bag-house  Particle monitor  Acid scrubber to clean amine  Oil mist filter  Plan for reduction of VOC  Lid on coating vessel  Use coating only when needed  Coal Dust replacement  Cleaning VOC and odour with biofilter  Cleaning VOC odour with RTO BAT Candidates Noise emission  Tipping scrap indoors  Noise reducing plan  Noise simulation  Silencers on ventilation systems  Additional insulation of fans and ducts BAT Candidates Chemicals Chemicals responsibility BAT Candidates Waste and residues  Recycling management  Equipment for mechanical sand regeneration  Reuse of rest sand outside the foundry  Bricketing BAT Candidates Simulation  Simulation  Optimized inlet and gating system  Simulation  Optimized inlet and gating system Nordic Foundries 11 BAT Candidates Energy  Energy audit  Blasting raw material before melting  Crushing inlet and gating system  Insulated ladles  Use of lid on furnace and ladle  Flameless ladle heating BAT Candidates Metal and melting  Scrap handling  Using deoxidiser  Flushing ladle with argon  Inoculation control  Automatized in-stream inoculation BAT Candidates High and Low pressure die casting  Maintenance of die tool  Preheat die tool  IR camera  Use of vacuum dosing furnace BAT Candidates Management and general  Circular economy  Material Flow Cost Accounting (MFCA)  Management system  Digitalization  Environmental communication BAT Candidates Energy Nordic Foundries 12 Emerging techniques  New release agent for High pressure die casting  Ignition of VOC at the sand mould  New inorganic sand binder  Reclaim green sand to core production  New release agent for High pressure die casting Nordic Foundries Nordic Foundries 13 Preface The Nordic Council of Ministers, the BAT Group under the Working Group for sustainable consumption and production, has commissioned to the Swedish foundry and casting research institute Swerea SWECAST AB to prepare a Best Available Techniques (BAT) report for foundries in the Nordic countries. The information provided by this report is intended to be used by operators, environmental consultants and environmental authorities with relevant competence. This report may also be used as Nordic input for the Technical Working Group in the revision of the BAT conclusions for the “Smitheries and Foundries Industry” according to the Industrial Emission Directive (IED). The foundry sector is very complex with a lot of different metals, different ways of melting, different ways to do the casting. It is difficult to make one report describing all different variants and this report is therefore a simplified picture of the sector. 46 potential BAT candidates and an three emerging technique are included in the report which addresses measures to reduce air emissions, noise emission and production of waste at foundries. This work couldn’t have been done without all the foundries in the Nordic countries that have answered a lot of questions. Thanks for all help. The following consultants have contributed to the report:  Martin Wänerholm, Swerea SWECAST AB (Project Manager).  Martin Wänerholm, Swerea SWECAST AB (Project Manager). Support team: Support team:  Henrik Borgström, Swerea SWECAST AB (Sweden)  Håkan Fernström, Swerea SWECAST AB (Sweden)  Lennart Elmquist, Swerea SWECAST AB (Sweden)  Lennart Sibeck, Swerea SWECAST AB (Sweden)  Marie Fredriksson, Swerea SWECAST AB (Sweden)  Markus Börrisson, Swerea SWECAST AB (Sweden)  Raul Carlsson, Swerea SWECAST AB (Sweden)  Roger Svenningsson, Swerea SWECAST AB (Sweden)  Sten Farre, Swerea SWECAST AB (Sweden)  Ulf Gotthardsson, Swerea SWECAST AB (Sweden)  Niels Skat Tiedje, Technical University of Denmark (Denmark)  Juhani Orkas Aalto University (Finland) The BAT project has been followed on by the Nordic BAT Group. The members of the BAT Group are:  Kaj Forsius, Finnish Environment Institute.  Susanne Särs, Environmental and Health Protection Agency of the Aland Islands.  Birgitte Holm Christensen, Danish Environmental Protection Agency.  Anne Kathrine Arnesen, The Norwegian Environment Agency.  Maria Enroth, Swedish Environmental Protection Agency.  Kristina Svinhufvud, Swedish Environmental Protection Agency.  Sigurdur Ingason, Environment Agency of Iceland.  Lena Ziskason, Environment Agency of Faroe Island.  Kaj Forsius, Finnish Environment Institute. Preface  Kaj Forsius, Finnish Environment Institute.  Kaj Forsius, Finnish Environment Institute.  Susanne Särs, Environmental and Health Protection Agency of the Aland Islands.  Birgitte Holm Christensen, Danish Environmental Protection Agency.  Anne Kathrine Arnesen, The Norwegian Environment Agency.  Maria Enroth, Swedish Environmental Protection Agency.  Kristina Svinhufvud, Swedish Environmental Protection Agency.  Sigurdur Ingason, Environment Agency of Iceland.  Lena Ziskason, Environment Agency of Faroe Island. Nordic Foundries Nordic Foundries 16 Introduction The current EU BREF documents are from 2005 and are focused on smitheries and foundries. The first part of this project was to investigate which foundries in the Nordic countries meet the requirements of the industrial emissions directive IED and if there are any smitheries that meet the requirements. When it comes to smitheries the requirement is “operation of smitheries with hammers the energy of which exceeds 50 kilojoules per hammer, where the calorific power used exceeds 20 MW.” Industry associations for forging, authorities in Nordic countries, and a number of possible smitheries were contacted. There are a number of forging facilities that meet the first requirement that the hammer energy exceeds 50 kilojoules per hammer, but no smitheries have been identified that at the same time fulfil the demand for calorific power used. It cannot be excluded there may be some companies in the Nordic countries that meet both requirements, but such has not been identified in this project. After this finding, it was decided that the rest of this work would focus solely on foundries. The way to find out which foundries are affected by the directive was made in the same way as for forging plants. Contact was made with industry associations and authorities and lists of company names were compared. The different sources listed almost the same companies, but the authorities’ lists generally contained some more companies than the industry associations’ lists. Among other things, there were some companies that cast but not melt the metal, companies only melting and companies no longer existing. On the industry associations’ lists were some companies that were not on the authorities’ lists. This may give reasons for the authorities and industry associations in each country to meet to discuss this discrepancy, and to obtain consensus. List of abbreviations BAT Best Available Techniques BREF Best Available Technology Reference Document EMS Environmental management systems HPDC High pressure die casting IED The Industrial Emissions Directive (2010/75/EU) KEI Key environmental indicators LPDC Low pressure die casting RTO Regenerative Thermal Oxidation VOC Volatile organic compounds XRF X-ray fluorescence BAT Best Available Techniques BREF Best Available Technology Reference Document EMS Environmental management systems HPDC High pressure die casting IED The Industrial Emissions Directive (2010/75/EU) KEI Key environmental indicators LPDC Low pressure die casting RTO Regenerative Thermal Oxidation VOC Volatile organic compounds XRF X-ray fluorescence BAT Best Available Techniques BREF Best Available Technology Reference Document EMS Environmental management systems HPDC High pressure die casting IED The Industrial Emissions Directive (2010/75/EU) KEI Key environmental indicators LPDC Low pressure die casting RTO Regenerative Thermal Oxidation VOC Volatile organic compounds XRF X-ray fluorescence Methodology The companies identified received a questionnaire about techniques used today, if the company itself considers this to be BAT, or if there is knowledge of some better technology. This questionnaire is in an annex to this report. The questionnaire was followed up with more questions to the companies that were considered to be in the forefront of technological development, based on their questionnaire replies. Swerea SWECAST is a research institute based in Sweden specializing on foundry and casting. Experts in different fields and subjects within the institute have thereby also contributed with facts and knowledge about different steps in foundry techniques and the casting process. The combined knowledge from questionnaires, interviews and expert input has been used to produce the list of BAT. Moreover, benchmark and harmonization discussions have been held with partners in Denmark and Finland. The final decision of what to be KEI and BAT was made by the experts. Some of the BAT listed needed a more detailed explanation. This report describes techniques used including technology but also way of working that can be at least as important. 1.1 Finland There are 30 foundry members in the Association of Finnish Foundry Product Industries. Foundries are usually small and medium size with wide customer base and wide variety of cast products and materials. Typical castings are cast components to diesel motors, paper machines, telecommunication, heavy vehicles (tractors, earthmovers, forest machines), lifts and power production applications. The production of iron and steel castings 2016 was 58,000 tons and non-ferrous metals 5,000 tons. Finnish foundries are employing 1,600 people. Number of IED foundries 10–15. 1.2 Åland Island There is no foundry relevant to the IED on the Åland Islands. The objectives The objectives when compiling this report have been to: The objectives when compiling this report have been to:  Provide an overview of the foundry sector in the Nordic countries.  Present currently used and potential environmental techniques in foundries in the Nordic countries.  Present the Key environmental issues with the perspective of foundries in the Nordic countries.  Present and describe techniques that shall be included in the considerations of representing BAT in foundries. Nordic Foundries Nordic Foundries 20 1.6 Iceland There is no foundry relevant to the IED on Iceland. 1.4 Norway The foundry production in Norway 2016 was nearly 30,000 tons iron. 50% of this was sold on export. For aluminium the production was just over 6,000 tons and 90% of this was sold to other countries. The statistics is based on 17 reporting members in the Norwegian foundry association. The total number employee is 700–800. Number of IED foundries 5–10. 1.3 Denmark The Association of Danish Foundries, (Danske Støberiers Brancheforening) has 14 member companies (7 ferrous and 7 non-ferrous foundries). Not all of these foundries are IED. The members were in 2015 employing 1,144 people, and the produced tonnage was 78,000 ton of iron-castings and 4,000 tons of non-ferrous castings (total 82,000 tons). Produced parts are mainly for following sectors: Pumps, Agriculture, Automotive, Marine, Off-shore, Hydraulics. Outside the Association of Danish Foundries are a number (5 or 6) of HPDC companies working mainly in Al-alloys. The largest is the in-house plant at Grundfos. There is also one relatively large producer of Zn parts, Linimatic. Further there are several small (1–2 employees) founders that mainly serve artists and museums. These are not operating on industrial scale. Number of IED foundries 5–10. 1.5 Sweden In Sweden there are total of 100 foundries, 40 casting iron or steel and 60 casting nonferrous metal. Foundries are usually small and medium size with wide customer base and wide variety of cast products and materials. Swedish foundries are employing 8,000 people. The production in 2016 of iron castings was 210,000 tons, steel 21,000 tons, aluminum and magnesium 47,000 tons, copper 4,000 tons and zink 8,500 tons. The numbers is based on statistics from the Swedish Foundry Association. Number of IED foundries 15–20. Number of IED foundries 15–20. 1.7 Faroe Islands There is no foundry relevant to the IED n Faroe Islands. Nordic Foundries 22 2.1 Finland The IED is implemented in Finland as part of the renewed Environmental Protection act (527/2014) and Decree (713/2014). Environmental permits for IED companies are reviewed case by case and shall fulfil the BAT conclusions under the IED. 2. Brief regulatory review The BREF document on Smitheries and Foundries Industry will soon be revised according to the IED. The current BREF is from 2005. Annex I of the IED includes: Annex I of the IED includes:  “Operation of smitheries with hammers the energy of which exceeds 50 kilojoule per hammer, where the calorific power used exceeds 20 MW.”  “Operation of ferrous metal foundries with a production capacity exceeding 20 tonnes per day.”  “Melting, including the alloyage, of non-ferrous metals, including recovered products and operation of non-ferrous metal foundries, with a melting capacity exceeding 4 tonnes per day for lead and cadmium or 20 tonnes per day for all other metals.” 2.2 Åland Islands Åland has its own provincial laws in a number of important areas based on its autonomous position. The environmental licensing for industrial plants is outlined in the Provincial law on environmental protection (ÅFS 2008:124, changed ÅFS 2015:14) and decree (ÅFS 2008:130, changed ÅFS 2015:15). 2.4 Norway The IED is implemented in Norwegian legislation by the Pollution Regulations and Waste Regulations. Environmental permits for IED companies are reviewed case by case and shall fulfil the BAT conclusions under the IED. 2.6 Iceland The environmental regulations for industry licensing are based on the IED directive since the first of June 2017, when IED implemented in Iceland. 2.5 Sweden The regulations of the IED are implemented in Swedish law by general binding rules, mainly in the Ordinance on Industrial Emissions (2013:250). The General Rules of Consideration and the provisions on the licensing process in the Swedish Environmental Code are not changed due to the implementation of the IED. BAT conclusions are implemented as a parallel system through general binding rules which are recurrently updated due to the publication of new BAT conclusions. The environmental licensing for industrial plants outlined in the Swedish Environmental Code is based on case-by-case assessments of environmentally hazardous activities and taking into account the local conditions. Licensing of large and medium-size installations takes place by courts or County administrative boards. 2.3 Denmark IED is implemented in Denmark and included in the Executive Order for environmental permitting of companies (BEK nr 1517 af 07/12/2016) which was made effective recently and is a part of the environmental regulation system. Environmental permits for IED companies are reviewed case by case and shall fulfil the BAT conclusions under the IED. 3.1 Introduction In metalworking, casting means a process, in which liquid metal is poured into a mould that contains a hollow cavity of the desired shape. When the shape is filled, the metal is allowed to cool and solidify. The solidified part is ejected or broken out of the mould to complete the process. Casting is most often used for making complex shapes that would be difficult or uneconomical to make by other methods. There are a number of methods for casting, but these can be generally divided into permanent and non- permanent moulds. To determine which casting method to apply, the first step is to determine the technical requirements on the product one wants to cast, and from this determine the appropriate metal. Choice of metal then to some extent controls the casting method to be used. It is therefore not relevant to say that one casting method is BAT and another is not. However, within each method there is good or less good practice. The design of a product and the choice of casting method are often outside the foundry’s influence and therefore not discussed more in this document. Faroe Islands The Faroe Islands have their own laws in a number of important areas based on their autonomous position. The environmental legislation for industrial plants is outlined in the Act on environmental protection from 1988. Nordic Foundries 24 3. Casting 3.2 Non-permanent moulds Non-permanent moulds are often based on sand. When producing a sand mould a first important step is to make a simulation of the casting. There are a number of different parts of the process that can be simulated, but the most common and most necessary is to simulate how the mould should be designed for the filling of the metal to be as good as possible, to avoid cavities or pores that can result in poor quality of the casting. The experiences from these simulations may even influence the design of the cast product. Based on the design and simulation a model of the cast product is made. The model is often made of wood, metal or plastic. Generally the foundry does not make this model themselves. Therefore, it is considered outside the scope of this document to discuss what is BAT for model making. Figure 1: Part of a model made of wood One common method for mould making is to place the model in an iron frame, mould flask, and pack sand around it. Cavities are created in the finished casting by placing a sand core in the mould. For the sand in the mould and in the core to hold together different types of binders used. When the model has formed an imprint into the sand the model is removed and reused for the next mould. Figure 1: Part of a model made of wood One common method for mould making is to place the model in an iron frame, mould flask, and pack sand around it. Cavities are created in the finished casting by placing a sand core in the mould. For the sand in the mould and in the core to hold together different types of binders used. When the model has formed an imprint into the sand the model is removed and reused for the next mould. One common method for mould making is to place the model in an iron frame, mould flask, and pack sand around it. Cavities are created in the finished casting by placing a sand core in the mould. For the sand in the mould and in the core to hold together different types of binders used. When the model has formed an imprint into the sand the model is removed and reused for the next mould. 3.2 Non-permanent moulds Figure 2: Mould flask waiting to be filled with sand Figure 2: Mould flask waiting to be filled with sand Nordic Foundries 26 Figure 3: Core placed in a mould of green sand Another way is to print the sand mould in a sand printer with additive manufacturing. This is a new method and there is no need for a wood or plastic model which shortens the time from idea to finished casting. It is also possible to make more complicated moulds then with normal sand moulds. Today the method is mostly used for prototypes and not series production. Figure 3: Core placed in a mould of green sand Figure 3: Core placed in a mould of green sand Another way is to print the sand mould in a sand printer with additive manufacturing. This is a new method and there is no need for a wood or plastic model which shortens the time from idea to finished casting. It is also possible to make more complicated moulds then with normal sand moulds. Today the method is mostly used for prototypes and not series production. Another way is to print the sand mould in a sand printer with additive manufacturing. This is a new method and there is no need for a wood or plastic model which shortens the time from idea to finished casting. It is also possible to make more complicated moulds then with normal sand moulds. Today the method is mostly used for prototypes and not series production. Figure 4: Sand printer at Swerea SWECAST Figure 4: Sand printer at Swerea SWECAST Nordic Foundries Figure 5 is a simple picture of foundry using sand. Text in red is environmental impacts described in chapter 4. Figure 5 is a simple picture of foundry using sand. Text in red is environmental impacts described in chapter 4. 3.2 Non-permanent moulds Figure 5: The foundry non-permanent moulds (example sand) e 5: The foundry non-permanent moulds (example sand) Figure 5: The foundry non-permanent moulds (example sand) Raw material: Sand, Ingot, Alloy metals Scrap, Binder Model Preparing core sand+ binder + coating Pouring the melt to the mould Shake-out Waste: Metal, sand, slag Noise Transport Dust, VOC Remove gating system and further processing Melting Energy use Simulation Transport Emissions air Heat loss Management THE FOUNDRY NON-PERMANENT MOULDS (EXAMPLE SAND) Sand Preparing mould sand+ binder VOC Product Dust Dust, VOC Noise Noise THE FOUNDRY NON-PERMANEN MOULDS (EXAMPLE SAND) THE FOUNDRY NON-PERMANENT MOULDS (EXAMPLE SAND) Management Raw material: Sand, Ingot, Alloy metals Scrap, Binder Preparing mould sand+ binder Pouring the melt to the mould Shake-out Product Nordic Foundries Nordic Foundries 28 Other methods than sand 3.2.1 A special version of casting is a model made of styrofoam, and sand is formed around this. The styrofoam model is never removed but burned when the melt is poured into the mould, which may give rise to some smoke. This is not a very common method in the Nordic countries. Another method is investment casting were a wax model is formed. The wax model is dipped in slurry of sand and binder and formed into a hard shell around the wax. Next, this is heated, and the wax melts and flows out and may be used to form a new model. What remains is a shell where the casting takes place. This is not a very common method in the Nordic countries. Green sand For green sand water, bentonite clay and a carbonaceous material (coal dust) or a replacement for this is added. This is a very common way to make moulds. Other ways to hold the sand together Other ways to hold the sand together There are also several other methods to keep the sand together. One example is vacuum, where a binder is not needed. Creating the vacuum requires much energy. Chemical binders There are also a number of chemical binders. Most chemical binders used consist of organic compounds, but also inorganic compounds are possible. The binder system consists of two different parts that react in the sand to create a fixed mould or core. A binder resembles the curing of plastics; the foundry buys sand and binder and mixes it at site. Inorganic binders are good from an environmental perspective as they do not give rise to emissions to air. But they do not work in all applications. They consist of sodium silicate, which is cured in different ways. Among the organic binders there are a variety of options to choose from. A further alternative is a method wherein the sand already at delivery is coated with a binder. Different types of binders for the sand There are several ways to hold the sand in the mould and core together and most sand foundries use some kind of binder. There are advantages and disadvantages of each type of binder and it is difficult to say which to consider as BAT. Coating To avoid that the sand sticks on the cast product a protective coating is sometimes used. The coating often contains graphite and some kind of thickener dissolved in water or alcohol. It establishes a protective layer between the sand and the metal. When Nordic Foundries 29 alcohol is used, the emissions to air need to be minimized. For water based coating energy-use for the drying of the water instead needs to be minimized. If the foundry ch Figure 6: Coating of mould with a brush. Previously published in Gjuteriet / Gjuteriinformation AB Figure 6: Coating of mould with a brush. Previously published in Gjuteriet / Gjuteriinformation AB oses water or alcohol based coating often depends of technical reasons. oses water or alcohol based coating often depends of technical reasons. The sand is normally circulated and re-used in the foundry several times. But for technical reasons, such as wearing down of the sand grains and contamination with other dusts, a certain proportion of new sand is added at each circulation. This means that the same amount of sand that is added at each circulation also must be sent away from the foundry at each circulation. For reasons of cost, efficiency and waste minimization foundries try to achieve an as high rate of circulation as possible, while avoiding to compromising any quality aspects of the products. For environmental reasons most foundries tries to achieve a good secondary use for the mould sand, so that it not only ends up in landfills. Different sand for example different level of silica can affect what secondary use that is possible. 3.3 Permanent moulds There are several ways to make casting with permanent moulds. Figure 7 is a simple picture of foundry using high pressure die casting. Text in red is environmental impacts described in chapter 4. Nordic Foundries 30 Figure 7: The foundry permanent moulds (example high pressure diecasting) THE FOUNDRY PERMANENT MOULDS (EXAMPLE HIGH PRESSURE DIECASTING) 3.3.1 Gravity die casting Gravity die casting is a repeatable casting process used for non-ferrous alloy par i ll l i i i d b ll Raw material: Ingot, Oil, Alloy metals Waste: Metal, slag, oil Energy use Noise Transport Die tool Water Emission Remove gating system and further processing Melting Energy use Simulation Heat loss Management ( ) Dust, Oil mist Spraying release agent and cooling Pouring the melt to the mould. If HPDC use of high pressure Product Dust Transport Emissions air Noise Noise Dust Management Spraying release agent and cooling Product Transport Noise Transport Gravity die casting 3.3.1 Gravity die casting is a repeatable casting process used for non-ferrous alloy parts, typically aluminium, zinc and copper base alloys. Gravity die casting is a repeatable casting process used for non-ferrous alloy parts, typically aluminium, zinc and copper base alloys. The mould is made of iron or steel. First the mould is preheated and then gravity is used to fill the mould with the liquid alloy. The method is suited to medium to high Nordic Foundries 31 volumes products and typically parts are of a heavier sections than high pressure die casting (see below), but thinner sections than sand casting. A variation of this is a method where the mould is tilted from horizontal to vertical position during filling which gives a better filling. 3.3.3 Centrifugal casting or rotocasting is a casting technique that is typically used to cast thin- walled cylinders or rollers. In centrifugal casting, a permanent mould is rotated continuously about its axis at high speeds as the molten metal is poured. The molten metal is centrifugally thrown towards the inside mold wall, where it solidifies after cooling. Continuous casting Continuous casting 3.3.2 Low Pressure Die Casting (LPDC) is a method used for metals as aluminium and brass where the melt is pressed from below into the metal mould. The advantage is less turbulence that gives a product with better structure and less cassation. The mould is normally made from two steel halves mounted in the casting machine. The metal is melted either in a central furnace and then transported to each casting machine in a movable casting crucible or the liquid metal is transported from a smelter next door to the receiving furnace for future transport by the metal launder system to the holding furnaces next to each caster and casting crucible. There are advantages and disadvantages with both options. LPDC means that molten metal is pressed with low speed into the Mould, leading to a very smooth and sound filling of the space between the two steel halves named cavity. The melt gives off heat to the steel Mould and the metal solidifies. The steel Mould would be cooled either by water or pressure air. The Mould opens and the cast product is taken out. Thanks to low speed filling the casting get very sound with high strength even at thin wall casting by use of sand cores and complicated geometries. There are two basic types of LPDC casting machines; either with movable casting crucible using long fill tubes or fixed casting crucible with movable casting machine using several short fill tubes for continuous casting like the vacuum & pressure riser-less casting. The advantage is a cast product nearly free of porosity with good structure and low cassation. Low Pressure Die Casting Low Pressure Die Casting 3.3.6 Release agent Release agent Continuous casting 3.3.4 In continuous casting the liquid metal is poured through a graphite nozzle forming tubes or metal rods. The method is used for copper and iron. The product is free from porosities, sand and other inclusions. Nordic Foundries 32 High pressure die casting 3.3.5 The most commonly used permanent mould is used for High pressure die casting (HPDC). The HPDC permanent moulds are made of steel, referred to as die tool. HPDC is used for magnesium, zinc, aluminium and some other metals. The die tools are made from two steel halves mounted in a die casting machine. The metal is melted either in a furnace adjacent to each die casting machine, or is melted in a central furnace and are then transported to each machine. There are advantages and disadvantages with both options. HPDC means that molten metal is pressed at high speed into the die tool, leading to an almost instantaneous and complete filling of the space between the two steel halves. The melt gives off heat to the tool parts and the metal solidifies. The tool opens and the cast product is taken out. Thanks to high-speed injection and high injection pressures the melt can be extruded into very thin sections and be able to fill complicated geometries. There are two basic types of die casting machines; hot-chamber machines, and cold-chamber machines. The difference is how the melt is fed into the machine. The two types of casting machines have different areas of application. The principle of cold chamber die casting is seen in Figure 8. 33 Nordic Foundries Nordic Foundries 33 Figure 8: Principle of High pressure die casting. Note: a. The die tool is closed. Melt is poured into the filling chamber. Figure 8: Principle of High pressure die casting. Note: a. The die tool is closed. Melt is poured into the filling chamber. b. The shot piston presses the melt into the die tool cavity with great speed and pressure. The melt is subjected to pressure during solidification. c. When the melt has solidified the tool is opened and the ejector pins eject the finished casting from the movable die halves. Release agent is sprayed in to the halves of the tool. The tool is shut and a new cycle begins. Release agent 3.3.6 The die tool must be lubricated between each shot with a release agent, normally water with a small amount of oil or wax in it. There are some new methods that use a powder instead of water and oil but this one is not common today. The lubrication of the die tool is important both to achieve the intended casting quality, and to keep the lifetime of the die tool. The lubrication also has some effect on the cooling. The lubricant coating facilitates the form filling and it acts as an insulator between the casting alloy and the tool surface. Nordic Foundries 34 Vacuum 3.3.7 Some foundries now use a new type of HDPC, in which the melt is both pulled in by vacuum while also pushed by high pressure into the melting chamber. This combined force provides a more accurate filling, and also reduces oxygen to dissolve into the melt, which otherwise results in air bubble inclusions into the component, and consequential scrapping of goods. 3.4.2 Cupola furnace Some very large foundries use a cupola furnace. The construction of a conventional cupola consists of a vertical steel shell which is lined with a refractory brick. The charge consists of alternate layers of the metal to be melted, coke fuel and limestone flux. The fuel is burnt in air which is introduced through tuyeres positioned above the hearth. The hot gases generated in the lower part of the shaft ascend and preheat the descending charge. The cupola furnace is used for melting iron. The advantages are continuous melting, high melt rates, relatively low operating cost and ease of operation and there is no need for clean or dry raw material. . The disadvantages are the larger emissions than from induction furnace. 3.4 Melting and pouring Metal is melted in a furnace. There are several variants, and it is difficult to say that a certain type of furnace is BAT as it depends on a number of factors which furnace is used. Some furnaces use electricity and other use burning gas or coal which of course gives different emissions. Induction furnace The most common furnace for melting iron and steel is an induction furnace. In an induction furnace, the metal charge material is melted or heated by current generated by an electromagnetic field. 3.4.8 Holding furnace Sometimes the melt is poured from the melting oven to a holding furnace until all is set for pouring the metal in to mould. Sometimes the melt is also transported in heated metal launder system. 3.4.5 Electric resistance furnace Furnace only used for non-iron foundries. Common in small foundries. 3.4.6 Other types of furnace There are also other furnaces where electricity or burning gas is used. 3.4.7 Some foundries buy the melt on thermos directly from a smelter. Then there is no melting at the foundry, no energy consumption of melting and no air emissions from melting at the foundry but at the smelter. But on the other side there is a need of transportation of the melt. It is therefore difficult to say if this is BAT or if it is BAT to melt at the foundry. It depends on the performance of the melting facility and the actual distance for transportation. The aspect should be dealt with by the technical working group revising the BAT conclusions under the IED. Electric arc furnace 3.4.3 Electric arc furnace is used for melting steel. The furnace generally consists of a cylindrical steel shell which is lined with acid or basic refractories. The roof which can normally swing away to facilitate charging, generally contains three carbon electrodes operating on a high tension three-phase power supply. These electrodes protrude vertically through the roof and an electric current passes directly through them and into the metal bath. The distance between the electrodes and the metal bath is automatically controlled and determines the power input into the bath. The furnace have a lot of advantages. The disadvantages are the larger emissions than from induction furnace. Nordic Foundries 35 Shaft furnace 3.4.4 A furnace of upright form. The heating is made by burning gas. A furnace in the form of a vertical cylinder that is charged at the top and tapped at the bottom in which hot gas is forced upwards through the contained solids. It gives a good melt and is energy efficient as the combustion gases heat the scraped / unmelted metal on the way down to the melting chute and oxides from the scrap can be removed from the melting chute (at least in newer ovens). In smaller foundries, resistive furnaces are probably still dominant. 3.5.1 Sand 3.5.1 Sand When the casting is completed the sand is removed in a shake-out step. It is then important that the sand binder allows the sand to easily come off from the cast product. For the casting made in a permanent mould with sand core a core knock out and shaking units would be used to get a fast removal of the sand. The sand is usually reused to new moulds in the foundry and the casting is allowed to cool. A sand reclaiming unit will be used, this is either mechanical or using heat. 3.4.9 To ensure that the melt is distributed properly in the mould a so-called inlet and gating system is used. For most casting except HPDC the melt is poured into a ladle, which in turn is used to pour the molten metal into the finished moulds. In some foundries the melt is transferred from a larger ladle to a smaller ladle before the pouring into the mould. To compensate for the heat loss at each intermediary pouring it is common to start with an overheated melt. At each intermediary pouring there is a risk that oxygen dissolves into the metal. This can corrupt the properties of the cast component and give rise to higher cassation. This especially concerns steel. Nordic Foundries Nordic Foundries 36 3.4.10 Slag 3.4.10 If there is contamination in the melt, such as for example remainders of sand or dissolved gases, these normally float up to the surface of the melt, forming a slag. This slag is removed and later turns up as waste and scrap from the foundry. When pouring iron and steel and to avoid that the slag follows the melt into the mould a slag filter is normally used. Another way is to use a ladle which is emptied through the bottom of the ladle because the slag is floating on top of the melt. 3.5.2 General The resulting product from the casting procedure is generally a larger product than the metal component intended. Due to production necessities, the pouring of the molten metal, the gating system is added to the metal component. These have of course also solidified together with the component, and they need to be removed. Since they are pure metal, they can be removed and be melted again. After removal of gating system, the component is processed to remove sharp edges or sand that has stuck to the surface and to obtain its intended surface. Blasting is sometimes used. The resulting product from the casting procedure is generally a larger product than the metal component intended. Due to production necessities, the pouring of the molten metal, the gating system is added to the metal component. These have of course also solidified together with the component, and they need to be removed. Since they are pure metal, they can be removed and be melted again. After removal of gating system, the component is processed to remove sharp edges or sand that has stuck to the surface and to obtain its intended surface. Blasting is sometimes used. Small products of aluminium go to a barrel polish rotating with water and plastic or ceramic chips to get a good surface. From this process there is a sludge of aluminium particles and rest from the chips, this is sent away as waste. Some foundries also have a more advanced mechanical processing with drilling or grinding. This is more like a mechanical industry and not described more in detail in this document. But if the foundry has this process there is often metal chips that will be melted again in the process. There can be a rest of oil on the metal chips. Only some few foundries conduct surface coating, such as painting, which is therefore not further discussed in this report. For many casted products you use heat treatment to give the right properties. Sometimes this is done inside the foundry, sometimes the product is sent away. 37 Nordic Foundries 37 4. Key environmental issues (KEI) and environmental impact of foundry and casting The KEI in the scope of this study are (in non-prioritised order):  Air emissions (dust and VOC)  Noise emission and vibrations  Emissions to water and water use  Chemical use and risks  Waste/residues  Energy use  Raw material  Transports  Air emissions (dust and VOC)  Noise emission and vibrations  Emissions to water and water use  Chemical use and risks  Waste/residues  Energy use  Raw material  Transports In the next chapter, BAT are described within each of the listed KEIs. The environmental impacts for every KEI are listed below. These questions are often handled in some kind of management system for example ISO 14001. 4.1.1 Particles and dust A number of processes in a foundry cause emissions of dust and particles to air. The melt causes emissions of dust that contain metal oxides. The melt may also be treated with various additives, such as when adding magnesium to establish the properties needed for producing ductile iron. This addition results in a rather heavy momentary smoke of small particles. There is a lot of different way of treat the melt with magnesium and it is difficult to say what system is BAT. But often which method that is used depends on technical demands on the product. Other dust and particle sources are smoke associated with casting, dust from the shake-out when the sand moulds are opened and when the is removed from the castings, metal-containing dust from cleaning when inlet and gating system and sharp edges are removed, and dust from the final blasting of the components, that contains a mixture of metal and sand. For example it is difficult to collect all air from the shake-out, especially large cast components. Sometimes the emissions from the melt are not cleaned probably because the risks of sparks from the melt will end up in the filter and cause a fire. But cyclone can be used before the filter to prevent sparks to get into the filter. High pressure die casting High pressure die casting use release agents which normally consists of some percent oil or wax and the rest water, leading to an emission of oil mist. Sometimes there is a diffuse leakage of the oil mist leaving via the general ventilation. Low pressure die casting Low pressure die casting use Mould coating which normally consists of some percent graphite or silicate and the rest water, leading to a small emission. When use of sand core there is a diffuse leakage of phenol mist leaving via the general ventilation. 4.1.2 Foundries have some emissions of volatile organic compounds VOC. Some of these may be harmful and some may merely cause annoying odours for local residents. Sand casting Foundries using sand also have air emissions of VOC, mainly originating from the organic binders used to manufacture moulds and cores. When the hot melt is poured into the moulds many of the chemical substances contained in the binder are released or generated due to binder decomposition. One share of the chemicals goes immediately to the air during the casting, and some is released at the shake-out when the sand is removed from the product. Common air emissions resulting from organic binders are benzene and formaldehyde. Which substances that are actually released, and in which amounts, depends on type of binder used. For green sand there is carbonaceous material added and this can react and give rise of emissions. For one special binder system frequently used for the cores an amine is used as a catalyst for the chemical reaction of the binder. The amines used generally have a very unpleasant odour and the emissions require cleaning. Other binders also give rise to VOC in various degrees. The exception is the inorganic binder with sodium silicate hardened with carbon dioxide that from an environmental viewpoint is a very good binder and does not give rise to emissions of VOCs. But for technical reasons, it is not possible to use this binder in production of all kind of products. Other air emissions from sand foundries are associated with coating. The coating is added to get smoothen the surface between the sand and metal, and also to minimize the amount of sand that sticks to the metal. There are two types of coating, water or alcohol-based, which often have a content of isopropyl alcohol. Water based coatings would not give any VOC emissions but has other drawbacks, both technically and with Nordic Foundries Nordic Foundries 40 regards to energy needed for drying the coating. The water-based coating also contains biocides which may be a problem for the work environment. It is therefore not safe to say that the water based coating is simply better. There are different ways to attach the coating to the sand. Small surfaces are painted manually but larger surfaces may be spray painted or dipped into the coating in large vessels. Isopropanol is evaporating from the coated sand, normally this is burnt off. 4.3 Emissions to water and water use Foundries using sand rarely have significant amounts of contaminated process water. Foundries using high pressure die casting use a release agent that is sprayed on the tools between each shot. Today usually the release agent is composed of water with a small amount of oil or wax. Much of this evaporates but there will still be some oily water that flows down from the machines and have to be taken care of. Foundries using low pressure die casting and cooling of mould with water, often are using a closed water system and only a small amount of used water will go through oil separator before going to the public draining system. 4.2.3 The most common cause of noise nuisance for neighbours are otherwise fans and ventilation on the foundry roof. Other activities 4.2.4 There are also other activities causing noise for example blasting with steel sand or dry ice-blasting. For aluminium foundries barrel polishing give a high noise level. 4.2.2 4.2.2 The casting process itself is not so loud. The casting process itself is not so loud. The problem that can arise is that it is often very high temperatures inside a foundry and to get a decent working environment gates or windows are often open, which lead to noise from inside can spread into the surroundings. This is also a waste of heat that could been taken care of. Nordic Foundries Receiving the raw material There are several noisy operations in foundries and vibrations can also occur. Unloading of incoming metal scrap is often done by simply tipping the scrap off the truck, resulting in a very loud instantaneous sound. Foundries using aluminium normally receive their raw material as ingots and therefore have a more controlled unloading. Figure 9: Aluminium ingots Transport to and from the foundry can also be a source of disturbance. Sand foundries receive truck loads with sand that blows new sand into a sand silo. Such filling also causes noise disturbances. Figure 9: Aluminium ingots Transport to and from the foundry can also be a source of disturbance. Sand foundries receive truck loads with sand that blows new sand into a sand silo. Such filling also causes noise disturbances. Nordic Foundries 41 4.4 Chemicals use and risks Foundry industry is not chemical-intensive. However, there are some chemicals, mainly oils for machines release agents for high pressure die casting and binder to the sand. Some foundries also have their own metal processing with lathes and drilling, but this is not considered to be specific for the foundry industry and is therefore not addressed specifically in this report. The binders to the sand often consist of two different components mixed in the sand. These can react very intensive with each other if mixed outside the sand. For the LPDC process the ultrasonic cleaning is sometimes used for mould by use of caustic soda, which will be handled in closed system. The waste will be treated as hazardous waste. Some metals are complicated to handle. For example molten magnesium where there is a risk of fire and explosions. Therefore magnesium melt always is protected with 42 a protective gas. Before it was SF6 but this will not be used in the future because it is a greenhouse gas. Today SF6 is replaced by several other gases but most common is SO2. There are waste/residues of foundry production. There are waste/residues of foundry production. There are waste/residues of foundry production. The melt may contain oxygen and impurities from scrap or sand. These impurities are often floating to the surface and form a slag. The most common for iron and steel foundries is that the slag will be placed in a landfill, for which the foundry will need to pay a fee. When the slag is to be removed from the surface of the molten metal is always a risk that some pure metal is included. The slag from for example aluminium foundries is sent back to larger smelters. In sand foundries most of the sand is being internally recycled. Technical reasons however require that a certain amount of new sand continuously is added so that that basically the same amount of excess sand is generated as waste. Nordic foundries recycle about 6 million tonnes and the total waste generated is in the order of 400,000 tonnes of sand residue. Today about 65% of sand waste is used to build cover constructions on landfill, but the landfill sites will all be covered in the near future. So, instead of being used the sand will become waste. It is also important to take into account the effect of organic/inorganic material from for example binder etc. on the utilization of sand waste. From high pressure die casting there is some oil contaminated water that may be purified or sent away as waste. This comes mainly from the release agent sprayed on the tools between each shot, but also various types of oils to high pressure die cast machines. Other residues resulting from foundries is material t collected in cleaning equipment for air, such as fabric filters, containing dust with metal or sand or a mixture of the two or residues from cleaning the air from gas for example amine or SO2 used for some binders. There are also small amounts of binders and oils sent away as waste. 4.7 Raw material The foundries use a lot of different metals to make the optimal alloy. Some are not good from environmental point of view for example lead used in bronze, lead is also often a contamination in aluminium. Some other metals are very rare or come from areas with conflicts. The metals can be recycled again and again. Iron and steel foundries uses metal scrap and other foundries using recycled metals. This is very good but the metals are not always used in an optimal way. For example sometimes a foundry makes a very specific alloy with special metals. When recycled the specific alloy ends up at a foundry that don’t want these special metals in their casting. Instead they “dilute” the metal with clean metal to lower the percentage of the unwanted metal. 4.6 Energy use One of the foundries’ most important environmental aspects is the use of energy. There are many places where the input energy is lost as heat, for example when lids are not used on furnace and ladles. Energy savings can also be made on support processes, such as on air compression systems. In this report, however, the focus is on the energy- consuming steps that are specific for the foundry industry and not on the support systems. Wasting energy is both an economic and an environmental loss for the foundry. The choice of source for the electricity is of course important. A foundry normally uses 13–16% of the energy for heating, 8–9% for ventilation, 5–6% for compressed air, 1–4% for cooling, 60% for production process incl. melting and 5–11% for the rest. Nordic Foundries 43 To compensate for the temperature drop during filling and transportation normally melt is overheated in the furnace which needs a lot of extra energy. There is metal residue from the parts resulting from the inlet and gating system. This is often remelted directly inside the foundry but sometimes the foundry sends this away. The amount of inlet and gating system circulated is related to the energy efficiency of the foundry, since large inlet and gating system means that a large amount of non-component related metal needs to be melted. For many casted products heat treatment is used. There is example when a product is heated much longer than necessary. Energy efficiency is connected with material efficiency. Those both categories have to take into account when optimizing the total efficiency of the production. 5. BAT for foundries The overview of BAT at foundries is described in this chapter. Specific BAT candidates, which are selected as the most relevant to consider in preventing environmental impacts from foundries are described in more detail in the following chapters and mentioned with chapter reference in the list below. In chapter 6, the criteria used when identifying BAT candidates are listed. The list contains mostly techniques used in foundries in the Nordic countries but also some techniques from used in other industries or in foundries in Europe but can be used also in the Nordic countries. Potential environmental impacts from foundries, and also possibilities to mitigate the impacts, are strongly case dependent, for a number of reasons. The KEI and the sources of potential environmental impacts from foundries in different situations were discussed in the previous chapter. Some of the techniques described in the report are methods to make a better quality of the casted products. Better quality give less cassation which means less metal needed to be melted again saving energy and emissions. 4.8 Transports Foundries use a lot of transports both for raw material and products but also for waste and finished products. Sometimes the foundry has the influence to choose and manage the transport sometimes it is decided by supplier or customers. Foundries use a lot of transports both for raw material and products but also for waste and finished products. Sometimes the foundry has the influence to choose and manage the transport sometimes it is decided by supplier or customers. Castings from Nordic foundries are often exported all over the world but of course mainly to Scandinavia and the rest of Europe. Nordic Foundries 44 5. BAT for foundries 5.1 BAT for control of air emissions  For the sake of work environment and to avoid spreading dust, use vacuum cleaner instead of compressed air or broom to remove excessive waste sand.  Take measures to secure that any outdoor handling of sand does not generate airborne dust.  If an open cooling tower is used ensure there is no risk for legionella that can be spread to the surroundings. 5.1 BAT for control of air emissions  Use hoods and fans to collect emissions or encapsulate processes that may cause air emissions, such as dust, particles, gases or odours, so that the open air can be effectively cleaned from pollutions.  Work to minimise VOC and odour and if this is not possible use cleaning equipment. Example on cleaning equipment is Biofilter or RTO (this techniques are not used in the Nordic foundries today but in other countries). See 7.9, 7.13 and 7.14.  Design and use ventilation and exhaust hoods properly.  When changes are made to the ventilation systems, review the entire ventilation systems so that it is still works properly.  Use bag-house filter or better technology to clean dust from air. These can give very low emissions (normally 1–4 mg/m3 but requirements from authorities often 5 mg/m3). This cannot be used for all processes. For example foundries using cupola often use a wet abatement technique. See 7.1.  Adapt the filter media so that it is correctly chosen for the type and amount of dust.  Make sure the collection of dust from the filter does not leak or escape when emptying the filter.  Consider whether the dust collected in the filters can be reused or incinerated at some step in the process.  High pressure die casting should use oil mist filter. See 7.8.  Perform regular maintenance and service of filter systems and fans. This reduces the risk of pollution, reduces energy consumption and reduces noise.  Perform regular monitoring and measurement of filter equipment. See 7.3.  Install pressure drop gauges on the filter equipment. Establish procedures that describe how often the gauge should be checked and what is the normal level, as well as measures to take if something deviates from normal. See 7.2.  Use fluorescent powder to identify leakage in fabric filters. See 7.5.  For larger amounts of dust, use particle meter/monitoring that enables control of dust levels between services. There are different variants from simple alerts to system that constantly logs the exact amount of dust emission. See 7.6.  If continuous dust meter is used calibrate them periodically.  If there is a risk that the filter function deteriorates because of cold climate, the filter should be equipped with heating coils. See 7.4. BAT to prevent noise emissions and vibrations 5.2 Many of these things may be handle in a management system see 15.3 and 8.2. Many of these things may be handle in a management system see 15.3 and 8.2.  Try placing noisy activities shielded from nearby homes for example blasting with steel sand or dry ice-blasting.  If vibrations may cause problems, measure vibration impacts at nearby properties.  If there is a problem with vibration, check if the frequency of the vibrating machines can be adjusted or if the vibrating machines can be isolated from the floor.  Control working hours so vibrating activities do not take place when the neighbours are asleep. Figure 10: Forklift Figure 10: Forklift 5.3 BAT for control of wastewater emission Many of these things may be handle in a management system see 15.3 and 8.2.  Unload scrap indoors. See 8.1.  Perform shake-out indoors, as far as possible given the size of the casting.  Perform cleaning indoors, as far as possible given the size of the casting.  For LPDC using sand core, perform de-coring by core knock out indoors.  Measure the noise at nearby properties and if necessary use noise simulation. Especially important after changes in the foundry. See 8.3.  Carry out regular noise rounds, where somebody walks around and listens and measures on the company’s fans, exhausts, etc.  Prioritize regular maintenance of fans, motors, belts, bearings, etc.  Control working hours to minimise disturbances for neighbours.  Turn off fans, etc. when not in use.  Inform staff about noise requirements for the foundry.  Keep doors and windows closed whenever possible.  Measure the noise at nearby properties and if necessary use noise simulation. Especially important after changes in the foundry. See 8.3.  Carry out regular noise rounds, where somebody walks around and listens and measures on the company’s fans, exhausts, etc.  Prioritize regular maintenance of fans, motors, belts, bearings, etc.  Control working hours to minimise disturbances for neighbours.  Inform staff about noise requirements for the foundry.  Keep doors and windows closed whenever possible. Nordic Foundries 46  Educate forklift drivers in quiet driving.  Try placing noisy activities shielded from nearby homes for example blasting with steel sand or dry ice-blasting.  If forklift transports take place outdoors make sure the ground is level and paved.  Limit time slots for transportation.  Keep noise levels as a criterion when purchasing new equipment.  If the above measures do not help, carry out an investigation to identify potentials to reduce noise by installing silencer or shield sources of noise. See 8.4 and 8.5.  If silencer is used it is important to regularly check and clean the silencer if necessary.  If vibrations may cause problems, measure vibration impacts at nearby properties.  If there is a problem with vibration, check if the frequency of the vibrating machines can be adjusted or if the vibrating machines can be isolated from the floor.  Control working hours so vibrating activities do not take place when the neighbours are asleep.  Educate forklift drivers in quiet driving. 5.3 BAT for control of wastewater emission  Clean water from die spraying and other processes like mould cooling producing polluted waste water.  If it is possible reuse the water in some way for example recycling of water from barrel polish.  If contaminated scrap metal is stored outside store it under a roof or otherwise it is important to use cleaning equipment in the water drainage system.  To protect surface water drainage system for example at leakage of chemicals use a lid to the system or a closing valve. Nordic Foundries 47 BAT High pressure die casting BAT High pressure die casting 5.4  Avoid that the melt comes in contact with oxygen in the air for example by using vacuum dosing furnace instead of open scoop to take when taking melt from the furnace to the filling chamber in the machine. It also provides more precise dosing. See 14.4.  Optimize the amount of piston lubrication. Too much lubrication involves the risk that the piston lubrication leaks and comes in contact with the melt resulting in heavy smoke. Too little piston lubrication means the machine is faltering which can lead to lower production and more cassation.  The time from that the melt is taken from the furnace to the melt is inserted in the filling chamber in the machine should be as short as possible.  Automatize spraying of release agents and optimise the amount of release agent  Automatize spraying of release agents and optimise the amount of release agent  Use infrared camera or similar tool to ensure the temperature of the tool and  Use infrared camera or similar tool to ensure the temperature of the tool and adjust cooling accordingly. See 14.3.  Review whether the current release agent can be replaced with new variations.  Preheat the die tools for longer life. See 14.2.  Establish sufficient maintenance of die tools to prolong the lifetime, including also blasting and grinding of the surface of the die tool. See 14.1. Figure 11: Open scoop taking the melt from the furnace. Nordic Foundries 48 BAT sand and binders BAT sand and binders 5.5  If the green sand is used replace the normal carbonaceous material (coal dust) with one that reduces VOC emissions. See 7.12.  If green sand is used try to minimize the amount of carbonaceous material that is used to minimize VOC emissions.  Review the possibilities to change binders to one with a lower environmental impact, such as shifting to a process without binders.  Store the components of the binder system so that they cannot react with each other.  Sand should be reused in the process. For sand with chemical binders the foundry should at least have a mechanical sand reclamation. See 10.2.  Use as little binders as possible. Study and monitor the strength of the cores and moulds to determine how much binder is needed. BAT High pressure die casting  Perform maintenance of the sandmixer to ensure a good mixing and to avoid excessive use of binders.  Use as little sand as possible.  When using a binder system hardened with an amine, clean the air with an acid scrubber. An acid scrubber can give very low emissions normally under 1mg/m3 but the regulations from authorities can vary. This is important both for time when the core is made but also after the core is ready because amine evaporates from core sometime after they are produced. Some foundries also have the core in an oven for about one hour to remove residual gasses. See 7.7.  Investigate if it is possible to decrease the use of coating. See 7.11.  When drying water based coating seek to minimize energy consumption.  If an alcohol based coating is used, use lids on coating troughs. See 7.10.  If a trough with flowing coating is used ensure that the coating can be reused several times.  If an alcohol based coating is used burn the coating on the core. Of course it is important to be aware of risk with the use of open flame.  Optimise the mixing of coating and follow the quality of the coating by for example control the viscosity.  Make sure that the plant internal recycling rate of the sand is as high as possible. The maximum rate depends on type of binder used.  If the sand cannot be recycled internally the sand should preferably be used for some other purpose outside the foundry rather than end up in a landfill. The use of the sand for other purpose is often regulated and there is often a need of a chemical analysis of the content. See 10.3. Nordic Foundries 49  At shake-out try to separate the core sand from the moulding sand. This is particularly important if these have different binders. This may be difficult especially with automatic green sand moulding line.  Do not use cold sand. This is particularly important when using furan as a binder. When using furan sand it is especially important that the sand is also dry. Figure 12: Burning alcohol based coating. Previously published inGjuteriet / Gjuteriinformation AB BAT for chemicals handling and substitution  Correct labelling is important and should be done based on chemical legislation. Especially to avoid mixing the different components in the binder system since this can result in severe chemical reactions.  Have absorbent material available. Sand foundries can use sand.  Assign a person to have responsibility for the management and the control of new chemicals. See 9.1.  Store chemicals so there is no risk for spill to ground or water. Store liquid chemicals in collection containers that will collect any spill. 5.7 BAT for waste management and minimisation 7 BAT for waste management and minimisation The basis of this work is to minimise the up come of waste. The basis of this work is to minimise the up come of waste.  Minimise spilling sand that ends up as waste. Avoid for example spilling sand on the floor.  Maintenance of transport belts and elevators for sand is essential to reduce internal waste. Nordic Foundries 50  Sort waste to facilitate recycling.  Sort waste to facilitate recycling.  Proper sorting of recycled metal can increase the amount that can be recycled. See 10.1.  Avoid to store sand directly on ground outside to minimise dust blowing away in the wind.  If there is sand on the ground outside the foundry use a road sweeper to collect this to avoid dust blowing in the wind.  Investigate the possibilities to use some of the waste as a resource in other contexts. For example sand can be used for construction work, it can also be used to cover old landfills or used in concrete. Slag from iron and steel can sometimes be crushed and used in construction work. 5.8 BAT for construction simulation  Use simulation to optimize the casting process. Things that should be simulated are the flow, thermal solidification, cooling and defects. See 11.1 and 11.2.  Make several simulations to get better result.  Continuously verify the simulations compared to real data.  Properly executed and interpreted simulations reduce the risk of having to scrap castings, and thereby improve the yield.  Design and position inlet and gating system in such way that it can easily be broken off instead of need to use a saw that requires more energy.  When building new foundries or new production facilities it is possible to use production modelling successfully to find bottlenecks and other critical points in the manufacturing systems. Models can vary from relatively simple spreadsheets to use of advanced computer systems.  Close cooperation between buyer and constructor to have a product that is easy to cast which give lower cassation. 5.9 BAT for energy Many of these things may be handled in a management system see chapter 15.3 Many of these things may be handled in a management system see chapter 15.3  Handle raw material indoors to avoid exposing it to moisture.  Take advantage of waste heat from ventilation, cooling water from furnaces or compressors to heat and dry the raw material. Nordic Foundries 51  Pre-dry metal chips which may contain oil or oil-emulsion by for example centrifugation.  Crush scrap and return with a machine for fragmentation or ask the suppliers of scrap to deliver scrap in the right size. See 12.3.  To achieve energy efficiency, establish a short melting time, rapid overheating and immediate casting without unnecessary delay time. Sometimes this is not done because this gives a high cost from the electricity provider.  Pack the raw material as tightly as possible in the furnace.  Press briquettes of metal chips that are used as raw material. The briquettes reduce the charging time compared to loose metal chips and also provide some reduction in energy consumption during melting. Cannot be used for all furnace. Especially interesting for aluminium. See 10.4.  Use lid on the melting furnace, holding furnace and ladle as much as possible. If it’s not possible to use lid on furnace one alternative is to have a layer of slag at the top. See 12.5.  The use of highly insulating lining in ladle greatly reduces heat loss. Highly insulated ladles have possibly inferior mechanical strength so it is doubtful whether they are suitable for treatment of ductile iron. See 12.4.  Plan preheating of ladles and preparation of moulds so that they are ready in time when the melt is ready.  Use lids on the ladle or place it upside down between uses. See 12.5.  Try preheating ladle with newer technology than conventional gas burner for example with a flameless system or electrical heating (Not used in Nordic foundries today but in other countries). See 12.6.  Use the right ladle size with respect to the melt amount.  Make practical tests to find out what it costs to overheat the melt kWh / degree / tonne and make everyone in the personal adware of this.  Use the maximum electric power as early as possible and then throughout the melting process to provide the fastest and most energy-efficient melting process. 5.9.2 Induction furnace  Add easily digestible materials such as pig iron in the bottom of the furnace.  Add easily digestible materials such as pig iron in the bottom of the furnace.  The furnace energy efficiency increases if it has a melt early to work with  Add easily digestible materials such as pig iron in the bottom of the furnace.  The furnace energy efficiency increases if it has a melt early to work with.  The furnace energy efficiency increases if it has a melt early to work with.  Charge the furnace in such a way that the residual heat is utilized. Fill hot furnace at the end of the shift, for example in the evening.  Large amount of slag indicates that improvements can be made, both in terms of energy consumption and metal recovery.  If necessary the charging material should be blasted before use to remove sand and rust. See 12.2. 5.9 BAT for energy Sometimes this is not done because this give a high cost from the electricity provider.  Use meter showing furnace energy consumption for each charge and note this together with temperature and the charge weight for each melt. If the comparison is made with the previous melts, operators can detect the variations in energy consumption and directly reduce energy consumption. Nordic Foundries 52  To avoid excessive melting time it is important to quickly arrive at the right alloy composition of the melt. It is therefore important to weigh the different compositions. Weighing is also important to determine the energy consumption per ton of melt and find energy efficient practices. For monitoring production it is also good to weigh the amount of tapped melt and slag weight.  Measure the temperature often instead of guessing.  Measure the temperature often instead of guessing.  After the use of temperature lance replace the probe tip and let the lance tip drop in temperature before the next measurement.  If there is waste heat from the process, streamline the process first. Once this is done the waste heat should be used either internally or sent to external recipients, alternatively stored for use at a later time.  Recycle heat from the cooling water and from the cooling of castings.  Optimizing heat treatment so that the goods are not heat treated unnecessarily.  Lower temperature if the furnace is not in use and lower ventilation if not needed.  Conduct energy audit. See 12.1.  Use heat exchanger to handle excess heat from the processes.  When collecting heat in the process for reuse, do not mix heat with high temperature (for example directly after pouring) with low temperature (for example at the end of the cooling area).  When collecting heat in the process for reuse, do not mix heat with high temperature (for example directly after pouring) with low temperature (for example at the end of the cooling area). 5.9.3  Make sure to have the correct carbon content from the beginning by keeping track of the carbon content in the various raw materials. To redeem more carbon in the already molten material takes time and often require elevated temperatures. This requires excessive energy use. An iron-carbon alloy has the lowest melting point at 4.3% carbon but often it is the desired end result that controls how much carbon is used.  Calculate which combination that gives the right carbon content in the melt. There is software available for this  Calculate which combination that gives the right carbon content in the melt. There is software available for this. Nordic Foundries 53 5.10 BAT metal and melting 5.10 BAT metal and melting 5.10.1 Iron and steel  Use feeders that do not cause problems in sand contents. For example use of fluorinated feeders can give a higher level of fluorine in the waste sand if the residues of the feeder end up in the sand.  Set quality requirements on purchased scrap. Pure raw material gives less slag. See 13.1.  Inspect the quality and purity of the received scrap.  Optimise the composition, the alloy content and the shape of the scrap.  Use modern techniques and tools to charge optimization, such as computer program for calculating the composition of the charge.  Large ladles for steel can be flushed with argon to effectively minimize the risk of entrapped oxygen within the melt. Entrapped oxygen can cause defects and cassation. See 13.3.  When iron is treated with magnesium to produce ductile iron ensure that added magnesium is kept within the melt.  If possible use a method for producing ductile iron which give less smoke to the air or try to minimise the smoke emitted to the air for example by using methods where the melt is treated in ladle with lid so the smoke can be collected or that the magnesium is added down in the melt and not to the surface.  If inoculation is added directly to the cast beam ensure that the inoculant ends up into the melt and is not somehow ejected besides the melt.  Measure that the right amount of inoculant is in the melt after inoculation. See 13.4.  Use automated inoculation if possible. See 13.5.  When using a cupola furnace add 2–3% extra oxygen, then the amount of combustion gases and the amount of coke can be reduced. 5.10.2 General 5.10.2 General  Calibrate the instrument for measuring temperature.  Use quick and reliable process control of the melt to avoid incorrect mixture that could lead to cassation or unnecessary overdose of substances, such as of magnesium for ductile iron treatment.  Use deoxidisers/degassing to remove oxygen/hydrogen and other gases in the melt to avoid cassation. Can be used for steel and other metals but not used for iron. See 13.2. Nordic Foundries 54  Use only the amount of deoxidiser/degassing agent that is really required to regulate gas levels.  When a holding furnace is used, procedures for correct maintenance are important to avoid oxides that can build up and overtime can influence melt quality. 5.11 BAT for environmental management These techniques are not specific for the foundry industry and sometimes more used in other industry but can help also foundries to work better.  Work with lean manufacturing or similar systems.  Use standardized working operations so all operators use the best method.  Use FMEA (Failure modes and effects analysis) design and production. FMEA means to assess each production step and analyse what can go wrong, the severity if something goes wrong, how often it happens, or can happen, how likely it is that the failure is discovered, as well as to form preventive actions.  Evaluate before new technology is purchased.  Evaluate before new technology is purchased.  Plan internal and external transports and logistics and review the internal process flows.  Use a standardized management system, such as a ISO 9001, ISO 14001, or ISO 50001. See 15.3.  Assess the foundry’s environmental aspects, such as waste management, suppliers’ environmental performance, and apply life cycle thinking of the supply chains (even if it does not include a complete lifecycle assessment).  Develop a policy to establish what environmental decisions are allowed to cost, as well as take environmental aspects into account when purchasing goods and services.  Try to apply circular economy. This may mean trying to extend the lifetime of equipment used throughout the production, by repairing or by improved maintenance. Other examples may be to considering remodelling the business model of the company, to establish a take back system of products for recycling valuable alloys, for product refurbishment, or for establishing an environmentally based relationship with the customers. See 15.1.  Check for other practices, methods, tools and standards that may be useful to improve or assess the environmental performance of the business. Examples are life cycle assessment, described in the international ISO 14040 family of standards, or the ISO 14060 family standards providing guidance about how to measure, mitigate and adapt to greenhouse gases and climate change. See 15.4, and 15.5. Nordic Foundries 55  The environmental manager or coordinator should be supported in their work either internally from different people in the organization or externally.  Use material flow cost analysis MFCA or similar to map the economic costs of spill and wasting of material and energy resources. See 15.2.  Have procedures for all operations, such as for melting, how to buy chemicals, how to handle and manage waste. 5.11 BAT for environmental management  Energy, noise, chemicals and waste are some factors to consider when purchasing new equipment. 56 Nordic Foundries Nordic Foundries 6. BAT Candidates 6. BAT Candidates In the following chapters the identified BAT candidates are described. They were chosen according to the following priorities:  BAT, which addresses the identified Key Environmental Indicators  BAT, which addresses the identified Key Environmental Indicators  BAT, which is assessed to have a significant reduction of emissions and impact  BAT, which is economically and technically viable taking into consideration the cost and advantages E BAT did t i d ib d ith h dli  BAT, which is assessed to have a significant reduction of emissions and impact  BAT, which is economically and technically viable taking into consideration the cost and advantages Every BAT candidate is described with seven headlines: Every BAT candidate is described with seven headlines:  Introduction  Applied process and techniques. Short description of the techniques  Environmental benefits  Applicabiity. Which foundries can use this  Cross media effect If the use of the technique influence something else for  Introduction  Applied process and techniques. Short description of the techniques  Environmental benefits  Cross-media effect. If the use of the technique influence something else for example use of energy  Economics. If there is any information about the cost to use the techniques. This is a very rough estimation  Reference. Some examples of foundries using the technique today. A lot more foundries may use the technique This is a selection of course there can be other techniques that also may be BAT candidates. The technical and economic applicability of BAT candidates differ a lot case-by- case. It is also noteworthy that the selection of techniques is not always in the hands of the foundry. All techniques cannot be used by all foundries. The BAT Candidates have been plotted with their number in green in the following pictures Figure 13 and Figure 14. Figure 13: The foundry non-permanent moulds (example sand) Raw material: Sand, Ingot, Scrap, Alloy Metals, 7.7 Binder Model Preparing core sand+ binder + coating Pouring the melt to the mould Shake-out Waste: Metal, sand, slag Noise Transport Dust, VOC Remove gating system and further processing Melting Energy use Simulation Transport Emissions air Heat loss Management THE FOUNDRY NON-PERMANENT MOULDS (EXAMPLE SAND) Sand Preparing mould sand+ binder VOC Product Dust Dust, VOC Noise Noise 7.1–7.7 8.2–8.5 7.13 8.1 7.14 7.9–7.12 9.1 10.1 10.2 10.3 11.1 11.2 12.1 12.2 12.3 12.4–12.6 13.1 13.2–13.5 15.1–15.5 16.2–16.4 THE FOUNDRY NON-PERMANENT MOULDS (EXAMPLE SAND) THE FOUNDRY NON-PERMANENT MOULDS (EXAMPLE SAND) Management Model Preparing mould sand+ binder Pouring the melt to the mould Product Nordic Foundries 58 Figure 14: The foundry permanent moulds (example high pressure diecasting) Figure 14: The foundry permanent moulds (example high pressure diecasting) Raw material: Ingot, Alloy metals, Oil Waste: Metal, slag, oil Energy use Noise Transport Die tool Water Emission Remove gating system and further processing Melting Energy use Simulation Heat loss Management THE FOUNDRY PERMANENT MOULDS (EXAMPLE HIGH PRESSURE DIECASTING) Dust, Oil mist Spraying release agent and cooling Pouring the melt to the mould. 7.1.1 Introduction Dust abatement by bag-house filters is an effective method to clean particles from gases. Thanks to the research and development of filter media, there is a wide range of different filtering solutions, coupled with other modern components in the filter system, the valves and the electronic equipment. Every BAT candidate is described with seven headlines: If HPDC use of high pressure Product Dust Transport Emissions air Noise Noise Dust 7.1–7.6 7.8 8.2–8.5 9.1 10.1 10.4 11.1–11.2 12.1 12.3 12.5 13.2 14.1–14.4 15.1–15.5 16.1 THE FOUNDRY PERMANENT MOULDS (EXAMPLE HIGH PRESSURE DIECASTING) Die tool Spraying release agent and cooling Product Nordic Foundries 59 7.1 Bag-house filter 7.1 Bag-house filter 7.1.5 There is need of electricity and compressed air. A right customised filter systems with continuous service, lower operating expenses. Electricity and compressed air consumption is reduced by a canal system in balance, and if the filter media are changed with the correct range for maintaining dimensioned pressure drop in the system. The use of frequency converters for fans is saving electricity when parts of the plant requires less ventilation or is not used. The equipment generats noise. 7.1.2 The system is based on negative pressure. Hoods and ventilation channels lead dust into a bag-house filter. Figure 15: Bag-house filter. Figure 15: Bag-house filter. Environmental benefits 7.1.3 Depending on the type of process dust, these modern filter systems give a high degree of separation. Many applications have low emissions to the external environment (normally 1–4 mg/m3 but requirements from authorities often 5 mg/m3). Right adapted effective hoods provides a cleaner internal working environment with health benefits, and extends service intervals on mechanical equipment with less wear and tear, caused by dust. 7.1.4 Applicability In principle, most foundries use this technology. The differences lie in the individual adaptation of the system to the needs of the process. Many factors influence the choice of technology for filtering. Examples affecting; Temperature, type of dust, moisture, pressure requirements, amount of dust, particle size, ATEX-classification, etc. Some process cannot use this, for example cupola furnace often use a wet abatement technique. 7.1.6 Economics You cannot make a “general price list” when needs vary from individual small machine 2,000 m³/h, large complex systems of several hundred thousand m³/h, and cost of several hundred thousand euro. 7.1.7 Reference 7.2.2 One way to have a better control of the filter is to use a pressure drop gauge connected to the filter. The gauge measures the difference in pressure between the inside and outside of the filter bag. If there is a variation in pressure difference it could indicate that something is wrong with the filter. Applicability Applicability A pressure drop gauge can be used for a lot of different filters. If a pressure drop gauge is used it is important that the staff controlling the gauge know what a normal value is and at value they should react. 7.2.5 Cross-media effect No cross-media effect. No cross-media effect. Pressure drop gauge 7.2 7.2.1 Introduction It is often difficult to see if a fabric filter is working properly or not. There can be a small tear in the filter media that a person cannot see with her bare eye but that may result in higher emission of dust. 7.1.7 Reference Several foundries in the Nordic countries also commonly used in several countries in Europe. Nordic Foundries 62 7.2.3 Earlier detection of dust abatement problems. 7.2.6 Economics There are simple analog for a few hundred euros, to advanced electronic for some thousands euro. Dust filter measurements 7.3 7.3.2 An external consultant is commissioned to perform measurements of the throughput from the filter (m3/h) as well as dust content (mg/m3), and sometimes also the amount of different metals compared to the total amount of dust (%). It is important to use standars for performing the measurements. 7.3.3 To achieve the intended environmental protection from the filter, it is important that the filter works properly or whether corrective actions need to be taken. Applicability All foundries should do this but the measurement frequency may vary between different foundries depending on how much the filter is used. No cross-media effects. 7.3.1 Introduction Emissions of dust particles are removed from the air by dust filters. The regular maintenance of these filters is important for the filters to work properly. It is also important to check that the filter works by measuring the throughput and the dust emissions. 7.2.7 Several foundries both in the Nordic countries and the rest of Europe. 63 Nordic Foundries 7.3.6 Economics A measurement of one single filter may cost from EUR 3,000. The actual cost depends on how many filters needs to be measured and how far the consultant has to travel. It is often more economic to measure several filters at the same time to reduce the cost of travel for the consultant. Big foundries normally don’t measure every filter every year but has a rolling schedule. 7.3.7 Reference 7.5 Using fluorescent powder to check for problem in bag-house filter 7.5.1 Introduction 7.4.4 Applicability Applicability Applicability This should only be used if there can be a problem with the filter because of cold weather. 7.4.7 Reference Not very common. 7.3.7 Several foundries both in the Nordic countries and the rest of Europe. Nordic Foundries 64 7.4 Filter heating 7.4.1 Introduction In the Nordic countries there is a risk that the filter will not work properly in the winter because of the cold weather. 7.4.2 Applied process and techniques There are several techniques but normally there is some heating loops built in to the filter. 7.4.3 Environmental benefits Filter heating ensures the functionality of the filter in the cold winter. 7.4.4 Applicability This should only be used if there can be a problem with the filter because of cold weather. 7.4.5 Cross-media effect Electricity is used for heating. 7.4.6 Economics Not available. 7.4.7 Reference Not very common. 7.5 Using fluorescent powder to check for problem in bag-house filter 7.5.1 Introduction It is often difficult to see if a fabric filter is working properly or not. There can be a small tear in the filter media that a person cannot see directly but will give a higher emission of dust. 7.4.2 Applied process and techniques There are several techniques but normally there is some heating loops built in to the filter. Filter heating ensures the functionality of the filter in the cold winter. 7.4.1 Introduction In the Nordic countries there is a risk that the filter will not work properly in the winter because of the cold weather. 7.5.7 Several foundries in the Nordic countries for example Arvika Gjuteri, a foundry in Sweden. 7.5.5 Need of fluorescent powder. 7.5.1 Introduction It is often difficult to see if a fabric filter is working properly or not. There can be a small tear in the filter media that a person cannot see directly but will give a higher emission of dust. 65 Nordic Foundries Applied process and techniques 7.5.2 One technique to identify small tears in the filter media is to use a fluorescent powder that is sprayed on the filter bag. By illuminating with a UV lamp even small damage on the filter can be detected. 7.5.4 Applicability This technique can be used on all fabric filters. Sometimes the staff at the foundry can do this otherwise an external company can do this when they are doing maintenance of the filter. 7.5.3 There is an earlier detection of problems with the dust abatement. 7.7 Acid scrubber to clean amine Introduction Some of the binders are catalysed with an amine with a very unpleasant odour. The air therefore needs to be cleaned before being emitted. An acid scrubber is used for this. Economics There are different variants, ranging from smaller system at around EUR 3,000, up to larger system at around EUR 20,000. Several foundries in the Nordic countries for example SKF, a foundry in Sweden. Environmental benefits 7.6.3 Dust abatement problems are detected early. A particle monitor can be used in a lot of different filter systems but often for bigger filter systems To function properly particle monitors need to be calibrated regularly. 7.6.1 Introduction Measuring is important, since without measuring it is impossible to know if the performance is alright, within limits and towards targets and goals. Unfortunately, measurements of particle emissions are done very seldom, maybe not even once a year. Significant dust sources may need to be measured more frequently, and therefore different automatic particle monitors can be used. Nordic Foundries 66 Applied process and techniques 7.6.2 A particle monitor is monitoring the dust emission in real time. There are different variants, ranging from smaller systems that set off an alarm at a predefined level of dust, up to large monitoring systems that continuously log the amount of particle emissions. Environmental benefits Applied process and techniques The air from the core making where the amine is used is collected and transferred to the acid scrubber. In the acid scrubber normally sulfuric acid but also other acid is used. The acid is sprayed into a chamber where it meets the air with amine. The amine is dissolved in the acid and can then be removed. The acid is sent away as waste. In Germany the acid is sent for reuse but this possibility is not available in the Nordic countries. Nordic Foundries 67 Environmental benefits 7.7.3 An acid scrubber is very effective and gives a very low remainder of amine in the cleaned air and there is no problem with odour after this process. An acid scrubber can give very low emissions normally under 1mg/m3 but the regulations from authorities can vary. An acid scrubber is very effective and gives a very low remainder of amine in the cleaned air and there is no problem with odour after this process. An acid scrubber can give very low emissions normally under 1mg/m3 but the regulations from authorities can vary. Applicability All foundries using amine as a part of the binder system for sand. There is a need of acid and electricity to make the process running. The acid is sent away as waste. 7.9.1 Introduction 9.1 Introduction Foundries using sand normally have a binder holding the sand together. If this is an organic chemical binder there is a risk it will lead to emissions of VOC when the hot melt is poured into the mould. Mould or core preparation may also cause VOC emissions. 7.8.7 Several foundries in the Nordic countries using HPDC. 7.8.6 Not available. Reference Reference Applied process and techniques There are good oil mist filters that can be connected to each HPDC machine. The functionality of the filter results in very good air quality, with often so low concentration of oil mist that the air can be recirculated into the foundry without health risks. 7.8.3 Nordic Foundries 68 Applicability 7.8.4 Most foundries using HPDC could use an oil mist filter. However, sometimes the process give very low emission of oil mist so a filter is not needed. It is as always therefore important to first perform measurements of the air quality. In order to make the most out of the oil mist filter it is also important to have a hood that collects all oil mist from the machine. There may be a problem with magnesium where it is a risk for magnesium coming into the filter causing fire. 7.8.5 Cross-media effect There will be waste from the filter. There will be waste from the filter. 7.9.2 One way to handle VOC is to use a cleaning technique for the emissions. But before investing in such techniques, other possibilities should be investigated. Every foundry should have a plan for this. This plan should include at least:  Measurements of the scale of VOC emission, and types of emissions.  Investigate the binders may be substituted with some other technology.  Investigate whether carbonaceous material (coal dust) in green sand may be substituted to additives with lower emissions. 69 Nordic Foundries  Investigate whether it is possible to reduce the amount of carbonaceous material in green sand.  Investigate whether it is possible to reduce the amount of binders used, without reducing quality of the core and the mould? reducing quality of the core and the mould? Figure 16: Preparing measurements on a roof of a foundry 7.9.3 Environmental benefits Reduction of VOC emissions, which is good for both the working environment and the outdoor environment. Figure 16: Preparing measurements on a roof of a foundry Figure 16: Preparing measurements on a roof of a foundry 7.9.3 Environmental benefits 7.9.3 Environmental benefits 7.9.3 Reduction of VOC emissions, which is good for both the working environment and the outdoor environment. Reduction of VOC emissions, which is good for both the working environment and the outdoor environment. Reduction of VOC emissions, which is good for both the working environment and the outdoor environment. All foundries using sand binders can have a plan to lower the emissions of VOC. 7.10.1 Introduction To avoid that the sand sticks on the cast product a protective coating is sometimes used. The coating often contains graphite and some kind of thickener dissolved in water or alcohol. It establishes a protective layer between the sand and the metal. Sometimes the whole sand core or mould is dipped in to the coating. If an alcohol is used for the coating it will evaporate to the air. 7.10.2 Applied process and techniques 7.9.6 7.9.6 Economics Decreasing the amount of carbonaceous material or binder may save money, but substituting carbonaceous material or binder might increase costs. Nordic Foundries 70 7.10 Lid on coating vessel with alcohol based coating 7.10.2 Applied process and techniques If the whole core or mould is dipped into the coating it is important to use a lid of the container with coating. 7.11.4 Applicability Every foundry using coating should be aware of this. 7.11.5 Cross-media effect No cross-media effects. 7.11.1 Introduction To avoid that the sand sticks on the cast product a protective coating is sometimes used. The coating often contains graphite and some kind of thickener dissolved in water or alcohol. It establishes a protective layer between the sand and the metal. When alcohol is used, the emissions to air need to be minimised. For water based coating energy-use for the drying of the water instead needs to be minimised. The coating is often bought ready to use but after a while there is a need of add some more water or alcoholic caused by evaporation. 7.11.2 Applied process and techniques Sometimes coating is used by old habit, even though it is not needed. The first step is to overlook where coating really is needed. Of course if coating is needed it should be used otherwise it can cause cassation. Next is to have a controlled mixing of the coating with a regular control of the viscosity to ensure the right composition. 7.10.3 Environmental benefits Lid prevents some emission of alcohol to the air, it also lower the risk of open fire. The lid cannot be used when the dipping is done but all the time when dipping is not going on. 7.10.6 Economics A lid is not very expensive and in the same time the need of extra alcohol is minimised. Lid also gives a better working environment. Several foundries in the Nordic countries. Several foundries in the Nordic countries. Nordic Foundries 71 7.11.3 Environmental benefits If use of coating can be reduced there is an environmental benefit from the resource savings. If too much alcohol is used more will evaporate to the air. If too much water is used it will take longer time for the drying using more energy. It is important to have the right viscosity to get the right effect of the coating. 7.11.4 Applicability 7.12 Coal dust replacement 7.12 Coal dust replacement 7.12.3 Environmental benefits Lower emissions of VOC, which is good both for the working environment and the outdoor environment. 7.12.1 Introduction Foundries are using so called green sand that use bentonite to hold the sand together and carbonaceous material (coal dust) to get smooth surface. The problem with some of the carbonaceous materials are that it gives rise to emissions of volatile organic compounds, VOC, when the melt is poured into the mould. 7.12.2 Applied process and techniques There are different types of carbonaceous material. 7.12.4 Applicability Foundries using green sand with carbonaceous material can try this. It may not work for all foundries. Substituting carbonaceous material with one with lower emissions can give initial product quality problems, so it is recommended it is introduced gradually. 7.12.5 Cross-media effect No cross-media effects. 7.11.6 Economics If use of coating can be reduced there is a cost saving. If use of coating can be reduced there is a cost saving. A viscosity meter is not so complicated and not so expensive. The saving is the use of less alcohol or less heat to dry the core with water based coating. 7.11.7 Reference Several foundries. Nordic Foundries 72 7.12.6 Economics It can be more expensive than normal carbonaceous material. 7.13.3 Lower emissions of VOC and compounds causing odour. 7.13 Cleaning VOC and odour with biofilter 7.13 Cleaning VOC and odour with biofilter 7.13 7.13.1 Introduction Odour may be a problem especially foundries that use binders for sand forms. First one should try to reduce the source of the odour. If that is not possible there are some techniques for cleaning the air before emission. 7.13.4 Applicability The applicability is limited to large series casting foundries with almost continuous production, as biofilters need very stable conditions throughout the year. It is an air cleaning system for emissions with low VOC content. Advantages  Suitable for clean large flows with relatively low levels of organic substances  Cleaning efficiency 70–90%  Disadvantages  Requires large areas  Requires constant access to VOCs, long break in the foundry production may not occur  High cost  Suitable for clean large flows with relatively low levels of organic substances  Suitable for clean large flows with relatively low levels of organic substances  Requires constant access to VOCs, long break in the foundry production may not occur 7.13.3 Environmental benefits 7.13.3 Environmental benefits 7.12.7 Reference 7.12.7 Reference Several foundries in the Nordic countries and the rest of Europe, for example Arvika Gjuteri, a Swedish foundry. Several foundries in the Nordic countries and the rest of Europe, for example Arvika Gjuteri, a Swedish foundry. Nordic Foundries 73 7.13.2 Applied process and techniques Biofiltration is one possible odour abatement technique. The function of biofilter is based on micro-organisms (bacteria), which is laid on organic carrier material (bed material) like wood, chips, peat, bark etc. Biofiltration consists of a sorption phase of pollutants on a carrier surface and a subsequent degradation phase by micro- organisms, which are settled in an aqueous phase on the carrier substance. Main factors affecting the sizing of a biofilter system are the volumetric flow rate of the waste gas stream, the type of waste gas constituents and their concentration as well as the type of filter media. To make optimum use of available space, several enclosed biofilter modules may be vertically stacked. 7.13.6 Economics 7.13.6 It is difficult to say the exact cost because the size of the facility may vary a lot between different foundries but in general it is an expensive method. There is a theoretical calculation of an example foundry with  106,000 m3 air /h to be cleaned  45 ton VOC/year  3,620 ton production of casting/year  Biofilter volume 981 m3 Then the investment cost is calculated to EUR 1,168,000 and the total annual netto cost is EUR 170,000/year. Another example: Based on 33,000 m³/h air flow, 0% humidity, 30 °C temperature; production time: 1,920 h/year, 240 days; 1 m³ water (without wastewater) = EUR 3.0/m3, 1 m³ wastewater = EUR 7.0/m3, 1 kWh electricity = EUR 0.1/kWh Investment costs for biofilters, scrubber system, fan, control unit, internal gas and water piping and engineering EUR 325 000 and a total yearly cost EUR 24 000 Another example: Based on 33,000 m³/h air flow, 0% humidity, 30 °C temperature; production time: 1,920 h/year, 240 days; 1 m³ water (without wastewater) = EUR 3.0/m3, 1 m³ wastewater = EUR 7.0/m3, 1 kWh electricity = EUR 0.1/kWh production time: 1,920 h/year, 240 days; 1 m³ water (without wastewater) = EUR 3.0/m3, 1 m³ wastewater = EUR 7.0/m3, 1 kWh electricity = EUR 0.1/kWh Investment costs for biofilters, scrubber system, fan, control unit, internal gas and water piping and engineering: EUR 325,000 and a total yearly cost EUR 24,000. EUR 3.0/m3, 1 m³ wastewater = EUR 7.0/m3, 1 kWh electricity = EUR 0.1/kWh Investment costs for biofilters, scrubber system, fan, control unit, internal gas and water piping and engineering: EUR 325,000 and a total yearly cost EUR 24,000. 7.14.1 Introduction Odour may be a problem from foundries, especially foundries using binders for sand moulds. First it is important to try to reduce the source of the odour and if that is not possible there are some techniques for cleaning the air before emission. 7.13.5 Cross-media effect Requires some humidity,a controlled temperature and continuous operation. Nordic Foundries 74 7.13.7 Reference No Nordic foundries use this method today. The method is used in some larger foundries in Germany. 7.14 Cleaning VOC and odour with RTO 7.14 Cleaning VOC and odour with RTO 7.14.6 Economics An example based on 30,000 Nm³/h air flow, 0% humidity, 50 °C temperature; VOC 100 mg/Nm³, production time: 1,920 h/year, 240 days; 1 kWh / 10.5 Nm³ natural gas = EUR 0.03/kWh, 1 kWh electricity = EUR 0.07/kWh. Investment costs for a regenerative thermal oxidiser, fan, control unit, raw gas and clean gas piping, stack, transport, engineering installation and commissioning: EUR 530,000 (plant capacity: 30,000 Nm3 per hour) and a total yearly cost EUR 49,000. 7.14.5 Cross-media effect 7.14.5 Cross-media effect If the VOC concentration is very low (<< 100 mg/Nm³) preferably a pre-concentration with a zeolite rotary concentrator or a fixed bed should be used in combination with the RTO. Otherwise there is a need to put in extra fuel. Lower emissions of VOC and compounds making odour. 7.14.4 Applicability RTO is a process for emissions with high VOC concentration. 7.14.2 Applied process and techniques One technique that is used in some foundries in Europe is Regenerative Thermal Oxidation, RTO where the VOC is incinerated. The process begins with that the waste gas is fed through the regenerative heat exchanger, and is preheated to near combustion temperature. In the combustion chamber, the contaminants are oxidized at temperatures around 800 °C. The resulting combustion heat reduces the necessary burner power in relation to the level of contaminants. For concentrations near autothermal level the burner can be switched to ignition flame. 75 Nordic Foundries 75 Environmental benefits Environmental benefits 7.14.3 Lower emissions of VOC and compounds making odour. Lower emissions of VOC and compounds making odour. 7.14.7 Reference No Nordic foundries use this method today. The method is used in some larger foundries in Europe. Nordic Foundries 76 8.1.5 Cross-media effect There maybe be a need of a new building, some electricity to the lighting in the building, and maybe also some extra heat transportation equipment (fans for ventilation channels or pumps for heat exchangers, etc.) if the scrap should be dried or preheated in the indoor scrapyard. 8.1.4 Applicability All foundries can use this technique. The scrapyard housing can be more or less complex. Examples are how much insulation to use to reduce the noise disturbance, and whether the excess heat from the foundry processes should be used to dry and/or preheat the scrap. Noise disturbance from tipping will also decrease if the doors can be kept closed. Environmental benefits Noise disturbances from tipping decrease. The scrap is held dry. Dry and warm scrap requires less energy to melt. Wet scrap charged in the furnace may cause explosions. 8.1.1 Introduction Iron and steel foundries use a lot of scrap to the melt. Delivery is done by truck and it is not unusual that the trucks simply tip the load of scrap on the yard, resulting in a very high instantaneous source of noise. 8.1.2 Applied process and techniques Indoor scrapyards is a good idea to reduce noise disturbances, and also to keep the scrap dry. 8.2.1 Introduction One of the most important key environmental issues for foundries is the problem with noise disturbing neighbours. Noise is not only a disturbing factor it may also cause health problem for example high blood pressure. Some foundries use silencer on the ventilations and fan and that is a good technique to use but also rather expensive. It is better to start with some simpler things that can help a lot. The most important is to have a plan for how to handle noise problem and how to work with this questions in the ordinary work. 8.1.6 The economics may vary widely, from pure noise walls to isolated housing constructions with heat exchange solutions. Reference Reference 8.1.7 Several foundries in the Nordic countries for example SKF, a Swedish foundry. 8.2.2 Applied process and techniques A plan for working with noise may include some of these steps: A plan for working with noise may include some of these steps:  Measure the noise at nearby properties and if necessary use noise simulation. Especially important after changes in the foundry. In some situations the continuous noise measurements can be used especially near housing.  Carry out regular noise rounds where somebody goes around and listen and measures on the company's fans exhaust etc.  Prioritize regular maintenance of fans, motors, belts, bearings, etc.  Control working hours so noisy activities do not take place when the neighbours are asleep.  Turn off fans, etc. when not in use.  Inform the staff about the noise requirements for the foundry.  Keep doors and windows closed whenever possible.  Try placing noisy activities shielded from nearby homes.  Educate truck drivers in quiet driving  If truck transport takes place outdoors, make sure the ground is level and paved.  Limit times for transportation.  Keep noise levels as a criterion when purchasing new equipment.  If the above measures do not help should an investigation be carried out showing potential to reduce noise by installing silencer or shield sources of noise.  If silencer is used, it is important to regularly check and clean the silencer if necessary.  Inform the staff about the noise requirements for the foundry.  Try placing noisy activities shielded from nearby homes.  If silencer is used, it is important to regularly check and clean the silencer if necessary. Nordic Foundries 78 Figure 17: Shield on roof to prevent noise from fan spreading to the surroundings Figure 17: Shield on roof to prevent noise from fan spreading to the surroundings 8.2.3 Environmental benefits A lower noise level. 8.2.5 Cross-media effect No special cross-media effect. 8.2.4 Applicability All kind of foundries can work in this way. However Iron and steel foundries that use scrap metal are more challenged than foundries (Al, Mg and Bronze) that have raw metal supplied on pallets. If they cannot do all steps the can start with some. 8.2.5 Cross-media effect 8.2.6 Economics The economics depends on how many of the steps the foundry will use. A simple noise measurement may cost EUR 1,000. Have a person walking around the foundry to listen maybe one hour per month or week and to turn of fans when not in use or plan the production in a better way will not cost very much. Nordic Foundries 79 Reference 8.2.7 Several foundries in the Nordic countries. 8.3.1 Introduction Noise disturbances may be very subjective. Therefore, before solving noise issues by for example investing in new equipment, it is important to acquire an objective overview of the noise situation. One way to do this, is to understand how the foundry contributes to the total noise level of the neighbourhood, as well as to understand which measures are effective and cost efficient. Measurement of noise levels can often be enough to provide an understanding of the situation, but if the noise environment is complicated, with roads, railroads or other industries nearby, noise simulation may be a good tool. Applicability All foundries can use this. Since the modelling is time consuming, however, it may only be recommended for foundries having problem with noise disturbances, and where ordinary noise measurements does not give conclusive results, possibly because of other disturbing activities as roads or other industries. It is important to test the model to decide the accuracy. Applied process and techniques 8.3.2 In a noise simulation software tool all noise sources, such as fans, doors, ventilation blowout etc. is measured. Buildings and the surroundings is drawn up in the software virtual world. A special function calculates and models noise levels at different distances from the foundry. A result is presented on a map where for example different noise levels are represented as different colours. This makes it possible to for example see which sources give the highest contribution to the total noise level at different locations on the map. The model allows testing for example what happens if a fan is moved, if a silencer is installed on a blowout or what is the result if there will be a new fan on the roof. Before any investments are made any model results should of course be validated with real measurements. Nordic Foundries 80 Figure 18: Example noise simulation. Different colours represent different noise levels. 8.3.3 Environmental benefits A visual simulation tool makes it possible to see the noise distribution landscape, and to more easily prioritize which sources to prioritize and which measures to take. Figure 18: Example noise simulation. Different colours represent different noise levels. Environmental benefits A visual simulation tool makes it possible to see the noise distribution landscape, and to more easily prioritize which sources to prioritize and which measures to take. A visual simulation tool makes it possible to see the noise distribution landscape, and to more easily prioritize which sources to prioritize and which measures to take. A visual simulation tool makes it possible to see the noise distribution landscape, and to more easily prioritize which sources to prioritize and which measures to take. 8.4.3 Environmental benefits Silencers decrease the noise emissions to the surroundings. Silencers decrease the noise emissions to the surroundings. 8.4.2 Applied process and techniques Silencers can be installed on the ventilation canals / pipelines. Silencers should be installed as closely as possible to the noise source, such as at the outlet or the inlet, to avoid that the sound waves propagates through the ventilation ducts and spreads and amplifies before reaching the silencer. Different types of ventilation silencers are:  Reactive  Absorptive  Combinations of these 8.3.6 Economics It is time consuming to measure all fans, ventilations etc. and also to build the model in the computer. The cost therefore depends on number of noise sources to measure and model. It may cost around EUR 5,000 to several EUR 10,000 to noise model a site. This may be well spent if it results in pinpointing an elusive noise source It is time consuming to measure all fans, ventilations etc. and also to build the model in the computer. The cost therefore depends on number of noise sources to measure and model. It is time consuming to measure all fans, ventilations etc. and also to build the model in the computer. The cost therefore depends on number of noise sources to measure and model. It may cost around EUR 5,000 to several EUR 10,000 to noise model a site. This may be well-spent, if it results in pinpointing an elusive noise source. Nordic Foundries 81 Reference 8.3.7 Several foundries in the Nordic countries for example Union Electric Åkers, a Swedish foundry. Several foundries in the Nordic countries for example Union Electric Åkers, a Swedish foundry. 8.4.1 Introduction Fans and air streams are causes of noise from ventilation systems. Ventilation noise can be emitted from the plant via inlets, outlets and from cabinets. Air must of course be able to pass the inlet and outlet, and therefore these openings of the ventilation systems are also sources where noise can be emitted to the surroundings. Noise can also escape through different pipes / ducts. 8.4.7 Reference Several foundries in the Nordic countries for example Arvika Gjuteri, a Swedish foundry. There is a lot more information about noise in the publication from the Nordic Council of ministers: “Bullerdämpning av externt industribuller från livsmedelsindustri och hamnar”. Though it was not specifically compiled for the foundry sector much can also be applied on foundries. 8.5.1 Introduction The noise radiates from ventilation ducts. The noise levels strongly depend on the design of duct. 8.4.4 Applicability 8.5.2 Applied process and techniques If noise radiates from a duct or a connected hood, an that goes into and through a duct, additional sound 8.5.3 Environmental benefits Insulation lowers the noise levels in the surroundin Cross-media effect 8.4.5 If the silencer is installed in a ventilation channel with a dust stream it is important to regularly clean the inside of the silencer. 8.4.4 Applicability A silencer can be used in a lot of different ventilation outlet. A silencer can be used in a lot of different ventilation outlet. Reactive silencers are tuned to attenuate sound at a particular frequency, such as blade frequency. Reactive silencers are relatively expensive and unusual in the ventilation context. Absorptive silencers suppress a broad spectrum of sound. They are relatively cheap and common in ventilation context. 82 Nordic Foundries 82 8.4.5 Cross-media effec If the silencer is installed in a regularly clean the inside of t 8.4.6 Economics An absorption silencer has s price of an absorption silenc high, about EUR 70–1,300. Big absorptive, or cylind flows. They are often custom the entire ventilation system From a total cost point o silent fan than to have a custo made when considering vent 8.4.7 Reference Several foundries in the Nord There is a lot more informati of ministers: “Bullerdämpni hamnar”. Though it was not s be applied on foundries. 8.5 Additional insula 8.5.1 Introduction The noise radiates from ven design of duct. 8.5.2 Applied process an If noise radiates from a duct or that goes into and through a d 8.5.3 Environmental ben Insulation lowers the noise le 8.4.5 Cross-media effect If the silencer is installed in a ventilation channel regularly clean the inside of the silencer. 8.4.6 Economics An absorption silencer has simple construction a price of an absorption silencer, for example, cyli high, about EUR 70–1,300. Big absorptive, or cylinder baffle silencers, a flows. They are often custom made, and the cost i the entire ventilation system. From a total cost point of view, it is often be silent fan than to have a custom made silencer. Th made when considering ventilation silencers. 8.4.7 Reference Several foundries in the Nordic countries for exam There is a lot more information about noise in the of ministers: “Bullerdämpning av externt indus hamnar”. Though it was not specifically compiled be applied on foundries. 8.5 Additional insulation of fans and 8.5.1 Introduction The noise radiates from ventilation ducts. The design of duct. 8.4.6 Economics An absorption silencer has simple construction and can be produced efficiently. The price of an absorption silencer, for example, cylindrical silencers, is therefore not so high, about EUR 70–1,300. Big absorptive, or cylinder baffle silencers, are often used in large or very large flows. They are often custom made, and the cost is a significant part of the total cost of the entire ventilation system. From a total cost point of view, it is often better to choose a more expensive and silent fan than to have a custom made silencer. This cost optimization should always be made when considering ventilation silencers. 8.5.2 Applied process and techniques If noise radiates from a duct or a connected hood, and if it is not possible to reduce the noise that goes into and through a duct, additional sound insulation may be a very good option. 8.5.5 Cross-media effect No cross-media effect. No cross-media effect. 8.5.6 Economics This method is costly for long or many ventilation ducts. It is most commonly used to silence ducted fans, since the total cost will not be too high. Insulation of a large ducted fan or duct, cost at around EUR 135 /m2. Environmental benefits Environmental benefits Insulation lowers the noise levels in the surroundings. Insulation lowers the noise levels in the surroundings. 83 Nordic Foundries Applicability 8.5.4 Additional insulation of ducts can be an alternative to the silencer ducts if the ducts are not as long or where the silencers cannot be used. Additional insulation of ducts can be an alternative to the silencer ducts if the ducts are not as long or where the silencers cannot be used. The effect of noise damping that can be achieved depends on the thickness of the insulation and on the type of insulating material. Commonly achieved noise reduction is between 5–30 dB. Additional insulation can of course be bulky and difficult to implement in small volumes and when ducts, pipes and equipment are close together. 8.5.7 Reference 8.5.7 8.5.7 There is a lot more information about noise in the publication from the Nordic Council of ministers: “Bullerdämpning av externt industribuller från livsmedelsindustri och hamnar”. Though it was not specifically compiled for the foundry sector much can also be applied on foundries. Nordic Foundries 84 9.1.1 Introduction Foundry industry is not chemical-intensive. However, there are some chemicals, mainly oils for machine, release agents for high pressure die casting and for binders to sand core and sand casting. 9.1.3 Decreased chemicals risks to health and environment. L f h d h i l Decreased chemicals risks to health and environment. Less use of hazardous chemicals. Decreased chemicals risks to health and environment. Less use of hazardous chemicals. 9.1.4 Applicability All foundries can work with this. 9.1.2 Applied process and techniques Have a person responsible of the use chemicals in the foundry. This person work with approving new chemicals before use, help the different departments focusing on how to minimise the risk of chemical use or how to replace chemicals not good for the environment. To facilitate the chemicals responsibility it may be good to use some IT-tool, such as top procure some chemicals management data base service or software. Environmental benefits 9.1.6 Economics Difficult to say how much this will cost. If some chemicals management data base service or software is used the cost may depend on number of chemicals used in the foundry. It will be a cost for the person responsible for the chemicals management, but whether this cost is higher than not having such an assigned responsibility or not is difficult to say. Reference 9.1.7 Several foundries in the Nordic countries for example Arvika Gjuteri and SKF, two Swedish foundries. Nordic Foundries Nordic Foundries 86 10.1.1 Introduction The foundry processes produces some waste, for example dust, slag or sand. Less waste. 10.1.4 Applicability All foundries can do this. 10.1.5 Cross-media effect 10.1.3 Environmental benefits Less waste. 10.1.2 Applied process and techniques To decide the recycling possibilities, first all waste produced in the factory need to be identified and see what can be done to minimise the amount (see also Material flow cost accounting). Next step is to consider if the waste can be used as a resource somewhere else. To facilitate using waste as resource it is important to sort the waste into different useful fractions. 10.1.3 Environmental benefits Less waste. 10.1.4 Applicability All foundries can do this. 10.1.5 Cross-media effect There is a need for space to make a good sorting of the waste. 10.1.6 Economics Waste is often associated with some cost, and recycling may therefore save money. 10.1.7 Reference Several foundries in the Nordic countries. 10.2.5 Cross-media effect There is a need of electric power for the mechanical sand regeneration. It may generate more dust. 10.2.3 The sand can be used more times. 10.2.1 Introduction When a chemical binder is used to hold the sand together there is often remainders of the binder in the sand after shake-out. This remainder makes it difficult to reuse the sand again in the foundry. 10.2.4 Applicability All sand foundries should investigate the possibility to reuse the sand, as well as their need for mechanical sand regeneration. 10.1.5 Cross-media effect There is a need for space to make a good sorting of the waste. Waste is often associated with some cost, and recycling may therefore save money. 10.1.7 Reference Several foundries in the Nordic countries. Several foundries in the Nordic countries. Several foundries in the Nordic countries. 10.2.2 Applied process and techniques To be able to reuse the sand the foundry needs to use a sand recovery equipment. The sand to be purified contains lumps of mould and / or core sand, and certain metal residues. Sand lumps are first crushed in a lump crusher. Then a magnetic separation is used to rid the sand of metal residues. Then, if the sand contains some moisture and other material than sand grains, the sand is sifted and then dried. The next is a mechanical step, done to remove the binder residues from the sand. It can be done by scrubbing or by compressed air. It is possible to also use thermal cleaning when the sand is heated, but this process is seldom needed for to be able to reuse the sand for new moulds. Even chromite sand separation can be a good idea. 10.3.1 Introduction Foundries using sand often recycle the sand several times in the foundry. But for technical reasons there is a need to replace some of the used sand with new sand at each cycle. The foundry therefore needs to get rid of the replaced and used sand. Used foundry sand may be a good resource to use in different applications throughout the society instead of using virgin sand. Whether the sand can be used to replace virgin sand in different applications is not up to the foundry to decide, but to avoid sending sand to waste it is important for the foundries to find good second uses of their used form sand. Foundries using sand often recycle the sand several times in the foundry. But for technical reasons there is a need to replace some of the used sand with new sand at each cycle. The foundry therefore needs to get rid of the replaced and used sand. Used foundry sand may be a good resource to use in different applications throughout the society instead of using virgin sand. Whether the sand can be used to replace virgin sand in different applications is not up to the foundry to decide, but to avoid sending sand to waste it is important for the foundries to find good second uses of their used form sand. 0.3 Reuse of rest sand outside the foundry 10.3 10.3.2 Applied process and techniques Where the used sand can be applied depends on what kind of binder it contains. One area cannot be generally application for all used sand of the same kind. Instead every sand foundry need to analyse their used sand with regards to constituents, such as hazardous substances. Normally there are no hazardous substances in the used sand. Sand has been used for:  Cover old landfill, especially sand holding bentonite which forms a very dense layer  Use as material to build on, roads, buildings or parking areas. . Before utilization the environmental quality of the sand must be analysed  Use as material to build on, roads, buildings or parking areas. . Before utilization the environmental quality of the sand must be analysed  Build walls against noise  Manufacturing of bricks  To mix with other material to make good soil for cultivation  Use in concrete or cement 10.2.6 Economics The sand system is different at every foundry and there is also a great variation how much sand is circulating in the system, so it is difficult to say the cost for the equipment. 10.2.7 Reference Several foundries in the Nordic countries. 10.2.7 Reference Several foundries in the Nordic countries. Several foundries in the Nordic countries. Nordic Foundries 88 10.4.1 Introduction 10.4.1 Introduction Sometimes foundries use small size raw material such as metal chips or metal powder. There are some difficulties with charging the furnace with such material because of the risk that the material will blow away. Small sized raw material often originate from somewhere in the process, and is to be melted back into the production again. 10.3.6 Economics 10.3.6 Economics Economics 10.3.6 Normally an industry has to pay to get rid of waste. In this case the foundries do not have to pay more than maybe the transport. In Denmark foundries pay EUR 80/ton to get rid of waste sand. 10.3.7 Reference Several foundries in the Nordic countries. 10.3.3 Environmental benefits The sand does not need to be waste on a landfill. Sand is a natural resource taken from sand pits. By increasing the second use of used foundry sand, the sand in natural sand pits can be saved and the expansion of sand pits can go slower. 10.3.5 Cross-media effect Less need of new sand. 89 Nordic Foundries 10.4.5 Cross-media effect The machine needs electricity. Sometimes some chemical is needed to hold the briquettes together. 10.4.4 Applicability Briquetting can be used to make briquettes of a lot different materials but mostly used for aluminium. Cannot be used for all furnace for example can there be a problem with induction furnace. 10.4.2 Applied process and techniques Small metal chips can be pressed together into briquettes to be used as raw material. Briquettes reduce the charging time compared to loose metal chips. Figure 19: Briquettes of aluminium 10.4.3 Environmental benefits Briquettes reduce energy consumption during melting. If the foundry can remelt its own recycled material instead of sending it away there is less need of transportation. Figure 19: Briquettes of aluminium Figure 19: Briquettes of aluminium 10.4.3 Environmental benefits Briquettes reduce energy consumption during melting. If the foundry can remelt its own recycled material instead of sending it away there is less need of transportation. Nordic Foundries 90 10.4.6 Economics The bricketing machine cost between EUR 40,000 and EUR 250,000. 10.4.7 Reference 11.1.3 Simulation is a tool for make better quality in the products and less cassations of the material. Less cassations leads to less metal to be remelted which means less use of energy and material for making moulds and so on. 11.1 Simulation 11.1 Simulation 10.4.7 Reference Several foundries in the Nordic countries. Nordic Foundries Nordic Foundries 91 11.1.2 Applied process and techniques Most methods for the production of castings can be simulated with the simulation program occurring on the market. The simulation gives an almost exact image of the mould filling and solidification. Simulation can also provide a better understanding of the process and how the different parameters affect the quality of the final casting. 11.1.1 Introduction Casting is a complex method with a lot of parameters. If somethings goes wrong in the process the foundry may need to remelt the product. To avoid the costs and other consequences of this simulation is used. Simulation is used as a tool to improve, optimize and get a better understanding of the process. Another important aspect is that simulation is a good way of increasing the knowledge about the influence of different material, production and product parameters. It is important that the foundries work with simulation in a systematic way in order to improve the precision of the results. The need of physical testing is also minimized by use of simulations. 11.1.4 Applicability All foundries can work with simulation. There is different software for simulation. In order to fully benefit from the use of simulation the company needs to dedicate resources to build up their knowledge. It might not be good enough to just occasionally perform simulations. 11.1.5 Cross-media effect No cross-media effects. 11.1.5 Cross-media effect 11. BAT Candidates Simulation 11.1 Simulation 11.1 Simulation 11.1.5 Cross-media effect No cross-media effects. 11.1.6 Economics The foundry may need to purchase simulation software EUR 45,000 to EUR 150,000 + a yearly fee. For maintenance and support and person who know how to use the software. It may be considered rather expensive. But use of simulation will also save money for the foundry with less cassation. 11.1.7 Reference Several foundries in the Nordic countries. 11.1.7 Reference 11.1.7 Reference Several foundries in the Nordic countries. 11.2.1 Introduction When the melt is poured in to the mould there is an inlet and gating system with some amount of extra cast metal to ensure the mould is filled completely. This extra metal will not be used in the finished product but will be removed and melted again. Sometimes there is a need of a saw to take the inlet system away from the rest of the product. 11.2.2 Applied process and techniques The first step is to design the inlet and gating system so it is as small as possible but still works effectively. By a right design of the inlet and gating system it is also possible to easily turn or break off the inlet instead of using a saw. 11.2.3 Environmental benefits Minimise the inlet and gating system to reduce the amount metal needed to be melted again. There is less need of after processing energy when it is possible to break of the inlet instead of using a saw. 11.2.4 Applicability All foundries should work with this. 11.2.5 There is a need of computer simulation. Nordic Foundries 94 11.2.6 Economics It will take some time to make the simulation but less time is needed to take the inlet system away and less metal needs to be melted again. See also the cost of simulation software. 12.1.1 Introduction A foundry uses a lot of energy for all processes. This is an environmental issue, but for the foundry it is also an economic issue. 12.1.6 Economics The cost depends on the size of the foundry but it often starts at EUR 5,000 and above. 11.2.7 Reference 11.2.7 Reference Several foundries in the Nordic countries, for example Holsbyverken a Swedish foundry. Several foundries in the Nordic countries, for example Holsbyverken a Swedish foundry. Nordic Foundries Nordic Foundries 95 12.1.3 12.1.4 Applicability Can be done by all foundries. 12.1.5 Cross-media effect No cross-media effects. 12.1 Energy audit 12.1.1 Introduction 12.1.2 Applied process and techniques Many different things can be done to reduce the energy use. But to do this systematically a good knowledge of the process is needed. This may done by performing an energy audit. The aim of an energy audit is to answer how much energy is supplied to the organization and that is used to power the company’s operations, including buildings and transport, on an annual basis. It shows how the energy is distributed in different parts of the business. The audit is done by performing actual measurements and calculations for the costs for the energy. An energy audit also includes suggestions on how the business can be more energy efficient. 12.1.3 Environmental benefits Potential to save energy. 12.1.4 Applicability Can be done by all foundries. 12.1.5 Cross-media effect No cross-media effects. 12.1.6 Economics The cost depends on the size of the foundry but it often starts at EUR 5,000 and above. 12.1.7 Reference Several foundries in the Nordic countries for example Union Electric Åkers and Arvika.Gjuteri, two Swedish foundries. 12.1.3 Environmental benefits Potential to save energy. 12.1.4 Applicability Can be done by all foundries. 12.1.3 Environmental benefits Potential to save energy. 12.2.2 Applied process and techniques 12.2.2 Applied process and techniques Blasting is used to remove sand from the material that will be melted again. Blasting is used to remove sand from the material that will be melted again. 12.2.1 Introduction Often some of the sand sticks to the finished cast product if sand is used in the casting process. The risk is that such sand will follow the material into the melt, since some faulty casts will be taken to cassation and will be re-melted. If these parts include sand it will both lead to that the melting demand more melting energy and a slag will be formed which will have to be sent to waste. To avoid this it is important to clean the material. 12.1.7 Reference Several foundries in the Nordic countries for example Union Electric Åkers and Arvika.Gjuteri, two Swedish foundries. 12.2.4 Electricity is needed for the blasting. There is a waste produced. Several foundries in the Nordic countries for example Holsbyverken, a Swedish foundry. 12.3 Crushing inlet and gating system 12.3.1 Introduction 12.2.3 Environmental benefits Less sand to the melt will give a better melt, will not need so much energy and less waste. 12.2.4 Applicability Foundries using sand. 12.4.1 Introduction Conventional lining of casting or transport ladles are not only there to protect the ladle itself. They also provide insulation against heat loss during filling and transport. The lining of ladles is made by conventional bricks or cement with a thickness of about 100 mm or above. The ladle is normally pre-heated with a gas burner. The melt is often overheated because there is some heat loss when the melt is in the ladle. There are different machines for crushing the raw material. 12.3.3 Environmental benefits 12.3.7 12.3.7 Reference Used by some foundries in the Nordic countries. 12.3.1 Introduction Inlet and gating system may be melted again after it is broken off from the cast product. These are sometimes rather large and may therefore be difficult to put into the furnace. They can also have sand stuck to them, which means higher energy consumption and slag forming. Nordic Foundries Nordic Foundries 98 Applied process and techniques Applied process and techniques 12.3.2 There are different machines for crushing the raw material. 12.3.3 Environmental benefits Improved furnace efficiency which lowers energy consumption. Separation of great deal of the sand during crushing which lower energy consumption and give less waste. 12 3 4 Applicability Improved furnace efficiency which lowers energy consumption. Separation of great deal of the sand during crushing which lower energy consumption and give less waste. 12.3.4 Applicability All foundries with large raw material or large in-duct channels No information. 12.3.7 Reference Used by some foundries in the Nordic countries. 12.3.7 Reference Used by some foundries in the Nordic countries. 12.4.2 Applied process and techniques There are insulation materials that can be used in the ladle so that preheating is not necessary and the heat loss is minimised so that there is no reason to overheat the melt. 12.4.3 Environmental benefits Saving of energy. 12.4.3 Environmental benefits Saving of energy. Saving of energy. Nordic Foundries 99 12.4.4 Applicability Foundries using ladles. 12.4.5 Cross-media effect Have to use an insulation material. 12.4.5 Cross-media effect Have to use an insulation material. 12.4.6 Economics Some of these numbers are a few years old. From the present material change, the actual energy savings result to approximately EUR 168,000/year. The system operates only about six to seven times with the same ladle liner. Therefore, this technical solution is primarily for non- continuous processes. Such as single taps at high temperature levels can be found for example in steel foundries. Case study: 5 t ladle, 5 tons furnace, 1,500 °C tapping temperature. If a loss of temperature reduction by 30 °C in a 5 t pan 60 kWh of energy can be saved in the overheating process, this will corresponds to about EUR 6/melt. The energy cost savings alone do not justify the investment. A stable temperature may also give production and product quality advantages. 12.4.7 Reference Used by some foundries in the Nordic countries for example Österby Gjuteri, a Swedish foundry. 100 Nordic Foundries 12.5 Use of lid on furnace and ladle 12.5.1 Introduction Every foundry uses a lot of energy to make the melt from scrap metal or ingots. A lot of energy is lost from the furnace if lid is not used. 12.5.2 Applied process and techniques There are different types of lid that can be used on furnace but also on ladle. 12.5.3 Environmental benefits Lids save energy. 12.5 Use of lid on furnace and ladle 12.5.6 Economics In relation to the cost of a furnace or ladle it is a rather small cost that also saves money. Several foundries in the Nordic countries. Several foundries in the Nordic countries. 12.5.5 Cross-media effect No cross-media effects. Applicability 12.5.4 This is a very simple solution to save energy. It cannot be used when filling or empting the furnace or ladle but else the lid may be on, to save energy. But even if lids is a simple technique it is not used everywhere. This is a very simple solution to save energy. It cannot be used when filling or empting the furnace or ladle but else the lid may be on, to save energy. But even if lids is a simple technique it is not used everywhere. This is a very simple solution to save energy. It cannot be used when filling or empting the furnace or ladle but else the lid may be on, to save energy. But even if lids is a simple technique it is not used everywhere. Figure 20: Furnace with lid. 12.5.5 Cross-media effect No cross-media effects. Figure 20: Furnace with lid. 12.5.5 Cross-media effect No cross-media effects. Figure 20: Furnace with lid. 12.5.5 Cross-media effect 12.5 Use of lid on furnace and ladle 12.5.1 Introduction Introduction Introduction Every foundry uses a lot of energy to make the melt from scrap metal or ingots. A lot of energy is lost from the furnace if lid is not used. 12.5.2 Applied process and techniques There are different types of lid that can be used on furnace but also on ladle. Nordic Foundries 100 12.6.3 Potential energy savings in the process of up to 50 per cent due to lower heating up time and more efficient heat transfer. Lower emissions compared to natural gas burner with open flame. No hot spots during preheating giving longer life time of the lining in the ladle. 12.6.2 Applied process and techniques There are burners which function without open fire. For this purpose a geometrically adjusted and insulated cap is placed on the ladle. Inside the cap an integrated burner transmits the heat indirect to the ladle as infrared radiation. There are also electrical ways of preheat the ladle. 12.6.4 More expensive than using open burner. For optimal operation the “burner-stone” is shaped individually, which may cause the supply and storage of several shapes 12.6.5 Cross-media effect 12.6.5 Cross-media effect 12.6.1 Introduction Many foundries use conventional ladle preheating flares, based on natural gas or liquefied petroleum gas-air burners. These kinds of preheating procedures are, generally speaking, connected to the following disadvantages: they may cause hot Nordic Foundries 101 spots during preheating the ladles, cause high volume flow rates and require a long warm-up time. 12.6.5 Cross-media effect Gas is still used but in a lower amount. There are also electrical ways of preheat the ladle using electricity instead of gas. 12.6.6 Economics Saves a lot of fuel. Single station systems are available where the investment costs are between EUR 40,000 and EUR 50,000. Saves a lot of fuel. Single station systems are available where the investment costs are between EUR 40,000 and EUR 50,000. Not very common in the Nordic countries but used of some foundries in Europe. Nordic Foundries 102 13.1 Scrap handling Iron and steel foundries use metal scrap as raw material, and they are mixing different scrap fractions to produce the right alloy composition of the melt. 13. BAT Candidates Metal and melting 13.1 Scrap handling 13.1.1 Introduction Iron and steel foundries use metal scrap as raw material, and they are mixing different scrap fractions to produce the right alloy composition of the melt. 13.2 Using deoxidiser/degassing It is important to prevent oxygen and other gases from the air to dissolve into the melt. The gases can form cavities and inorganic inclusions which will lead to lower quality and more cassation. 13.1.2 Applied process and techniques The first important step is to have an agreement with the scrap supplier to deliver well defined scrap. Quality standards of scrap and auditions of scrap suppliers are important to use. Figure 21: Example of scrap delivered to an iron foundry When the scrap arrive to the foundry it is important to have an inspection of the truck with scrap so it is clean without oil and fulfil the requirements of the foundry and maybe also a chemical analyse of the scrap to assure the quality. Figure 21: Example of scrap delivered to an iron foundry When the scrap arrive to the foundry it is important to have an inspection of the truck with scrap so it is clean without oil and fulfil the requirements of the foundry and maybe also a chemical analyse of the scrap to assure the quality. Next step is to plan the melt. This should be done with a special computer program that takes into account the scrap that is present and which gives a recipe of how much of different scrap should be used to obtain the optimal melt. For example an XRF (X-ray fluorescence) can be used to sort the metals. 13.1.6 Economics It is not expensive to order the right scrap or to control the scrap at delivery. To make an optimal melt you need a special computer program. Several foundries in the Nordic countries. 13.1.4 Applicability 13.1.4 Applicability All foundries using scrap should work in this way. 13.1.3 Environmental benefits Clean scrap results in lower emissions of dust when melted. With the right composition of the melt from the beginning there is no need for adjustments. Adjustments otherwise demands the melt to be put on standby causing use of more energy. 13.2.2 Applied process and techniques To minimise gas dissolution, some deoxidiser/degassing agent is put in to the melt. The deoxidiser/degassing agent is different depending on metal used and gases the have to be removed. The deoxidiser/degassing agent reacts with the gases creating inorganic material floating to the surface of the melt as a slag. The slag can be removed. Nordic Foundries Nordic Foundries 104 Environmental benefits Environmental benefits 13.2.3 Less gases in the melt leads to less cassation. 13.2.5 Cross-media effect This technique requires deoxidiser/degassing agent and it is important to choose an environmental friendly variant It will produce more slag. 13.3.1 Introduction Especially for steel foundries it is important to avoid oxygen and other gases from the air to dissolve into the melt. The oxygen can lead to lower quality and more cassation. Sometimes also dissolved nitrogen is a problem in steel. 13.2.6 Economics No investments needed. This is consumables with not very high price. This technique produces more slag that can cost to get rid of but this offset by lower cost for cassation. 13.2.4 Applicability All foundries except iron foundries use this. All foundries except iron foundries use this. Almost all steel foundries use this technique but a lot of work can be done to optimise the process for example in what way the deoxidiser is added to the melt. 2 Applied process and techniques Argon is used to flush through the melt sometimes together with adding deoxidiser agent. Oxygen and other gases more easily solve into argon than into the melt and are then transported out from the melt. The argon also provides a good stirring of the melt helping the deoxidiser agent to react, and less amount of deoxidiser can be used. 13.3 Flushing ladle with argon 13.3.1 Introduction 13.3.7 For example Sandvik SRP, a Swedish foundry. 13.4.1 Introduction In cast iron production, inoculants are used to control the solidification structure, and by that the final properties. The inoculant is often added as late as possible by in-stream inoculation to avoid the risk of fading. It is important to have good control over inoculation. 13.3.3 Environmental benefits There is less amount of slag produced compared to traditional deoxidiser with aluminium. Nordic Foundries 105 Applicability 13.3.4 Steel foundries with large ladle can use this technique. 13.3.5 Cross-media effect Need of argon. 13.3.5 Cross-media effect Need of argon. 13.3.6 Economics There is a need of some tool to let the argon into the ladle, maybe a new ladle and argon. The cost for investment is estimated to start at EUR 10,000. Reference 13.4.2 Applied processes and techniques The effect of the inoculant is to control the solidification microstructure and by that the final properties. The effect and the yield of the addition of inoculant can be made by microstructure analysis and by different evaluations of material properties. Microstructure investigation is based on microscopy and evaluations of microstructural constituents. Material properties may be evaluated by for example thermal analysis. In that case the temperature is registered during the cooling of an iron sample in a sampling vessel, and the resulting cooling curve is analyzed. The cooling curve depends on the amount of added inoculant. This is done before the casting process so it is possible to adjust the melt. 2 Applied processes and techniques Automatized in-stream inoculation is done to maximize and control the effect of the inoculant. In-stream inoculation is done when pouring the iron into the mold, and an additional benefit is an optimized dissolution into the metal. 13.4.3 The yield of the addition is increased and overdosing can be avoided. Nordic Foundries 106 .4.4 Applicability 13.4.4 Applicability 13.4.4 In principle, inoculation control can be used by any cast iron foundry. 13.4.5 Crossmedia effect A controlled addition of the inoculant minimizes the risk for defects and scrapped castings. Scrapped castings are used as foundry returns but that also means the same amount of iron has to be melted more than once before ending up in a cast component, and that means unnecessary amounts of energy. 13.4.7 Reference Several foundries in the Nordic countries. 13.5.1 Introduction In cast iron production, inoculants are used to control the solidification structure, and by that the final properties. Inoculants are often added as late as possible. The reason is that the effect of the inoculation is fading quickly by time in the melt. 13.4.6 Economics Microstructure analysis and evaluation of final properties is already often used as for quality control. The investment can therefore be considered small and any additional costs can be expected to be balanced by the beneficial effects from an increased yield of the addition. 13.4.7 Reference 13.5.3 Environmental benefits In-stream addition minimizes the risk of effect fading from the inoculation, and therefore enables optimization of the effect of the addition, while at the same time minimizing the risk for formation of defects and quality deviations. By automation the yield of the addition can be increased and overdosing can be avoided. Nordic Foundries 107 Applicability 13.5.4 In principle, automatized in-stream inoculation can be used by any cast iron foundry. 13.5.5 Crossmedia effect Controlled and exact addition of inoculant as late as possible minimizes the risk for defects and scrapped castings. Scrapped castings are used as foundry returns but that also means the same amount of iron has to be melted more than once before ending up in a cast component, and that means unnecessary amounts of energy. 13.5.6 Economics The investment to install automatized in-stream inoculation system is foundry dependent but the cost can be expected to be balanced by the beneficial effects from an optimized and controlled addition. Reference 13.5.7 13.5.7 Nordic Foundries Nordic Foundries 108 14.1.1 Introduction The die tool is worn in the process. Maintenance is important to prolong the life of the tool. 14.1.5 Cross-media effect There can be a dust emission from the blasting. 14.1.4 Can be used of all foundries using HPDC and LPDC. Especially important for foundries using aluminium or magnesium. 14.1.7 Most of the foundries using HPDC and LPDC. 14.1.2 Applied process and techniques 14.1.2 Applied process and techniques Maintenance of die tools is used for longer lifetime, including blasting and grinding of the surface of the tool. Maintenance of die tools is used for longer lifetime, including blasting and grinding of the surface of the tool. Figure 22: The two halves of a die tool, waiting to be put into the HPDC-machine. Previously published in Gjuteriet/Gjuteriinformation AB 14.1.3 Environmental benefits Production of a new tool demands a lot of resources and energy. A worn tool can cause more cassation of the products. Less cassations leads to less metal to be remelted which means less use of energy and material. Figure 22: The two halves of a die tool, waiting to be put into the HPDC-machine. Previously published in Gjuteriet/Gjuteriinformation AB 14.1.3 Environmental benefits 14.1.3 Environmental benefits Production of a new tool demands a lot of resources and energy. A worn tool can cause more cassation of the products. Less cassations leads to less metal to be remelted which means less use of energy and material. Production of a new tool demands a lot of resources and energy. A worn tool can cause more cassation of the products. Less cassations leads to less metal to be remelted which means less use of energy and material. Applicability 14.1.6 Economics Not performing maintenance will cause shorter lifetime of the die tool and the production of a new tool may be considered a large investment. 14.2.1 Introduction The die tool is worn in the process the result is lower quality on the products. A new tool is also a big cost. Therefore it is important to prolong the life of the tool. 14.2.2 Applied process and techniques 14.2.2 Applied process and techniques There are channels in the tool. These channels are used to preheat the tool with warm oil or water before casting. The oil or water is heated electrically. The channels are also used to cool the mould after every casting circle. Production of a new tool demands a lot of resources and energy. A worn tool can cause more cassation of the products. Less cassations leads to less metal to be remelted which means less use of energy and material. 14.2.4 Nordic Foundries 110 14.2.5 Cross-media effect There is a need of electricity to heat and circulate the oil or water. 14.3.1 Introduction Foundries using High pressure die casting, HPDC, need to cool the die tool between every shot. Often this is done by spraying water on the tool. But often the tool does not have the same temperature over the whole area and it is therefore difficult to get the right amount of water. Today the calculation of the amount of lubricant that is needed often is based on experience and test measuring. 14.3 IR camera 14.3 IR camera 14.2.7 Reference Most of the foundries using HPDC and LPDC. 14.3.3 Environmental benefits To produce a new tool demands a lot of resources and energy. A more even temperature on the die tool maybe prolong the life time of the tool. A worn tool can cause more cassation of the products. Less cassations leads to less metal to be remelted which means less use of energy and material. 14.3.2 Applied process and techniques There is an increased use of IR camera in the foundry industry and through them come more opportunities to indirectly control the lubrication of the die, with the objective of the tool surface temperature is kept as constant as possible. A more even die temperature means that the die can run more cycles. The camera gives a picture of what areas need extra or less cooling. 14.2.6 Economics Not to do preheat the tool will cause shorter lifetime of the die tool and the production of a new tool may be considered a large investment. 14.2.7 Reference Most of the foundries using HPDC and LPDC. 14.2.7 Reference 14.3.4 Applicability 14.3.4 Can be used of many foundries using HPDC. The foundry can buy one camera and use it at several machines when necessary. Nordic Foundries 111 14.3.5 Cross-media effect No cross-media effect. 14.3.5 Cross-media effect No cross-media effect. No cross-media effect. 14.3.6 Economics There are different types of IR-cameras from very simple to very complex. It is enough to have one camera per foundry. Not to do this may cause shorter lifetime of the die tool and the production of a new tool is a big investment. 14.4.1 Introduction During High pressure die casting for aluminium a scoop is often used to take the melt from the open furnace to the machine. The problem with this is that the melt is exposed a longer time for oxygen in the air. The oxygen can dissolve into the melt causing extra slag or causing defect in the cast product. 14.3.7 Reference Several foundries in the Nordic countries for example Ankarsrum Die Casting, a Swedish foun Figure 23: Picture from computer program connected to IR-camera. Picture showing different temperatures in a die tool dry. dry. Nordic Foundries 112 Energy is needed to create the vacuum. Energy is needed to create the vacuum. 14.4.6 Economics No information. 14.4.6 Economics No information. 14.4.6 Economics No information. 14.4.2 Applied process and techniques Avoid that the melt comes in contact with oxygen in the air for example if it is possible by a lid on the furnace and take out the right amount melt using vacuum ceramic container instead of open scoop to take melt to the machine. Figure 24: Vacuum dosing furnace 14.4.3 Environmental benefits The method gives lower loss of heat in the melt. If there is less oxygen in the melt it could to some extent lead to less slag. Figure 24: Vacuum dosing furnace Figure 24: Vacuum dosing furnace Figure 24: Vacuum dosing furnace 14.4.3 Environmental benefits 14.4.3 Environmental benefits The method gives lower loss of heat in the melt. If there is less oxygen in the melt it could to some extent lead to less slag. The method gives lower loss of heat in the melt. If there is less oxygen in the melt it could to some extent lead to less slag. All foundries using high pressure die casting for aluminium can use this method. 113 Nordic Foundries 14.4.5 Cross-media effect 14.4.7 Reference Some foundries in the Nordic countries for example Ankarsrum Die Casting, a Swedish foundry. Nordic Foundries Nordic Foundries 114 15.1.3 Environmental benefits The main environmental benefits of circular economy are to save resources by maintaining values in produced goods. It shall be stressed that in order to actually achieve the potential environmental benefits of a circular business model, lifetime prolongation, re-use, refurbishment, recycle and recycle logistics also need to be environmentally efficient. 15.1.1 Introduction 15.1.1 Introduction Circular economy intends to achieve positive economic development without increased resource use. 15.1.2 Applied process and techniques A key mechanism to establish a circular economy is to transform the business model of a company or of several companies in a value chain. The business models need to be changed from material-based businesses, to businesses satisfying needs and offering performance values regardless of material and resource consumption. The practical aspects of the circular economy include prolonged efficient lifetime by optimized design or service intervals, re-use and refurbishment, closed loop recycling to maintain alloys and generally resource efficient recycling systems. 15.2.2 Applied process and techniques An MFCA is performed as a practical investigation of the amounts of the material and the energy entering a production step and of the energy and the material leaving the production step, and all materials are assigned its economic cost. The result of the investigation is a list of costs for the different spills and where these costs occur. The information is provided to the production management, who may take different decisions to minimize or avoid the costs. MFCA is an international standard ISO 14051:2011. 5.1.5 Cross-media effect 5 Cross-media effect 15.1.5 A circular business model does not immediately change the production process of the individual products of a foundry, but in the long run they may lead to fewer items produced, due to reuse and longer product life-time, or to a change in designs for longer life cycles. A circular business model does not immediately change the production process of the individual products of a foundry, but in the long run they may lead to fewer items produced, due to reuse and longer product life-time, or to a change in designs for longer life cycles. 15.1.7 Reference Some foundries in the Nordic countries for example Union Electric Åkers, a Swedish foundry. Material Flow Cost Accounting (MFCA) MFCA is a standardized method for counting the costs for material and energy spill in a production unit. 15.1.6 Economics Much of today’s metal industry is to a large part already based on recycling of metals, and to a smaller degree also on reuse and refurbishment of metal components, the transition from today’s economic models into a circular economy is not a major leap. Cast components may gradually be designed and marketed for a circular business model, or may gradually become components of circular business models for compound products. 15.1.4 Applicability Any foundry can be part of a circular economy business model, either by themselves changing their business model into some form of a circular material handling, or by becoming part of a customer’s or supplier’s circular value chain. 15.1.5 Cross-media effect A circular business model does not immediately change the production process of the individual products of a foundry, but in the long run they may lead to fewer items produced, due to reuse and longer product life-time, or to a change in designs for longe life cycles. 15.1.6 Economics Much of today’s metal industry is to a large part already based on recycling of metals and to a smaller degree also on reuse and refurbishment of metal components, the transition from today’s economic models into a circular economy is not a major leap Cast components may gradually be designed and marketed for a circular business model, or may gradually become components of circular business models fo compound products. 15.1.7 Reference Some foundries in the Nordic countries for example Union Electric Åkers, a Swedish foundry. 15.2 Material Flow Cost Accounting (MFCA) 15.2.1 Introduction MFCA is a standardized method for counting the costs for material and energy spill in a production unit. 15.2.2 Applied process and techniques An MFCA is performed as a practical investigation of the amounts of the material and the energy entering a production step and of the energy and the material leaving the production step, and all materials are assigned its economic cost. The result of the investigation is a list of costs for the different spills and where these costs occur. The information is provided to the production management, who may take different decisions to minimize or avoid the costs. MFCA is an international standard ISO 14051:2011. 15.2.3 Environmental benefits The intended environmental benefits of MFCA is to save resources, as well as saving al 15.3.1 Introduction A management system supports an organization to coordinate its decisions and actions towards its goals, in line with its policy and vision. There are many ways to organize a management system. Here we will refer to the management systems as published by the international standardization organization ISO. To facilitate world trade ISO has published two certifiable management systems that are related to management of the foundry’s environmental aspects within the context of this report;  ISO 14001 – Environmental management systems  ISO 50001 - Energy management systems  ISO 14001 – Environmental management systems  ISO 50001 - Energy management systems  ISO 50001 - Energy management systems It may be argued that also the quality management systems, as specified in ISO 9001, relates to environmental aspects, since it intends to minimize waste, mistakes and failure to meet customer requirements. 15.2.7 Reference Used in other industries not so common in Nordic foundries yet. 15.2.3 Environmental benefits The intended environmental benefits of MFCA is to save resources, as well as saving all other environmental loads associated with producing and transporting the material that is ending up as spill. Nordic Foundries Nordic Foundries 116 Applicability Applicability 15.2.4 Any foundry may gain economic and environmental benefits from regularly performing an MFCA. Can be difficult for subcontractors. 15.2.5 Cross-media effect MFCA leads to waste minimization, an energy and other resource savings. 15.2.6 Economics MFCA is an investigation technique, with costs similar to Value Stream Assessment or Risk Assessment. 15.3.5 A management system is needed to exploit the environmental benefits of other environmental techniques. 15.3.3 Environmental benefits Environmental benefits of a managements system are that the organization has a consistent overview of its policies and targets, and that the day-to-day operation and decisions can be validated and benchmarked to targets. Since emission, waste or energy reduction goals are decided by the organization’s management team it will not be omitted due to pressing economic or production related matters without a conscious decision. 15.3.2 Applied process and techniques A management system according to the mentioned ISO standards is a continual process, iterating through the Plan, Do, Check and Act-stages. At the Plan-stage, the organization decides on what to do and how to do it. At the Do-stage, the organization does what it has planned. At the Check-stage the organization follows up on how its intentions have been met. At the Act-stage the organization takes Nordic Foundries 117 corrective actions towards yet another loop of Plan-Do-Check-Act and so on. Parts of the Plan-stage is a strategic view of the organisation’s stakeholders, its risks and opportunities, and a (re-)formulation of its policy. One very important thing to get a well-functioning management system is to base the work on measurements. For example the foundry need to know how much energy it is using or how much waste is produced. 15.3.4 Applicability 15.3.4 Applicability All foundries can apply a management system with a structured Plan-Do-Check-Act approach for their environmentally related responsibilities. However, smaller foundries may need help to scale their management system to appropriately reflect their needs. 15.3.5 Cross-media effect 15.4.1 Introduction 15.4.1 Introduction Here digitalization addresses the ability to use computers to automate process control. The level of digitalization and automation still varies widely in the foundry industry. 15.4.2 Applied process and techniques There are no known limits to how to apply digitalization, but here tree environmentally related techniques for digitalization will be mentioned:  Automation: Monitoring of temperature to avoid over heating that results in too high energy use, or to under heating, that results in quality problems and consequential need to repeat the production.  Logging: Collection of production spill and quality loss data for for example MFCA- assessments, to be used for calculating production spill costs.  Management: Easy access to relevant performance values in relation to for example energy reduction targets, to provide easy feedback to operators. 15.4.3 Environmental benefits Environmental benefits with digitalization are to support foundry managers and operators to meet environmental goals without having to focus on them. Much may even be automated, as part of the equipment. 15.4.4 Applicability Digitalization is relevant for any foundry. SMEs may find digitalized documentation and automated individual machines to be within reach. Large foundries may consider strategic paths towards fully automated factories, with automated control of waste and emissions. 15.4.5 Cross-media effect 15.4.5 Cross-media effect 15.3.6 Economics There is a cost for introducing a management system in an organization. The highest cost relates to the organizational cultural change, to change from reactive day-to-day way of working towards working with a systematic Plan-Do-Check-Act driven organization. An organization that is already ISO 9001-certified finds it easier to start working also with environmentally related aspects of their organization. 15.3.7 Reference Several foundries in the Nordic countries. 15.3.7 Several foundries in the Nordic countries. Nordic Foundries 118 15.5.1 Introduction 15.5.1 Introduction Environmental communication is a wide term to describe how an organization exchanges environmentally relevant information with external stakeholders. Environmental communication often is a precondition to get paid for environmental investments, and may also provide the incentives for further environmental improvements. Here we focus on four types of environmental communication; The environmental report, Environmental communication with customers, Environmental communication with suppliers, and Environmental communication with the community. 15.4.7 Reference Different foundries work with digitalization and automation in different ways. Economics 15.4.6 Digitalization ranges from very small to very high costs. It is recommended that a thorough cost-benefit analyses is made in order to avoid digitalization that does not also lead to benefits. 15.4.7 Reference 15.4.5 Cross-media effect Digitalization is not limited to environmental benefits, but may be installed to facilitate environmental efficiency and environmental information within the factory. 119 Nordic Foundries Environmental communication is an enabler for environmental benefits The environmental report: Financiers and insurance companies can provide better conditions for companies that consciously manage their risks, such as risks for higher waste management costs, higher energy costs, transport costs, etc. Environmental communication with customers and suppliers Communicating about environmentally beneficial improvements is no guarantee, but environmentally conscious customers may favour your offer, hence replacing a less environmentally friendly alternative. Customers may not be aware of that their environmental footprint can be improved by combining efforts with their suppliers, hence a dialogue about alternatives may lead to environmentally favourable decisions. Environmental communication with suppliers may in particular save on transport. If this is combined with MFCA (see separate section) it may also lead to less spill and waste. 15.5.2 Applied process and techniques The environmental report (which may be part of the yearly economical report or a separate report) summarizes the company’s environmental aspects, in terms of quantifications and in terms of risks and opportunities, and also how the company handles these aspects. For example environmental report, which include eco balance analysis gives good general information. Environmental communication with customers may be done in several different ways: Environmental communication with customers may be done in several different ways  Labelling or declaration: Presenting environmental facts about the production and products.  Sales dialogue: Presenting and discussing different material- and production options with different environmental consequences together with a customer representative.  Environmental procurement guidelines: Agreement with customer to follow a specific guideline for environmental aspects of product and production. Nordic Foundries 120 Environmental communication with suppliers is similar to communication with customer, except for the different role:  Requesting environmental information: Requesting that the supplier disclose information which shows that they operate environmentally responsibly.  Supplier dialogue: Establish a dialogue with the supplier, both to learn if there may be environmental benefits from changing anything in how the contract is run, and to learn from each other.  Environmental procurement guidelines: Agreement with supplier to follow a specific guideline for environmental aspects of product and production. This approach may be favourable if a customer requests the same guidelines. Environmental communication with the local community is part of a responsible relationship with neighbours, local government and local NGOs. A good communication with the local community can save time and costs and avoid environmental consequences before they occur or at an early stage. 15.5.3 Environmental benefits Environmental communication is an enabler for environmental benefits. 15.5.5 Cross-media effect 15.5.5 No cross media-effect. No cross media-effect. 15.5.6 Economics As mentioned above environmental communication is part of an organization’s management system, and some of the communication costs does not require more costs than meetings with stakeholders. However, costs for different information material may be in the following ranges (SME-Large company):  Environmental report: EUR 2,000–15,000  Environmental label or declaration: EUR 5,000–50,000  Customer and supplier dialogue (additional information and training): EUR 2,000–25,000  Environmental procurement guideline (learn to apply an existing guideline): EUR 2,000–10,000  Environmental dialogue with community (prepare and hold meeting): EUR 2,000–10,000  Requesting information from supplier: EUR 2,000–50,000  Environmental communication with suppliers: EUR 2,000–15,000 15.5.4 Applicability Any foundry can apply environmental communication, but such communication needs to be well-established in the organizations management system, for stakeholder dialogues to be an integral part of the organization’s long term strategy and behaviour. Nordic Foundries 121 15.5.5 Cross-media effect No cross media-effect. 15.5.7 Reference Different foundries work with communication in different way. Danish companies have to deliver a “Green balance” annually together with the financial statement. Different foundries work with communication in different way. Danish companies have to deliver a “Green balance” annually together with the financial statement. Nordic Foundries 122 16. Emerging techniques There are a lot of emerging techniques but this report focus on available techniques and therefore this chapter only includes a few examples. 16.1.4 Applicability Difficult to say something about applicability today. Difficult to say something about applicability today. 16.1.2 Applied process and techniques There are two types of new release agents. One use a powder instead of the water and the other use more concentrated oil/wax without water. 16.1.1 Introduction Today high pressure die casting uses a release agent with a small amount oil or wax mixed with water that is sprayed on the die tool between each shot. 16.1.2 Applied process and techniques 16.1.3 Environmental benefits There will be less oil mist to clean. There will also be less water with oil/wax that has to be cleaned or sent as waste. 16.2.1 Introduction The sand binder used today can give emissions of VOC when the melt is poured into the mould. One way is to change binder. If this is not possible another way minimise the emission is to clean the outgoing air. It would be better if the air could be cleaned close to the source. 16.1.5 Cross-media effect There may be a need of a new type of die tool with better cooling system because the water that is sprayed today also have a cooling effect. 16.1.6 Economics Not known. Reference 16.1.7 Tested of different foundries both in the Nordic countries and in the rest of Europe but no one in the Nordic countries regulary use this technique.. 16.2.2 In a research project a method has been tested where a metal frame is placed over the sand mould where there is a VOC emission. High voltage is used to make an ignition flame which will make the VOC to start burning giving some kind of combustion of the VOC. 124 Nordic Foundries 16.2.3 Environmental benefits Less VOC emission to air. 16.2.4 Applicability Rather large sand moulds. 16.2.5 Cross-media effect Use energy to make the ignition. 16.2.6 Economics Not possible to buy today. 16.2.7 Reference Only test have been done with a pilot in LIFE+ research project Odourless casting. 16.2.3 Environmental benefits Less VOC emission to air. 16.2.7 Reference Only test have been done with a pilot in LIFE+ research project Odourless casting. 16.2.7 f Only test have been done with a pilot in LIFE+ research project Odourless casting. Only test have been done with a pilot in LIFE+ research project Odourless casting. Nordic Foundries 124 16.3.1 Introduction There is one common inorganic sand binder today, sodium silicate but it cannot be used for all casting purposes. Instead most foundries use an organic binder giving rise to emissions of VOC to the air. 16.3.2 Applied process and techniques 16.3.2 Applied process and techniques Instead of the organic binders used today an inorganic variant is used. Applicability Today cores in green sand foundries are manufactured with new sand and therefore some of the green sand is sent away as waste. Today cores in green sand foundries are manufactured with new sand and therefore some of the green sand is sent away as waste. 16.4.2 Applied process and techniques 16.4.2 Applied process and techniques 16.4.2 Applied process and techniques Mechanical reclamation, where coal dust and bentonite is grinded or rubbed off from the sand grain surfaces. Mechanical reclamation, where coal dust and bentonite is grinded or rubbed off from the sand grain surfaces. Nordic Foundries 125 16.4.3 Environmental benefits Sand that otherwise can be a waste will be used again in the foundry. 16.4.4 Applicability Not known which foundries that can use this in the future. 16.4.5 Cross-media effect Special equipment for the reclamation, and use of energy. 16.4.6 Economics Not known. 16.4.7 Reference Not used in the Nordic countries. Some foundries in Europe is testing this technique. 16.4.3 Sand that otherwise can be a waste will be used again in the foundry. Sand that otherwise can be a waste will be used again in the foundry. 4 Applicability Not known which foundries that can use this in the future. Special equipment for the reclamation, and use of energy. 16.4.6 Economics Not known. 16.4.7 Reference Not used in the Nordic countries. Some foundries in Europe is testing this technique. Not used in the Nordic countries. Some foundries in Europe is testing this technique. Not used in the Nordic countries. Some foundries in Europe is testing this technique. Nordic Foundries Nordic Foundries 126 References and contacts Ahlqvist K, Cannerborg M. Lukt och VOC från gjutprocesser. Swerea SWECAST report 081219. 2008. Andreasson A, Bengtsson A, Nilsson PÅ, Wigholm P, Johansson Ö. Bullerdämpning av externt industribuller från livsmedelsindustri och hamnar. TemaNord 2014:548. https://doi.org/10.6027/TN2014-548 p g The Association of Danish Foundries, contact with Søren Knudsen. The Association of Danish Foundries, contact with Søren Knudsen. Crepaz R, Renere produkter i støberibranchen. Teknologisk Institut Denmark, 2014. Environment Agency of Iceland, contact with Sigurður Ingason. European Commission, Establishing best available techniques (BAT) conclusions, under Directive 2010/75/EU of the European Parliament and of the Council, for the non-ferrous metals industries, 2016. European Commission, Reference Document on Best Available Techniques in the Smitheries and Foundries Industry, 2005. European Commission, Reference Document on Best Available Techniques in the Smitheries and Foundries Industry, 2005. Frees N, Olsen S, Tiedje N, Brancheindsats for jern- og metalstøberier. IPU, DTU Frees N, Olsen S, Tiedje N, Brancheindsats for jern- og metalstøberier. IPU, DTU 2002. Intelligent Energy Europe. Foundrybench. Good practice guide on energy saving potentials and opportunities for foundries. 2011. Intelligent Energy Europe. Foundrybench. Good practice guide on energy saving potentials and opportunities for foundries. 2011. Karlebo Gjuteriteknik, contact with Lars Blidfors, Richard Larsson, Roger Persson. Karlebo Gjuteriteknik, contact with Lars Blidfors, Richard Larsson, Roger Persson. LIFE. LIFE10 ENV/FI/059 Final report, Odourless casting. Odour and hazardous emission abatement of foundries 2014. LIFE. http://odorlesscasting.com/ Webpage for the life project Odourlesscasting. LIFE. Cost and energy efficiency assessment of odour abatement systems. Meehanite Technology Ltd and AX Consulting Ltd. 2014. Norsk industry, contact with Kari Rømcke. Norsk industry, contact with Kari Rømcke. Norsk industry, Støperistatistikk 2016. Norsk industry, Støperistatistikk 2016. Processfilter, contact with Yngve Traerup. Processfilter, contact with Yngve Traerup. Sommarin P, Arvidsson V, Värmelagring för energiintensiva SMF med fokus på svensk gjuteriindustri. Swerea SWECAST report 2011-006. Sommarin P, Arvidsson V, Värmelagring för energiintensiva SMF med fokus på svensk gjuteriindustri. Swerea SWECAST report 2011-006. Svensson A, Sommarin P, Bloom J, Lisell R, Energieffektiv smältning. Swerea SWECAST report 2012-010. Swedish Foundry Association. www.gjuterihandboken.se Swedish Foundry Association, contact with Peter Naystrom and Diana Bogic. Wänerholm M, Exponering av gasformiga ämnen och partiklar. Swerea SWECAST report 2011-010. Ålands miljö- och hälsoskyddsmyndighet, contact with Susanne Särs. Ålands miljö- och hälsoskyddsmyndighet, contact with Susanne Särs. Sammanfattning Nordiska ministerrådets BAT-grupp har beslutat att genomföra ett projekt om Bästa tillgängliga teknik (BAT) för gjuterier och smidesanläggningar i de Nordiska länderna. Inga smidesanläggningar uppfyller kriterierna i industriutsläppsdirektivet (IED) och därför har den här rapporten fokuserat enbart på gjuterier. gj Målen vid sammanställningen av den här rapporten har varit att:  ta fram en översikt över gjuteribranschen i de Nordiska länderna  beskriva använda och potentiella miljötekniker i gjuterier i de Nordiska länderna  beskriva viktiga miljöiaspekter ur de nordiska gjuteriernas perspektiv  identifiera och beskriva tekniker som hänsyn bör tas till när beskriver BAT för gjuterier. Arbetet har innefattat gjuterier som använder permanenta och icke permanenta formar. Fokus har varit på processer specifika för gjuteriindustrin, från simulering av gjutning till de gjutna produkterna, och i mindre omfattning några generella processer som är speciellt relevanta för gjutning. Den beskrivna informationen kan användas av gjuterier, miljökonsulter och miljömyndigheter. Projektet kan också bli användbart som ett nordiskt inspel till den tekniska arbetsgruppen i arbetet med revisionen av BAT-slutsatser för ”Smitheries and Foundries Industry” i enlighet med IED. Metallgjutning har en lång tradition och har tidigare varit en stor industri i de Nordiska länderna, men antalet gjuterier har minskat. Idag har Sverige flest gjuterier av de Nordiska länderna. De viktigaste miljöaspekterna som behandlas i denna rapport är (inte prioriterad ordning):  Utsläpp till luft (partiklar och VOC)  Buller och vibrationer  Utsläpp till vatten och vattenanvändn  Användning av kemikalier och risker  Avfall/restprodukter  Energianvändning  Råvaror  Transporter  Utsläpp till luft (partiklar och VOC)  Buller och vibrationer  Utsläpp till vatten och vattenanvändning  Användning av kemikalier och risker  Avfall/restprodukter  Energianvändning  Råvaror  Transporter Beskrivna BAT för gjuterier innefattar:  BAT kontroll av emissioner till luft  BAT för att förhindra buller och vibrationer  BAT kontroll av utsläpp till vatten  BAT pressgjutning  BAT sand och bindemedel  BAT kemikaliehantering och substitution  BAT avfallshantering och minimerade avfallsmängder  BAT simulering  BAT energi  BAT metall och smälta  BAT miljöledning Teknisk användbarhet, ekonomiska kostnader och miljöfördelar för de olika BAT varierar kraftigt från fall till fall. Det är också värt att betona att på grund av befintliga processer, produkter eller utformning av gjuterierna är de inte alltid fria att välja BAT. Presenterade BAT-tekniker listas i nedan. 130 Nordic Foundries BAT kandidater utsläpp till luft 130 Nordic Foundries BAT kandidater utsläpp till luft  Textila spärrfilter  Använda tryckfallsmätare  Stoftmätning  Uppvärmda filter  Använda fluorescerande pulver för att kontrollera textila spärrfilter  Stoftmätare  Syraskrubber för att rena amin  Oljedimfilter  Plan för att reducera VOC  Lock på blackkar  Använda black endast vid behov  Sotersättningsmedel  Rena VOC och lukt med biofilter  Rena VOC och lukt med RTO  Textila spärrfilter  Använda tryckfallsmätare  Stoftmätning  Uppvärmda filter  Använda fluorescerande pulver för att  Stoftmätare  Syraskrubber för att rena amin  Oljedimfilter  Plan för att reducera VOC  Lock på blackkar  Använda black endast vid behov  Sotersättningsmedel  Rena VOC och lukt med biofilter  Rena VOC och lukt med RTO  Textila spärrfilter  Använda tryckfallsmätare  Stoftmätning  Uppvärmda filter  Använda fluorescerande pulver för att kontrollera textila spärr  Stoftmätare  Syraskrubber för att rena amin  Oljedimfilter  Plan för att reducera VOC  Lock på blackkar  Använda black endast vid behov  Sotersättningsmedel  Rena VOC och lukt med biofilter  Rena VOC och lukt med RTO  Textila spärrfilter 130 BAT kandidater buller  Tippa skrot inomhus  Bullerreduceringsplan  Bullersimulering  Ljuddämpare på ventilationen  Tilläggsisolera fläktar och ventilationskanaler BAT kandidater kemikalier  Kemikalieansvarig BAT kandidater avfall och restprodukter  Kartlägga avfallsflöden  Mekanisk sandåtervinning  Använda restsand utanför gjuteriet  Brikettering BAT kandidater simulering  Simulering  Optimerat ingjutsystem BAT kandidater energi  Energikartläggning  Blästra råmaterial före smältning  Krossa ingjutsystem  Isolerade skänkar  Lock på ugnar och skänkar  Flamlös skänkförvärmning BAT kandidater kemikalier Nordic Foundries 131 BAT kandidater metall och smältning  Skrothantering  Användning av deoxidationsmedel  Spola skänk med argon  Ympkontroll  Automatisk ympning BAT kandidater pressgjutning  Underhåll av pressgjutverktyg  Förvärm pressgjutverktyg  IR kamera  Använd vakuumdoseringsugn Kommande tekniker  Nya släppmedel för pressgjutning  Antändning av VOC i sandform  Nya oorganiska bindemedel  Använda råsand för kärntillverkning. BAT kandidater metall och smältning  Skrothantering  Användning av deoxidationsmedel  Spola skänk med argon  Ympkontroll  Automatisk ympning Nordic Foundries 132 Appendix All IED foundries in the Nordic countries were contacted in the project with this questionnaire. The foundries also were asked to reflect if used technique is BAT or if the foundry knows some better technique. Air Air  What technology / working practices do you use to clean the outgoing air from dust, oil mist, odour and volatile organic compounds VOC?  What technology / working practices do you use to prevent emissions to air to arise at all?  What technology / working practices do you use to clean the outgoing air from dust, oil mist, odour and volatile organic compounds VOC?  What technology / working practices do you use to prevent emissions to air to arise at all? Chemicals (process chemicals and auxiliary chemicals)  How do you work to reduce the amount of sand that is spent?  What happens to the sand that you cannot reuse? Binder  How do you minimize the use of binders?  What are the possibilities out replace your binder with one more environmental friendly? Management 134 Nordic Foundries  Do you have a systematic improvement in production and logistics, such as a certified management system according to (ISO 9001, EMAS, ISO 14001, ISO 50001)? Waste  How do you store waste?  How are you working to minimize the amounts of waste in general?  How are you working to minimize the amounts of slag?  Is your waste used as a resource somewhere else, or could it be done? Simulation  How do you use simulation to reduce scrap?  Do you use simulation to verify process stability (e.g. temperature impact on shrink / suctions)?  In what way do you use the results from the simulation to make feedback to the customer and to influence the design?  Noise  What technologies / working practices are you taking to reduce noise from the foundry? Sand  Do you have sand reclamation, what kind of reclamation and what degree of recovery do you reach? Please comment both core and moulding sand  The sand that is recycled internally how it is used in the foundry?  How do you work to reduce the amount of sand that is spent?  What happens to the sand that you cannot reuse? Binder  How do you minimize the use of binders?  What are the possibilities out replace your binder with one more environmental friendly?  Which binders do you use? Waste Sand  Do you have sand reclamation, what kind of reclamation and what degree of recovery do you reach? Please comment both core and moulding sand  The sand that is recycled internally how it is used in the foundry?  How do you work to reduce the amount of sand that is spent?  What happens to the sand that you cannot reuse? Binder  How do you minimize the use of binders?  What are the possibilities out replace your binder with one more environmental friendly?  Which binders do you use?  How do you minimize the use of binders?  How do you minimize the use of binders? Nordic Foundries Chemicals (process chemicals and auxiliary chemicals)  How do you work to reduce the amount of chemicals used?  How do you work to replace hazardous chemicals?  How do you handle hazardous chemical substances?  How do you handle hazardous chemical substances? Energy  How do you ensure that the raw material (metal) is dry and free from oil, paint or sand?  How do you ensure optimal packing in the oven e.g. by crushing large material?  How do you work to optimize the melt (for example, apply packing density, melting time, to charge in the right order, to fill the oven while still hot for example after the end of the day)?  How are you working to reduce the need to overheat the melt?  Do you use holding furnace, and what do you do to minimize the time it is used?  Do you use a lid on furnaces / ladles during production / operating break?  What kind of preheat of the ladle do you use?  How do you reduce energy consumption for preheating ladle (e.g. insulation, plan melting so that you do not need to reheat unnecessarily, lid on ladles)?  How do you work to reduce the energy consumption for melting?  How do you work with taking care of waste heat? Management  Do you have a systematic improvement in production and logistics, such as a certified management system according to (ISO 9001, EMAS, ISO 14001, ISO 50001)? Waste  How do you store waste?  How are you working to minimize the amounts of waste in general?  How are you working to minimize the amounts of slag?  Is your waste used as a resource somewhere else, or could it be done? Simulation  How do you use simulation to reduce scrap?  Do you use simulation to verify process stability (e.g. temperature impact on shrink / suctions)?  In what way do you use the results from the simulation to make feedback to the customer and to influence the design?  Noise  What technologies / working practices are you taking to reduce noise from the foundry? Sand  Do you have sand reclamation, what kind of reclamation and what degree of recovery do you reach? Please comment both core and moulding sand  The sand that is recycled internally how it is used in the foundry? Chemicals (process chemicals and auxiliary chemicals)  What are the possibilities out replace your binder with one more environmental friendly?  Which binders do you use? Nordic Foundries 134 Water  How do you purify any water that occurs?  How do you minimize the amount of water that occurs? Die casting  How do you mixture the release agent?  How do you spray release agents?  How do you take care of release agent residues?  How do you minimize the use of release agents?  How do you work to extend the life of the tools? Blacking (coating of cores)  What kind of blacking/coating of cores do you use?  How do you do to reduce the amount of blacking/coating?  If you have water blacking how do you is dry it?  If you have alcohol-based black, how do you do to reduce emissions to the air?  Do you burn of alcohol-based black? Oven  What do you use for the type of furnace? Other  Do you quantify and describe the environmental impact of your products, raw materials or waste, using e.g. environmental labelling, life cycle analysis, or in any other way?  Do you resume your own products for remelting when customers no longer want to use them?  Something else we have not asked about but that you want to highlight? For Water  How do you purify any water that occurs?  How do you minimize the amount of water that occurs? Die casting  How do you mixture the release agent?  How do you spray release agents?  How do you take care of release agent residues?  How do you minimize the use of release agents?  How do you work to extend the life of the tools? Blacking (coating of cores)  What kind of blacking/coating of cores do you use?  How do you do to reduce the amount of blacking/coating?  If you have water blacking how do you is dry it?  If you have alcohol-based black, how do you do to reduce emissions to the air?  Do you burn of alcohol-based black? Oven  What do you use for the type of furnace? Other  Do you quantify and describe the environmental impact of your products, raw materials or waste, using e.g. environmental labelling, life cycle analysis, or in any other way? Chemicals (process chemicals and auxiliary chemicals)  Do you resume your own products for remelting when customers no longer want to use them?  Something else we have not asked about but that you want to highlight? For example, special techniques / working practices that you use. Water Die casting g ( g )  What kind of blacking/coating of cores do you use?  How do you do to reduce the amount of blacking/coating?  If you have water blacking how do you is dry it?  If you have alcohol-based black, how do you do to reduce emissions to the air?  Do you burn of alcohol-based black? Oven  What do you use for the type of furnace? Other  Do you quantify and describe the environmental impact of your products, raw materials or waste, using e.g. environmental labelling, life cycle analysis, or in any other way?  Do you resume your own products for remelting when customers no longer want to use them?  Something else we have not asked about but that you want to highlight? For example, special techniques / working practices that you use. Nordic Foundries 135 Nordic Foundries The Nordic Council of Ministers BAT Group under the Working Group for sustainable consumption and production, commissioned to Swerea SWECAST AB to prepare a Best Available Techniques (BAT) report for foundries in the Nordic countries. The objectives have been to: • Provide an overview of the foundry sector in the Nordic countries. • Present currently used and potent foundries in the Nordic countries. • Present the Key environmental issues with the perspective of foundries in the Nordic countries. • Present and describe techniques that shall be included in the • Present and describe techniques that shall be inc considerations of representing BAT in foundries. considerations of representing BAT in foundries. This report focuses only on foundries as no smitheries satisfies the criteria of the Industrial Emissions Directive (IED). Swerea SWECAST is a research institute based in Sweden specializing on foundry and casting.
https://openalex.org/W2159836597	https://advancesindifferenceequations.springeropen.com/counter/pdf/10.1186/1687-1847-2014-301	Latin	null	Dynamics of a two-dimensional competitive system of rational difference equations with quadratic terms	Advances in difference equations	2,014	cc-by	19,076	R ES EARCH Open Access ©2014 Hadžiabdi´c et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dynamics of a two-dimensional competitive system of rational difference equations with quadratic terms Vahidin Hadžiabdi´c1, Mustafa RS Kulenovi´c2* and Esmir Pilav3 *Correspondence: mkulenovic@mail.uri.edu 2Department of Mathematics, University of Rhode Island, Kingston, Rhode Island 02881-0816, USA Full list of author information is available at the end of the article Abstract We investigate global dynamics of the following systems of diﬀerence equations: ⎧ ⎨ ⎩ xn+1 = b1x2n A1+y2n , yn+1 = a2+c2y2n x2n , n = 0,1,2,..., where the parameters b1, a2, A1, c2 are positive numbers and the initial condition y0 is an arbitrary nonnegative number and x0 is a positive number. We show that this system has rich dynamics which depends on the part of a parametric space. We ﬁnd precisely the basins of attraction of all attractors including the points at ∞. MSC: Primary 39A10; 39A30; secondary 37E99; 37D10 Keywords: basin of attraction; competitive map; global stable manifold; monotonicity; period-two solution Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Theorem Consider system (). () Assume that β> Aand γA̸= α. Then there exist a set C ⊂R which is invariant and a subset of the basin of attraction of E. The set C is a graph of a strictly increasing continuous function of the ﬁrst variable on an interval (and so is a manifold) and separates R into two connected and invariant components, namely W– := {x ∈R\C : ∃y ∈C with x ⪯se y} and W+ := {x ∈R\C : ∃y ∈C with y ⪯se x}, which satisfy: which satisfy: lim n→∞(xn,yn) = (,∞) for every (x,y) ∈W–, lim n→∞(xn,yn) = (,∞) for every (x,y) ∈W–, and and lim n→∞(xn,yn) = (∞,) for every (x,y) ∈W+. lim n→∞(xn,yn) = (∞,) for every (x,y) ∈W+. () Assume that γA= α. Then system () can be decoupled as follows: xn+= βx n Axn + βγ , yn+=  β yn(A+ yn), n = ,,..., and every solution of this system (depending of the choice of the initial condition (x,y)) is either bounded and converges to an equilibrium point, or increases monotonically to inﬁnity. and every solution of this system (depending of the choice of the initial condition (x,y)) is either bounded and converges to an equilibrium point, or increases monotonically to inﬁnity. () Assume that β≤Aand γA̸= α. Every solution {(xn,yn)} of system (), with x> , y≥, satisﬁes lim n→∞xn =  and lim n→∞yn = ∞. lim n→∞xn =  and lim n→∞yn = ∞. Thus every solution of system () either converges to the unique equilibrium point or is asymptotic to one of the points at inﬁnity, precisely to either (,∞) or to (∞,). In all cases, either solution is eventually monotonic or the subsequences of even indexed and odd indexed terms are eventually monotonic. Introduction of quadratic terms into the system will substantially change the dynamics by introducing new equilibrium points (up to three) with diﬀerent local character and minimal period-two solutions (up to ). Again, most of the solutions of system () will be asymptotic to (∞,) or (,∞), but the separatrix between the two basins of attraction may consist of several global stable manifolds of either saddle point equilibrium points or non-hyperbolic equilibrium points or minimal period- two solutions. In one case, when there exists a unique non-hyperbolic equilibrium point, it is possible that this point will have a basin of attraction of positive Lebesgue measure. 1 Introduction In this paper we study the global dynamics of the following rational system of diﬀerence equations: ⎧ ⎨ ⎩ xn+= bxn A+yn , yn+= a+cyn xn , n = ,,,..., () () where the parameters b, a, A, care positive numbers and the initial condition yis an arbitrary nonnegative number and xis a positive number. where the parameters b, a, A, care positive numbers and the initial condition yis an arbitrary nonnegative number and xis a positive number. The related system of diﬀerence equations The related system of diﬀerence equations xn+= βxn A+ yn , yn+= α+ γyn xn , n = ,,..., () () where the parameters A, β, αand γare positive numbers and the initial conditions x> , y≥, was considered in [], where it was shown that this system has simple dynamics. Precisely, it was shown that system () has no equilibrium points if β≤Aand that it has a unique equilibrium point if β> A, in which case this equilibrium point is a saddle point. Furthermore, the following result describes the global dynamics of system (). where the parameters A, β, αand γare positive numbers and the initial conditions x> , y≥, was considered in [], where it was shown that this system has simple dynamics. Precisely, it was shown that system () has no equilibrium points if β≤Aand that it has a unique equilibrium point if β> A, in which case this equilibrium point is a saddle point. Furthermore, the following result describes the global dynamics of system (). Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 2 of 32 xn+= xn a + yn , yn+= yn b + xn , n = ,,..., () Theorem Consider system (). System () is a competitive system, and our results are based on recent results about competitive systems in the plane, see [, ]. System () can be used as a mathematical model for competition in population dynamics. The ﬁrst systematic study of a speciﬁc competitive system with quadratic terms was performed in [] where system of the form xn+= xn a + yn , yn+= yn b + xn , n = ,,..., () () Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 3 of 32 q http://www.advancesindifferenceequations.com/content/2014/1/301 http://www.advancesindifferenceequations.com/content/2014/1/301 where the parameters a,b > and the initial conditions x,y≥, was considered. It was shown that the dynamics of system () is very similar to the dynamics of the corresponding linear fractional system xn+= xn a + yn , yn+= yn b + xn , n = ,,..., with the same conditions on parameters and initial conditions. Both systems have nine parametric regions with diﬀerent dynamical behavior. As noted, the introduction of quadratic terms in system () dramatically changes the dy- namics. The techniques used to study system () were straightforward calculations, while the techniques which will be used to study system () are a combination of techniques for studying real algebraic curves and implicit function theorem as neither equilibrium points nor period-two solutions are explicitly computable. Some of our calculations are performed by using Mathematica and outputs are included in the Appendix. The paper is organized as follows. Section contains some necessary results on com- petitive systems in the plane. Section provides some basic facts about the equilibrium points and injectivity of the map associated with system (). Section contains local sta- bility analysis of both equilibrium solutions and minimal period-two solutions. Section  gives global dynamics in diﬀerent cases. 2 Preliminaries A ﬁrst-order system of diﬀerence equations A ﬁrst-order system of diﬀerence equations xn+= f (xn,yn), yn+= g(xn,yn), n = ,,,..., () () where S ⊂R, (f ,g) : S →S, f , g are continuous functions, is competitive if f (x,y) is non-decreasing in x and non-increasing in y, and g(x,y) is non-increasing in x and non- decreasing in y. If both f and g are non-decreasing in x and y, system () is cooperative. Competitive and cooperative maps are deﬁned similarly. Strongly competitive systems of where S ⊂R, (f ,g) : S →S, f , g are continuous functions, is competitive if f (x,y) is non-decreasing in x and non-increasing in y, and g(x,y) is non-increasing in x and non- decreasing in y. If both f and g are non-decreasing in x and y, system () is cooperative. Competitive and cooperative maps are deﬁned similarly. Strongly competitive systems of diﬀerence equations or strongly competitive maps are those for which the functions f and g are coordinate-wise strictly monotone. Competitive and cooperative systems have been investigated by many authors, see [, , –]. Special attention to discrete competitive and cooperative systems in the plane was given in [, , , , , , –]. One of the rea- sons for paying special attention to two-dimensional discrete competitive and cooperative systems is their applicability to mathematical models in biology and economics, the for- mer involves competition or cooperation between two species. Another reason is that the theory of two-dimensional discrete competitive and cooperative systems is very well de- veloped, unlike such theory for three and higher dimensional systems. Part of the reason for this situation is de Mottoni-Schiaﬃno theorem given below, which provides relatively simple scenarios for possible behavior of many two-dimensional discrete competitive and cooperative systems. However, this does not mean that one cannot encounter chaos in such systems as has been shown by Smith, see []. where S ⊂R, (f ,g) : S →S, f , g are continuous functions, is competitive if f (x,y) is non-decreasing in x and non-increasing in y, and g(x,y) is non-increasing in x and non- decreasing in y. If both f and g are non-decreasing in x and y, system () is cooperative. Competitive and cooperative maps are deﬁned similarly. 2 Preliminaries Strongly competitive systems of diﬀerence equations or strongly competitive maps are those for which the functions f and g are coordinate-wise strictly monotone. Competitive and cooperative systems have been investigated by many authors, see [, , –]. Special attention to discrete competitive and cooperative systems in the plane was given in [, , , , , , –]. One of the rea- sons for paying special attention to two-dimensional discrete competitive and cooperative systems is their applicability to mathematical models in biology and economics, the for- mer involves competition or cooperation between two species. Another reason is that the theory of two-dimensional discrete competitive and cooperative systems is very well de- veloped, unlike such theory for three and higher dimensional systems. Part of the reason for this situation is de Mottoni-Schiaﬃno theorem given below, which provides relatively simple scenarios for possible behavior of many two-dimensional discrete competitive and cooperative systems. However, this does not mean that one cannot encounter chaos in such systems as has been shown by Smith, see []. If v = (u,v) ∈R, we denote by Qℓ(v), ℓ∈{,,,} the four quadrants in Rrelative to v, i.e., Q(v) = {(x,y) ∈R: x ≥u,y ≥v}, Q(v) = {(x,y) ∈R: x ≤u,y ≥v}, and so on. Deﬁne the south-east partial order ⪯se on Rby (x,y) ⪯se (s,t) if and only if x ≤s and y ≥t. Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 4 of 32 Hadžiabdic et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Similarly, we deﬁne the north-east partial order ⪯ne on Rby (x,y) ⪯ne (s,t) if and only if x ≤s and y ≤t. For A ⊂Rand x ∈R, deﬁne the distance from x to A as dist(x,A) := inf{∥x – y∥: y ∈A}. By intA we denote the interior of a set A. It is easy to show that a map F is competitive if it is non-decreasing with respect to the south-east partial order, that is, if the following holds: x y ⪯se x y ⇒ F x y ⪯se F x y . For standard deﬁnitions of attracting ﬁxed point, saddle point, stable manifold, and re- lated notions, see [, ]. We now state three results for competitive maps in the plane. The following deﬁnition is from []. Deﬁnition Let S be a nonempty subset of R. 2 Preliminaries A competitive map T : S →S is said to satisfy condition (O+) if for every x, y in S, T(x) ⪯ne T(y) implies x ⪯ne y, and T is said to satisfy condition (O–) if for every x, y in S, T(x) ⪯ne T(y) implies y ⪯ne x. The following theorem was proved by de Mottoni and Schiaﬃno [] for the Poincaré map of a periodic competitive Lotka-Volterra system of diﬀerential equations. Smith gen- eralized the proof to competitive and cooperative maps []. Theorem Let S be a nonempty subset of R. If T is a competitive map for which (O+) holds, then, for all x ∈S, {Tn(x)} is eventually component-wise monotone. If the orbit of x has compact closure, then it converges to a ﬁxed point of T. If instead (O–) holds, then, for all x ∈S, {Tn(x)} is eventually component-wise monotone. If the orbit of x has compact closure in S, then its omega limit set is either a period-two orbit or a ﬁxed point. The following result is from [], with the domain of the map specialized to be the carte- sian product of intervals of real numbers. It gives a suﬃcient condition for conditions (O+) and (O–). Theorem Let R ⊂Rbe the cartesian product of two intervals in R. Let T : R →R be a Ccompetitive map. If T is injective and detJT(x) > for all x ∈R, then T satisﬁes (O+). If T is injective and detJT(x) < for all x ∈R, then T satisﬁes (O–). The following result is a direct consequence of the trichotomy theorem of Dancer and Hess, see [] and [], and is helpful for determining the basins of attraction of the equi- librium points. Corollary If the nonnegative cone of ⪯is a generalized quadrant in Rn, and if T has no ﬁxed points in Ju,uK other than uand u, then the interior of Ju,uK is either a subset of the basin of attraction of uor a subset of the basin of attraction of u. The next result is a well-known global attractivity result which holds in partially ordered Banach spaces as well, see []. Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 5 of 32 Hadžiabdi´c et al. 2 Preliminaries Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Theorem Let T be a monotone map on a closed and bounded rectangular region R ⊂R. Suppose that T has a unique ﬁxed point ¯e in R. Then ¯e is a global attractor of T on R. The following theorems were proved by Kulenović and Merino [] for competitive sys- tems in the plane, when one of the eigenvalues of the linearized system at an equilibrium (hyperbolic or non-hyperbolic) is by an absolute value smaller than , while the other has an arbitrary value. These results are useful for determining basins of attraction of ﬁxed points of competitive maps. Theorem Let T be a competitive map on a rectangular region R ⊂R. Let x ∈R be a ﬁxed point of T such that := R ∩int(Q(¯x) ∪Q(x)) is nonempty (i.e., x is not the NW or SE vertex of R), and T is strongly competitive on . Suppose that the following statements are true. (a) The map T has a Cextension to a neighborhood of x. (a) The map T has a Cextension to a neighborhood of x. (b) The Jacobian JT(x) of T at x has real eigenvalues λ, μ such that < \|λ\| < μ, where \|λ\| < , and the eigenspace E associated with λ is not a coordinate axis. Then there exists a curve C ⊂R through x that is invariant and a subset of the basin of attraction of x such that C is tangential to the eigenspace Eλ at x, and C is the graph of a strictly increasing continuous function of the ﬁrst coordinate on an interval. Any endpoints of C in the interior of R are either ﬁxed points or minimal period-two points. In the latter case, the set of endpoints of C is a minimal period-two orbit of T. The situation where the endpoints of C are boundary points of R is of interest. The following result gives a suﬃcient condition for this case. Theorem For the curve C of Theorem to have endpoints in ∂R, it is suﬃcient that at least one of the following conditions is satisﬁed. (i) The map T has no ﬁxed points nor periodic points of minimal period two in . (ii) The map T has no ﬁxed points in , detJT(x) > , and T(x) = x has no solutions x ∈. 2 Preliminaries (iii) For every x ∈W–, there exists n∈N such that Tn(x) ∈intQ(x) for n ≥n (iv) For every x ∈W+, there exists n∈N such that Tn(x) ∈intQ(x) for n ≥n. If T is a map on a set R and if x is a ﬁxed point of T, the stable set Ws(x) of x is the set {x ∈R : Tn(x) →x} and unstable set Wu(x) of x is the set x ∈R : there exists {xn} n=–∞⊂R s.t. T(xn) = xn+,x= x, and lim n→–∞xn = x . When T is non-invertible, the set Ws(x) may not be connected and made up of inﬁnitely many curves, or Wu(x) may not be a manifold. The following result gives a description of the stable and unstable sets of a saddle point of a competitive map. If the map is a diﬀeomorphism on R, the sets Ws(x) and Wu(x) are the stable and unstable manifolds of x. Theorem In addition to the hypotheses of part (B) of Theorem , suppose that μ > and that the eigenspace Eμ associated with μ is not a coordinate axis. If the curve C of Theorem  has endpoints in ∂R, then C is the stable set Ws(x) of x, and the unstable set Wu(x) of x is a curve in R that is tangential to Eμ at x and such that it is the graph of a strictly decreasing function of the ﬁrst coordinate on an interval. Any endpoints of Wu(x) in R are ﬁxed points of T. The following result gives information on local dynamics near a ﬁxed point of a map when there exists a characteristic vector whose coordinates have negative product and such that the associated eigenvalue is hyperbolic. This is a well-known result, valid in a much more general setting: we include it here for completeness. A point (x,y) is a subso- lution if T(x,y) ⪯se (x,y), and (x,y) is a supersolution if (x,y) ⪯se T(x,y). An order interval J(a,b),(c,d)K is the cartesian product of the two compact intervals [a,c] and [b,d]. Theorem Let T be a competitive map on a rectangular set R ⊂Rwith an isolated ﬁxed point x ∈R such that R ∩int(Q(x) ∪Q(x)) ̸= ∅. Suppose that T has a Cextension to a neighborhood of x. Let v = (v(),v()) ∈Rbe an eigenvector of the Jacobian of T at x, with associated eigenvalue μ ∈R. 2 Preliminaries (iii) The map T has no points of minimal period two in , detJT(x) < , and T(x) = x has no solutions x ∈. The next result is useful for determining basins of attraction of ﬁxed points of compet- itive maps. Theorem (A) Assume the hypotheses of Theorem , and let C be the curve whose existence is guaranteed by Theorem . If the endpoints of C belong to ∂R, then C separates R into two connected components, namely Theorem (A) Assume the hypotheses of Theorem , and let C be the curve whose existence is guaranteed by Theorem . If the endpoints of C belong to ∂R, then C separates R into two connected components, namely W– := {x ∈R\C : ∃y ∈C with x ⪯se y} and W+ := {x ∈R\C : ∃y ∈C with y ⪯se x}, such that the following statements are true. (i) W– is invariant, and dist(Tn(x),Q(x)) →as n →∞for every x ∈W–. (i) W– is invariant, and dist(Tn(x),Q(x)) →as n →∞for every x ∈W–. (ii) W+ is invariant, and dist(Tn(x),Q(x)) →as n →∞for every x ∈W+. (ii) W+ is invariant, and dist(Tn(x),Q(x)) →as n →∞for every x ∈W (ii) W+ is invariant, and dist(Tn(x),Q(x)) →as n →∞for every x ∈W+. Page 6 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 http://www.advancesindifferenceequations.com/content/2014/1/301 (B) If, in addition to the hypotheses of part (A), x is an interior point of R and T is Cand strongly competitive in a neighborhood of x, then T has no periodic points in the boundary of Q(x) ∪Q(x) except for x, and the following statements are true. (iii) For every x ∈W–, there exists n∈N such that Tn(x) ∈intQ(x) for n ≥n. (iv) For every x ∈W+, there exists n∈N such that Tn(x) ∈intQ(x) for n ≥n. (B) If, in addition to the hypotheses of part (A), x is an interior point of R and T is Cand strongly competitive in a neighborhood of x, then T has no periodic points in the boundary of Q(x) ∪Q(x) except for x, and the following statements are true. (iii) For every x ∈W–, there exists n∈N such that Tn(x) ∈intQ(x) for n ≥n. (iv) For every x ∈W+, there exists n∈N such that Tn(x) ∈intQ(x) for n ≥n. Lemma Let = –,a Ab c+ a  ,A b c + A  – a A b c + A c– b c  + ,a b – A b c + A c  and = –aA c+ a A+ A c – b c . = –aA c+ a A+ A c – b c . 3 Some basic facts In this section we give some basic facts which will be used later. The map T associated to system () is given by T(x,y) = f (x,y),g(x,y) = bx A+ y, a+ cy x . () () Let Let R = R + \ (,y) : y ≥ . R = R + \ (,y) : y ≥ . R = R + \ (,y) : y ≥ . 2 Preliminaries If v()v() < , then there exists an order interval I which is also a relative neighborhood of x such that for every relative neighborhood U ⊂I of x the following statements are true. (i) If μ > , then U ∩intQ(x) contains a subsolution and U ∩intQ(x) contains a supersolution. In this case, for every x ∈I ∩int(Q(x) ∪Q(x)), there exists N such that Tn(x) /∈I for n ≥N. (ii) If μ < , then U ∩intQ(x) contains a supersolution and U ∩intQ(x) contains a subsolution. In this case Tn(x) →x for every x ∈I. Page 7 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 3.1 Equilibrium points The equilibrium points (¯x, ¯y) of system () satisfy the equations b¯x ¯y+ A = ¯x, c¯y+ a ¯x = ¯y. () b¯x ¯y+ A = ¯x, c¯y+ a ¯x = ¯y. () By eliminating ¯x ̸= from (), we get By eliminating ¯x ̸= from (), we get By eliminating ¯x ̸= from (), we get ¯y+ A¯y– b c¯y+ A ¯y – ab = . () ¯y+ A¯y– b c¯y+ A ¯y – ab = . Similarly, we can eliminate variable ¯y from system () to obtain Similarly, we can eliminate variable ¯y from system () to obtain Similarly, we can eliminate variable ¯y from system () to obtain b¯x– A¯x– b c ¯x– bc(a– Ac)¯x – (a– Ac)= . () () Then the following statements hold: Then the following statements hold: (a) Consider equation (). Then all its real roots are positive numbers. Furthermore, equation () has one, two, or three real roots. (b) If > , then equation () has one real root and two pairs of distinct conjugate imaginary roots. (c) If < , then equation () has three distinct real roots and one pair of conjugate imaginary roots. (d) If = and ̸= , then equation () has one pair of conjugate imaginary roots and two real roots, one real root of multiplicity one and other one of multiplicity two. (e) If = and = , then equation () has one pair of conjugate imaginary roots and one real root of multiplicity three. Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 8 of 32 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 8 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Proof The proof of (a) follows from Descartes’ rule of signs. Let Proof The proof of (a) follows from Descartes’ rule of signs. Let ˜f (y) = y+ Ay– b cy+ A y – ab . ˜f (y) = y+ Ay– b cy+ A y – ab . ˜f (y) = y+ Ay– b cy+ A y – ab . The following matrix, called the discrimination matrix of ˜f (y) and ˜f ′(y) in [], is actually the Sylvester matrix of ˜f (y) and ˜f ′(y) with some permuted rows. Discr(˜f ) = ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝   A –b c A  –ab         A –b c A         A –b c A  –ab         A –b c A         A –b c A  –ab         A –b c A         A –b c A  –ab         A –b c A         A –b c A  –ab         A –b c A  ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠ . Let Dk denote the determinant of the submatrix of Discr(˜f ), formed by the ﬁrst k row and the ﬁrst k columns, for k = ,...,m. is invariant under the map T. Furthermore, the following holds: T x, bx– A¯x ¯x = (¯x, ¯y) for x ≥ A¯x b . T x, bx– A¯x ¯x = (¯x, ¯y) for x ≥ A¯x b . () If Ac> a, then T satisﬁes (O+), in which case {Tn(x,y)} is asymptotic to either (,∞) or (∞,), or to an equilibrium point, for all (x,y) ∈R. () If Ac< a, then T satisﬁes (O–), in which case {Tn(x,y)} is asymptotic to either (,∞) or (∞,), or to a period-two point, for all (x,y) ∈R. () If Ac< a, then T satisﬁes (O–), in which case {Tn(x,y)} is asymptotic to either (,∞) or (∞,), or to a period-two point, for all (x,y) ∈R. Proof () Assume that T(x,y) = T(x,y). Then we have Proof () Assume that T(x,y) = T(x,y). Then we have Proof () Assume that T(x,y) = T(x,y). Then we have Proof () Assume that T(x,y) = T(x,y). Then we have Abx – Abx + bx y – bx y  (A+ y )(A+ y ) , –ax + ax – cx y + cx y  x x  = (,). () () Equation () is equivalent to Equation () is equivalent to x  Ab+ by  – Abx – bx y = , () x  –a– cy  + ax + cx y = . () () () uation () implies Equation () implies x = x (A+ y ) A+ y  . () x = x (A+ y ) A+ y  . () By substituting this into equation (), we obtain By substituting this into equation (), we obtain x (y – y )(a– Ac) A+ y  = , from which it follows that y= ysince a̸= Ac. From () we have x= x, which com- pletes the proof of statement (a). from which it follows that y= ysince a̸= Ac. From () we have x= x, which com- pletes the proof of statement (a). () One can see that () One can see that T x, bx– A¯x ¯x – (¯x, ¯y) = , bc– ¯x¯y ¯x . T x, bx– A¯x ¯x – (¯x, ¯y) = , bc– ¯x¯y ¯x . Then the following statements hold: () If a= Ac, then the curve bx= ¯x A+ y 3.2 Injectivity, (O+) and (O–) 3.2 Injectivity, (O+) and (O ) Lemma Assume that (¯x, ¯y) is an equilibrium of the map T. Then the following hold: () If a̸= Ac, then T is injective. () If a= Ac, then the curve Lemma Assume that (¯x, ¯y) is an equilibrium of the map T. Then the following hold: () If a̸= Ac, then T is injective. bx= ¯x A+ y is invariant under the map T. Furthermore, the following holds: Then the following statements hold: So, by a straightforward calculation, one can see that D= , D= –A, D= –A – b c , D= b  –aA c+ a A+ A c – b c  = b , D= b  –,a Ab c+ a  ,A b c + A  – a A b c + A c– b c  + ,a b – A b c + A c  = b . D= –A, D= –A – b c , D= b  –aA c+ a A+ A c – b c  = b , D= b  –,a Ab c+ a  ,A b c + A  – a A b c + A c– b c  + ,a b – A b c + A c  = b . Assume that D> . The sign list of the sequence {D,D,D,D,D} is given by ,–,–,sign(D), , () ,–,–,sign(D), , ,–,–,sign(D), , () from which it follows that the number of sign changes of the revised sign list of list () is two. Now, statement (b) follows in view of Theorem []. Assume that D< . If D≥, then we obtain that ˜f (y) has three pairs of conjugate imaginary roots, which is a contra- diction. Hence, D< . The sign list of the sequence {D,D,D,D,D} is given by [,–,–,–,–], () () [,–,–,–,–], which implies that the number of sign changes of the revised sign list of () is one. Now, statement (c) follows in view of Theorem []. Similarly, one can prove statements (d) and (e). □ Page 9 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 http://www.advancesindifferenceequations.com/content/2014/1/301 3.2 Injectivity, (O+) and (O–) Lemma Assume that (¯x, ¯y) is an equilibrium of the map T. Then the following hold: () If a̸= Ac, then T is injective. () If a= Ac, then the curve bx= ¯x A+ y 3.2 Injectivity, (O+) and (O–) Lemma Assume that (¯x, ¯y) is an equilibrium of the map T. Then the following hold: () If a̸= Ac, then T is injective. is invariant under the map T. Furthermore, the following holds: Since a= Ac, equations () and () become Since a= Ac, equations () and () become c ¯y+ A A¯y + ¯y– b c =  () c ¯y+ A A¯y + ¯y– b c =  () in Diﬀerence Equations 2014, 2014:301 Page 10 of 32 ferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 10 of 32 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 10 of 32 q http://www.advancesindifferenceequations.com/content/2014/1/301 and () c– ¯x –A¯x+ b¯x– b c  = . From () we have A= b c–¯y ¯y . By substituting this into () we get From () we have A= b c–¯y ¯y . By substituting this into () we get (¯x¯y – bc)(b(c¯y + ¯x) + ¯x¯y) ¯y = , (¯x¯y – bc)(b(c¯y + ¯x) + ¯x¯y) ¯y = , which implies ¯x¯y – bc= , from which the proof follows. () The Jacobian matrix of the map T has the form which implies ¯x¯y – bc= , from which the proof follows. () The Jacobian matrix of the map T has the form JT = xb y+A – xyb (y+A) – (cy+a) x yc x . () JT = xb y+A – xyb (y+A) – (cy+a) x yc x . () The determinant of () at any point is equal to JT(x,y) = bxy(Ac– a) x(y+ A) . The proof of () and () follows from Theorem . Note that xg–(x) is always negative. Let xg(y) denote xg+(y). We consider only xf (y) and xg(y). Let 4 Linearized stability analysis The determinant of () at the equilibrium point is given by detJT(¯x, ¯y) = b¯y(Ac– a) ¯x(¯y+ A) . () detJT(¯x, ¯y) = b¯y(Ac– a) ¯x(¯y+ A) . () The trace of () at the equilibrium point is given by The trace of () at the equilibrium point is given by trJT(¯x, ¯y) = c¯y ¯x + . The characteristic equation has the form λ– λ c¯y ¯x +  + b¯y(Ac– a) ¯x(¯y+ A) = . Equilibrium curves Cf = {(x,y) ∈R : f (x,y) = x} and Cg = {(x,y) ∈R : g(x,y) = y} can be given explicitly as functions of y: Cf : xf (y) = A+ y b , Cg : ⎧ ⎨ ⎩ xg+(y) = + a+cy y , y > , xg–(y) = – a+cy y , y > . Cf : xf (y) = A+ y b , Cg : ⎧ ⎨ ⎩ xg+(y) = + a+cy y , y > , xg–(y) = – a+cy y , y > . Note that xg–(x) is always negative. Let xg(y) denote xg+(y). We consider only xf (y) and xg(y). Let Note that xg–(x) is always negative. Let xg(y) denote xg+(y). We consider only xf (y) and xg(y). Let ˜x(y) = xf (y) – xg(y). Page 11 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 11 of 32 http://www.advancesindifferenceequations.com/content/2014/1/301 Lemma Let T = (f ,g) be the map deﬁned by (). Then f ′ x(x,y) > , and the following is true: Lemma Let T = (f ,g) be the map deﬁned by (). Then f ′ x(x,y) > , and the following is true: sign ˜x(y) = sign y+ Ay– b cy+ A y – ab  . Proof The ﬁrst derivative of xf (y) is given by x′ f (y) = f ′ y(x,y) – f ′x(x,y) = y b > . Since f ′ y(x,y) < , we get f ′ x(x,y) > . Further, ′(x,y) < , we get f ′ x(x,y) > . Further, ˜x(y) = xf (y) – xg(y) = A+ y b – a+ cy y = √y(A+ y) – b a+ cy b√y . Now, the proof follows from √y A+ y– b a+ cy= y+ Ay– b cy+ A y – ab . 4 Linearized stability analysis □ □ Lemma Let T be the map deﬁned by (), and let Lemma Let T be the map deﬁned by (), and let JT(¯x, ¯y) = a b c d () JT(¯x, ¯y) = a b c d () be the Jacobian matrix of T at a ﬁxed point (¯x, ¯y). Then the Jacobian matrix () has real and distinct eigenvalues λand λsuch that \|λ\| < λand λ> . Furthermore, the following holds: be the Jacobian matrix of T at a ﬁxed point (¯x, ¯y). Then the Jacobian matrix () has real and distinct eigenvalues λand λsuch that \|λ\| < λand λ> . Furthermore, the following holds: sign ˜x′(¯y) = sign(– λ). sign ˜x′(¯y) = sign(– λ). Proof Implicit diﬀerentiation of the equations deﬁning Cf and Cg at (¯x, ¯y) gives Proof Implicit diﬀerentiation of the equations deﬁning Cf and Cg at (¯x, ¯y) gives x′ f (¯y) = f ′ y(¯x, ¯y) – f ′x(¯x, ¯y), x′ g(¯y) = – g′ y(¯x, ¯y) g′x(¯x, ¯y) . () () The characteristic equation associated with the Jacobian matrix of T at (¯x, ¯y) is given by p(λ) = λ– f ′ x(¯x, ¯y) + g′ y(¯x, ¯y) λ + f ′ x(¯x, ¯y)g′ y(¯x, ¯y) – f ′ y(¯x, ¯y)g′ x(¯x, ¯y) = λ– (a + d)λ + (ad – bc). Since the map T is competitive, then the eigenvalues of the Jacobian matrix of the map T at the equilibrium (¯x, ¯y) are real and distinct and \|λ\| < λ. By (), we have ˜x′(¯y) = x′ f (¯y) – x′ g(¯y) = f ′ y(¯x, ¯y) – f ′x(¯x, ¯y) – – g′ y(¯x, ¯y) g′x(¯x, ¯y) = b – a – – d c = –+ (a + d) – (ad – bc) c(– a) = –p() c(– a) = (– λ)(– λ) c(a – ) . Page 12 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 From trJT(¯x, ¯y) = λ+ λ> we get λ> . The map T is competitive, which implies c = g′ x(¯x, ¯y) < . In view of Lemma , we get a = f ′ x(¯x, ¯y) > , from which it follows that sign(˜x′(¯y)) = sign(– λ). □ □ Theorem Assume that > and λand λare eigenvalues of JT(¯x, ¯y). 4 Linearized stability analysis Then there exists the unique equilibrium point E = (¯x, ¯y) and the following hold: (a) If a< Ac, then E is a saddle point and < λ< , λ> . (a) If a< Ac, then E is a saddle point and < λ< , λ> . (b) Assume that a> Ac. Let (b) Assume that a> Ac. Let (¯y) = c¯y+ ¯y(Ac– a) + aA. (b) If (¯y) > , then E is a saddle point. Furthermore, the following hold: (b) If (¯y) < , then E is a repeller. Furthermore, the following hold: λ< –, λ> ; \|λ\| < λ. (b) If (¯y) = , then E is a non-hyperbolic equilibrium point. Furthermore, the following hold: λ= –, λ> . Proof In view of () and Lemma , we have that the function Proof In view of () and Lemma , we have that the function ˜f (y) = y+ Ay– b cy+ A y – ab  ˜f (y) = y+ Ay– b cy+ A y – ab  has one zero ¯y of multiplicity one. In view of Lemma , the map T has a unique equilibrium point. Since ˜f () = –ab < and limy→+∞˜f (y) = +∞, we have ˜f (y) < for y < ¯y and ˜f (y) > for y > ¯y. By Lemmas and from [], the equilibrium curves Cf and Cg intersect transversally at (¯x, ¯y), i.e., ˜x′(¯y) ̸= . In view of Lemma and by the continuity of function ˜x(y), there exists a neighborhood U¯y of ¯y such that ˜x′(y) > for y ∈U¯y, which implies has one zero ¯y of multiplicity one. In view of Lemma , the map T has a unique equilibrium point. Since ˜f () = –ab < and limy→+∞˜f (y) = +∞, we have ˜f (y) < for y < ¯y and ˜f (y) > for y > ¯y. By Lemmas and from [], the equilibrium curves Cf and Cg intersect transversally at (¯x, ¯y), i.e., ˜x′(¯y) ̸= . In view of Lemma and by the continuity of function ˜x(y), there exists a neighborhood U¯y of ¯y such that ˜x′(y) > for y ∈U¯y, which implies ˜x′(¯y) = x′ f (¯y) – x′ g(¯y) > . () () ˜x′(¯y) = x′ f (¯y) – x′ g(¯y) > . From () and Lemma we obtain λ< and λ> . 4 Linearized stability analysis From () and Lemma we obtain λ< and λ> . From () and Lemma we obtain λ< and λ> . From () and Lemma we obtain λ< and λ> . If a< Ac, then detJT(¯x, ¯y) = λλ> , which implies that λ∈(,). If a< Ac, then detJT(¯x, ¯y) = λλ> , which implies that λ∈(,). If a< Ac, then detJT(¯x, ¯y) = λλ> , which implies that λ∈(,). Now, assume that a> Ac. By using Now, assume that a> Ac. By using b= ¯y+ A ¯x , ¯x = c¯y+ a ¯y , one can see that one can see that p(–) = + detJT(¯x, ¯y) + trJT(¯x, ¯y) = c¯y+ ¯y(Ac– a) + aA (¯y+ A)(c¯y+ a) = (¯y) (¯y+ A)(c¯y+ a) = (¯y) (¯y+ A)(c¯y+ a) and and detJT(¯x, ¯y) = λλ= b¯y(Ac– a) ¯x(¯y+ A) , Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 13 of 32 http://www.advancesindifferenceequations.com/content/2014/1/301 where p(λ) = (λ – λ)(λ – λ). In view of () and p(–) = (λ+ )(λ+ ), we obtain state- ment (b) of the theorem. □ □ Lemma Suppose that all the assumptions of Theorem are satisﬁed. Let Lemma Suppose that all the assumptions of Theorem are satisﬁed. Let y± = a– Ac± –aAc+ a + A c  c . Then the following statements are true. Then the following statements are true. Then the following statements are true. (a) (¯y) > if and only if one of the following inequalities holds: Ac– a≥, Ac– a<  and –aAc+ a + A c < , Ac– a<  and –aAc+ a + A c ≥ and ˜f (y–) > or ˜f (y+) <  ; (b) (¯y) < if and only if the following hold: Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 (b) (¯y) < if and only if the following hold: Given points lie on the increasing curve then by (a) of Lemma we obtain that the map T has three equilibrium points that we denote by E, Eand E. Given points lie on the increasing curve xf (y) = A+ y b , xf (y) = A+ y b , which implies that the points are in the north-east ordering. Descartes’ rule of signs and () imply that detJT(x,y) > when a< Ac. In view of () and Lemma , we have that the polynomial ˜f (y) = y+ Ay– b cy+ A y – ab  has three zeros ¯yi, i = ,,, of multiplicity one. Since ˜f () = –ab < and limy→+∞˜f (y) = +∞, we have ˜f (y) < for y ∈(, ¯y) ∪(¯y, ¯y) and ˜f (y) > for y ∈(¯y, ¯y) ∪(¯y,+∞). has three zeros ¯yi, i = ,,, of multiplicity one. Since ˜f () = –ab < and limy→+∞˜f (y) = +∞, we have ˜f (y) < for y ∈(, ¯y) ∪(¯y, ¯y) and ˜f (y) > for y ∈(¯y, ¯y) ∪(¯y,+∞). By Lemmas and from [], the equilibrium curves Cf and Cg intersect transversally at E, Eand E, i.e., ˜x′(¯yi) ̸= , i = ,,. By this and Lemma and by the continuity of function ˜x(y), there exists a neighborhood U(i) ¯yi of ¯yi such that ˜x′(y) > for y ∈U(i) ¯yi for i = ,and ˜x′(y) < for y ∈U() ¯yi . Using this we get ˜x′(¯yi) >  for i = , and ˜x′(¯yi) <  for i = . Let JT(Ei) = ai bi ci di , i = ,,. In view of (), we have detJT(Ei) = λ(i) λ(i) > , i = ,,. By Lemma we obtain < λ(i) <  and λ(i) > for i = ,. Since ˜x′(¯y) < , by Lemma we have < λ() < λ() . This completes the proof. □ In view of (), we have detJT(Ei) = λ(i) λ(i) > , i = ,,. By Lemma we obtain < λ(i) <  and λ(i) > for i = ,. Since ˜x′(¯y) < , by Lemma we have < λ() < λ() . This completes the proof. □ Theorem Assume that = and ̸= Then there exist two distinct equilibrium points in the positive quadrant E= (¯x, ¯y) and E= (¯x, ¯y) such that E≪ne E. (b) (¯y) < if and only if the following hold: Ac–a<  and –aAc+a +A c ≥ and ˜f (y–) < and ˜f (y+) >  ; (c) (¯y) = if and only if Ac–a<  and –aAc+a +A c ≥ and ˜f (y–) = or ˜f (y+) =  . Proof The function ˜f (y) has one simple zero ¯y, which implies ˜f (y) < for ≤y < ¯y and ˜f (y) > for y > ¯y. Then (c) (¯y) = if and only if (c) (¯y) = if and only if ¯y > α if and only if ˜f (α) < , while ¯y < β if and only if ˜f (β) >  ¯y < β if and only if ˜f (β) >  ¯y < β if and only if ˜f (β) >  for some α,β ∈[,∞). Now the proof follows from the fact that ˜f (y) = has real roots a– Ac± –aAc+ a + A c  c , – a– Ac± –aAc+ a + A c  c if and only if if and only if Ac– a<  and – aAc+ a + A c ≥. □ Ac– a<  and – aAc+ a + A c ≥. □ Page 14 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Theorem Assume that < . Then there exist three distinct equilibrium points in the positive quadrant: E= (¯x, ¯y), E= (¯x, ¯y) and E= (¯x, ¯y) such that E≪ne E≪ne E and the following hold: (a) Eand Eare saddle points. If λ(i) and λ(i) are the eigenvalues of JT(Ei), i = ,, then < λ(i) < , λ(i) > . (a) Eand Eare saddle points. If λ(i) and λ(i) are the eigenvalues of JT(Ei), i = ,, then < λ(i) < , λ(i) > . < λ(i) < , λ(i) > . (b) The equilibrium point Eis a repeller. If λ()  and λ() are the eigenvalues of JT(E), then < λ() < λ() . Proof In view of Lemma , equation () has three positive roots of multiplicity one. Since Proof In view of Lemma , equation () has three positive roots of multiplicity one. Since ¯x = A+ ¯y b > , ¯x = A+ ¯y b > , then by (a) of Lemma we obtain that the map T has three equilibrium points that we denote by E, Eand E. (b) (¯y) < if and only if the following hold: Let λ(i)  and λ(i) be the eigenvalues of JT(Ei), i = ,. Then the following hold: (a) Exactly one of the roots ¯yor ¯yof () has multiplicity two. Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 15 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 (b) If ¯yis a root of () of multiplicity two, then the equilibrium point Eis non-hyperbolic and Eis a saddle point. Furthermore, λ() = , λ() > and < λ() < , λ() > . (c) If ¯yis a root of () of multiplicity two, then the equilibrium point Eis non-hyperbolic and Eis a saddle point. Furthermore, λ() = , λ() > and < λ() < , λ() > . Proof In view of Lemma , equation () has two positive zeros, one of multiplicity one and another one of multiplicity two, which implies statement (a). Since ¯x = (A+ ¯y)/b> , we obtain that the map T has two equilibrium points that we denote by Eand E. Descartes’ rule of signs and () imply that a< Ac⇒detJT(x,y) > . Now, we prove statement (b). Similarly as in the proof of Theorem , one can see that Eis a saddle point. In view of Lemmas and , from [] we have that ˜x′(¯y) = , since ¯yis the root of () of multiplicity two. By Lemma we obtain λ() = , λ() > . The proof of statement (c) is similar and we will skip it. □ Theorem Assume that = and = . Then there exists one equilibrium point in the positive quadrant E= (¯x, ¯y) which is non-hyperbolic. If λ() and λ() are eigenvalues of JT(E), then λ() = , λ() > . Proof In view of Lemma , ¯yis zero of () of multiplicity three. In view of Lemmas and , from [] we have that ˜x′(¯y) = . The rest of the proof is similar to that in Theorem  and we skip it. □ 4.1 Period-two solution Let T(x,y) = T T(x,y) = F(x,y),G(x,y) , where where F(x,y) = b x (A+ y)(acy+ a + Ax+ c y) F(x,y) = b x (A+ y)(acy+ a + Ax+ c y) d G(x,y) = (A+ y)(ac y+ a c+ ax+ c y) b x . F(x,y) = b x (A+ y)(acy+ a + Ax+ c y) d G(x,y) = (A+ y)(ac y+ a c+ ax+ c y) b x . and G(x,y) = (A+ y)(ac y+ a c+ ax+ c y) b x . Period-two solution {( ,),T( ,)} satisﬁes the system F( ,) = , G( ,) = , which is equivalent to which is equivalent to b  – A  A+ – A+  a+ c= , b  – a  A+ – c A+  a+ c= . () () Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 16 of 32 http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 16 of 32 q http://www.advancesindifferenceequations.com/content/2014/1/301 The Jacobian matrix of the map Tat (x,y) has the form JT(x,y) = ⎛ ⎝ xb (Ax+(cy+a)) (y+A)(Ax+(cy+a)) – xyb (Ax+(cy+a)(a+(y+A)c)) (y+A)(Ax+(cy+a)) – (y+A)(c y+a c+a(x+yc )) xb  y(y+A)(y(y+A)c +a c+a(x+(y+A)c )) xb  ⎞ ⎠. () The determinant of () at (x,y) is given by The determinant of () at (x,y) is given by detJT(x,y) = bxy(a– Ac)(a+ cy) (A+ y)((a+ cy)+ Ax). The trace of () at (x,y) is given by The trace of () at (x,y) is given by trJT(x,y) = ( b x((a+cy)+Ax) ((a+cy)+Ax) + ay(A+ y)(c (A+ y) + x)) b x(A+ y) + (a cy(A+ y)+ c y(A+ y)(A+ y)) b x(A+ y) . ()  + (a cy(A+ y)+ c y(A+ y)(A+ y)) b x(A+ y) . () () Lemma Let CF := {(x,y) : F(x,y) = x} and CG := {(x,y) : G(x,y) = y} be the period-two curves, that is, the curves the intersection of which is a period-two solution. Then, for all y > , there exist exactly one xF(y) > and exactly one xG(y) > such that F(xF(y),y) = x and G(xG(y),y) = y. Furthermore, xF(y) and xG(y) are continuous functions and x′ F(y) > . 4.1 Period-two solution By substituting this into () we obtain By substituting this into () we obtain By substituting this into () we obtain trJT( ,) = (A+ )(a(c (A+ ) + ) + a c+ c (A+ )) b   + – A  (a+ c)+ A > . trJT( ,) = (A+ )(a(c (A+ ) + ) + a c+ c (A+ )) b   + – A  (a+ c)+ A > . The rest of the proof follows from the fact that trJT( ,) = μ+ μ> , detJT( ,) = μμ> and Lemma . □ The rest of the proof follows from the fact that trJT( ,) = μ+ μ> , detJT( ,) = μμ> and Lemma . □ Theorem If the map T has a minimal period-two solution {( ,),T( ,)}, which is non-hyperbolic, then D(p) = , where D(p) is the discriminant of the polynomial p(y) := py+ py+ ··· + py + p, where the coeﬃcients pi, i = ,...,, are in the Appendix. If {( ,),T( ,)} and {( ,),T( ,)} are two minimal period-two solutions such that T has no other min- imal period-two solutions in J( ,),( ,)K = {(x,y) : ( ,) ⪯ne (x,y) ⪯ne ( ,)} and D(p) ̸= , then one of them is a saddle point and the other is a repeller. Proof Period-two solution curves CF = {(x,y) ∈R : ˜F(x,y) = } and CG = {(x,y) ∈R : ˜G(x,y) = }, where Proof Period-two solution curves CF = {(x,y) ∈R : ˜F(x,y) = } and CG = {(x,y) ∈R : ˜G(x,y) = }, where ˜F(x,y) = b x– Ax A+ y– A+ y a+ cy and ˜G(x,y) = b yx– ax A+ y– c A+ y a+ cy, are algebraic curves. By using software Mathematica, one can see that the resultant of the polynomials ˜F(x,y) and ˜G(x,y) in variable x is given by R(˜F, ˜G) = –b  A+ y a+ cy –ab + Ay+ A y – b cy+ y p(y) = –b  A+ y a+ cy˜f (y)p(y). Suppose that {( ,),T( ,)} is a non-hyperbolic minimal period-two solution. 4.1 Period-two solution Proof Since F(x,y) = x and G(x,y) = y if and only if Proof Since F(x,y) = x and G(x,y) = y if and only if b x– Ax A+ y– A+ y a+ cy= , b yx– ax A+ y– c A+ y a+ cy= , respectively, in view of Descartes’ rule of signs, we have that for all y > there exist exactly one xF(y) > and exactly one xG(y) > such that F(xF(y),y) = x and G(xG(y),y) = y. Taking derivatives of F(x,y) = x with respect to y, we get respectively, in view of Descartes’ rule of signs, we have that for all y > there exist exactly one xF(y) > and exactly one xG(y) > such that F(xF(y),y) = x and G(xG(y),y) = y. Taking derivatives of F(x,y) = x with respect to y, we get x′ F(y) = F′ y(x,y) – F′x(x,y). x′ F(y) = F′ y(x,y) – F′x(x,y). From F(x,y) = x we have that (A+ y)= b x (a+cy)+Ax, which implies From F(x,y) = x we have that (A+ y)= b x (a+cy)+Ax, which implies From F(x,y) = x we have that (A+ y)= b x (a+cy)+Ax, which implies F′ x(x,y) = xb (Ax+ (cy+ a)) (y+ A)(Ax+ (cy+ a))= – Ax (a+ cy)+ Ax> . □ Since F′ y(x,y) < , we get x′ F(y) > . Since F′ y(x,y) < , we get x′ F(y) > . Theorem If a≤Ac, then T has no minimal period-two solution. If a> Acand T has a minimal period-two solution {( ,),T( ,)}, then {( ,),T( ,)} is unstable. If μand μ(μ< μ) are the eigenvalues of JT( ,), then μ> and μ> . All period-two solutions are ordered with respect to the north-east ordering. Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 17 of 32 http://www.advancesindifferenceequations.com/content/2014/1/301 Proof If a≤Ac, the statement follows from Lemma . If a> Ac, then from the ﬁrst equation of () we have that Proof If a≤Ac, the statement follows from Lemma . If a> Ac, then from the ﬁrst equation of () we have that A+ = b   (a+ c)+ A . 4.1 Period-two solution This implies that ˜F( ,) = , ˜G( ,) = . By Theorem .[], ˜F and ˜G have a common non-constant factor if and only R(˜F, ˜G) = , which implies that system ˜F(x,y) = , ˜G(x,y) = has a solution if and only if R(˜F, ˜G) = . Since ˜f () ̸= , it must be p() = . Similarly Suppose that {( ,),T( ,)} is a non-hyperbolic minimal period-two solution. This implies that ˜F( ,) = , ˜G( ,) = . By Theorem .[], ˜F and ˜G have a common non-constant factor if and only R(˜F, ˜G) = , which implies that system ˜F(x,y) = , ˜G(x,y) = has a solution if and only if R(˜F, ˜G) = . Since ˜f () ̸= , it must be p() = . Similarly Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 18 of 32 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 18 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 as in Lemma , one can see that as in Lemma , one can see that x′ F() – x′ G() = F′ y( ,) – F′x( ,) – – G′ y( ,) G′x( ,) = f – e – – h g = –+ (e+ g) – (eh– fg) g(– e) = –p() g(– e) = (– μ)(– μ) g(e– ) , where p(μ) is the characteristic equation of the matrix where p(μ) is the characteristic equation of the matrix JT( ,) = e f g h . From Theorem we have that < μ< μand μ> . Since {( ,),T( ,)} is non- hyperbolic, we obtain that μ= , from which it follows that x′ F() – x′ G() = . Since R(˜F, ˜G) ̸≡, we have that CF and CG have no common component. By Lemmas and , from [], the curves CF and CG intersect transversally at ( ,) (i.e., y′ ˜F()–y′ ˜G() ̸= ) if and only if is zero of p(y) of multiplicity one. By Theorem .[], p(y) has zeros of multiplicity greater than one if and only if the discriminant D(p) of the polynomial p(y) is equal to zero, which proves the ﬁrst statement of the lemma. 4.1 Period-two solution Assume that {( ,),T( ,)} and {( ,),T( ,)} are two minimal period-two solutions such that T has no other minimal period-two solutions in J( ,),( ,)K = {(x,y) : ( ,) ⪯ne (x,y) ⪯ne ( ,)} and D(p) ̸= . From the previous discussion we have x′ F(i)–x′ G(i) ̸= , i = ,. Since xF(i)–xG(i) = , i = ,, it follows that (x′ F()– x′ G())(x′ F() – x′ G()) < . Indeed assume, for example, that x′ F() – x′ G() < and x′ F() – x′ G() < . Then there exists ϵ > such that xF(y) – xG(y) < for y ∈(,+ ϵ) and xF(y) – xG(y) > for y ∈(– ϵ,). Since xF(y) – xG(y) is a continuous function, this implies that there exists ∈(,) such that xF() – xG() = , which is a contradic- tion. The rest of the proof follows from the fact that ei > and gi < , i = ,. □ Notice that Notice that D(p) = –  p R p,p′ , where R(p,p′) = detSyl(p,p′), the determinant of the Sylvester matrix Syl(p,p′), see [, ], and where R(p,p′) = detSyl(p,p′), the determinant of the Sylvester matrix Syl(p,p′), see [, ], and Syl p,p′ = ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝ p p ··· p p  ···   p p ··· p p ···  ...  ··· p p p ··· p p p p ··· p   ···   p p ··· p  ···  ...   ··· p p p ··· p ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠ . Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 19 of 32 Theorem If a> Acand (¯y) < , then T has one equilibrium point E(¯x, ¯y), which is a repeller, and there exists at least one minimal period-two solution {(ψ,φ),T(ψ,φ)} which is non-hyperbolic or a saddle point. If T has no minimal period-two solutions which are non-hyperbolic, then (ψ,φ) ≪ne E ≪ne T(ψ,φ). Proof By Theorem we have that T has one equilibrium point E(¯x, ¯y), which is a re- peller. 4.1 Period-two solution This and Lemma imply that Tn(x,y) is asymptotic to either (,∞) or (∞,), or a minimal period-two solution, for all (x,y) ∈R. Let B(,∞) be the basin of attraction of (,∞), and let B(∞,) be the basin of attraction of (∞,). By using Theorem one can prove that int(Q(E)) ⊂B(,∞) and int(Q(E)) ⊂B(∞,). Let Sdenote the boundary of B(∞,) considered as a subset of Q(E), and let Sdenote the boundary of B(∞,) con- sidered as a subset of Q(E). It is easy to see that E ∈S, E ∈Sand T(R) ⊂int(R). Now we prove the following claim. Claim Let Sand Sbe the sets deﬁned as above. Then (a) If (x,y) ∈B(∞,), then (x,y) ∈B(∞,) for all (x,y) ⪯se (x,y). (b) If (x,y) ∈S∪S, then (x,y) ∈int(B)(∞,) for all (x,y) ≪se (x,y). (c) S∩int(Q(E)) ̸= ∅and S∩int(Q(E)) ̸= ∅. (d) T(S∪S) ⊆S∪S. Claim Let Sand Sbe the sets deﬁned as above. Then (a) If (x,y) ∈B(∞,), then (x,y) ∈B(∞,) for all (x,y) ⪯se (x,y). (b) If (x,y) ∈S∪S, then (x,y) ∈int(B)(∞,) for all (x,y) ≪se (x,y). (c) S∩int(Q(E)) ̸= ∅and S∩int(Q(E)) ̸= ∅. (d) T(S∪S) ⊆S∪S. (d) T(S∪S) ⊆S∪S. (e) (x,y),(x,y) ∈S∪S⇒(x,y) ≪ne (x,y) or (x,y) ≪ne (x,y). (e) (x,y),(x,y) ∈S∪S⇒(x,y) ≪ne (x,y) or (x,y) ≪ne (x,y). Proof (a) The statement follows from Tn(x,y) ⪯se Tn(x,y) ⪯se (∞,) and Tn(x,y) → (∞,) as n →∞. Proof (a) The statement follows from Tn(x,y) ⪯se Tn(x,y) ⪯se (∞,) and Tn(x,y) → (∞,) as n →∞. (b) The claim (b) follows from the observation that there exists a ball centered at (x,y) with the property that all its points (x,y) satisfy (x,y) ≪se (x,y). But one of these points necessarily lies in B(∞,), so by (a) there exists (x,y) ∈B(∞,). Furthermore, there exists a ball centered at (x,y) with the property that all its points (x,y) satisfy (x,y) ∈B(∞,), which implies (x,y) ∈int(B)(∞,). (c) Take y′ > ¯y arbitrary (but ﬁxed). Since T is strongly competitive, we have T(¯x,y′) ≪se T(¯x, ¯y), which implies T(¯x,y′) ∈int(Q(E)). This implies that there exists a ball Bε(T(¯x,y′)) with the property Bε(T(¯x,y′)) ⊂int(Q(E)). Since T is a continuous map on a set R + \ {(,y) : y ≥}, then there exists a ball Bδ(¯x,y′) such that T(Bδ(¯x,y′)) ⊂Bε(T(¯x,y′)) ⊂ int(Q(E)), which implies Tn(x,y) →(,∞) as n →∞for all (x,y) ∈Bδ(¯x,y′). 4.1 Period-two solution Similarly, one can prove that then there exists a ball Bδ(¯x + δ/, ¯y) such that Tn(x,y) →(∞,) as n →∞for all (x,y) ∈Bδ(¯x + δ/, ¯y). Let y′′ = sup{y : limn→∞Tn(¯x + δ/,y) = (∞,)}. It is easy to see that (¯x + δ/,y′′) ∈S∩int(Q(E)). The assertion concerning Sis proved in a similar fashion. (c) Take y′ > ¯y arbitrary (but ﬁxed). Since T is strongly competitive, we have T(¯x,y′) ≪se T(¯x, ¯y), which implies T(¯x,y′) ∈int(Q(E)). This implies that there exists a ball Bε(T(¯x,y′)) with the property Bε(T(¯x,y′)) ⊂int(Q(E)). Since T is a continuous map on a set R + \ {(,y) : y ≥}, then there exists a ball Bδ(¯x,y′) such that T(Bδ(¯x,y′)) ⊂Bε(T(¯x,y′)) ⊂ int(Q(E)), which implies Tn(x,y) →(,∞) as n →∞for all (x,y) ∈Bδ(¯x,y′). Similarly, one can prove that then there exists a ball Bδ(¯x + δ/, ¯y) such that Tn(x,y) →(∞,) as n →∞for all (x,y) ∈Bδ(¯x + δ/, ¯y). Let y′′ = sup{y : limn→∞Tn(¯x + δ/,y) = (∞,)}. It is easy to see that (¯x + δ/,y′′) ∈S∩int(Q(E)). The assertion concerning Sis proved in a similar fashion. (d) Take (x,y) ∈S∪S. Assume that T(x,y) /∈S∪S. Since S∪S= ∂B(∞,) = B(∞,)\int(B(∞,)), then either T(x,y) ∈int(B(∞,)) or T(x,y) /∈B(∞,). Assume that T(x,y) ∈int(B(∞,)). This implies that there exists a ball Bε(T(x,y)) with the property Bε(T(x,y)) ⊂int(B(∞,)). Since T is a continuous map on the set R + \{(,y) : y ≥}, then there exists a ball Bδ(x,y), δ > such that T(Bδ(x,y)) ⊂Bε(T(x,y)), which implies Bδ(x,y) ⊂ B(∞,). This is in contradiction with (x,y) ∈S∪S= ∂B(∞,). Hence T(x,y) ∈S∪S in this case. Similarly, one can prove that T(x,y) ∈S∪Sif T(x,y) /∈B(∞,). This implies that T(S∪S) ⊆(S∪S). Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 20 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 http://www.advancesindifferenceequations.com/content/2014/1/301 (e) Assume that (x,y),(x,y) ∈S∪S⇒(x,y) ⪯se (x,y) and (x,y) ̸= (x,y). Since T is strongly competitive, we get T(x,y) ≪ne T(x,y). This contradicts (e) and (b), which completes the proof. □ □ In view of Claim , we have that (S∪S,≪ne) is a totally ordered set which is invariant under T. If (x,y) ∈S∪S, then {T(n)(x,y)} is eventually component-wise monotone. 4.1 Period-two solution Then there exists a minimal period-two solution {( ,),T( ,)} ∈S∪S⊂Q(E) ∪ Q(E) such that T(n)(x,y) →( ,) as n →∞. By Theorem , {( ,),T( ,)} is a non-hyperbolic or a saddle point. Assume that T has no minimal period-two solu- tions which are non-hyperbolic points and, for example, that ( ,),T( ,) ∈Ssuch that E ≪ne ( ,) ≪ne T( ,). Since {( ,),T( ,)} is a saddle point, in view of Theorems , and , we have that the global stable manifolds Ws({( ,),T( ,)}) are the union of two curves Ws( ,) and Ws(T( ,)) whose endpoints are repeller points such that T(Ws( ,)) = Ws(T( ,)) and E ≪ne Ws( ,) ≪ne Ws(T( ,)). If Pand P(P⪯ne P) are endpoints of Ws( ,), then T(P) and T(P) are endpoints of T(Ws( ,)) and either T(P) ⪯ne T(P) or T(P) ⪯ne T(P). Assume, for example, that P≪ne Ws( ,) ≪ne P≪ne T(P) ≪ne Ws(T( ,)) ≪ne T(P). By Theorem be- tween two repellers Pand T(P), there exists a saddle point Swhere its stable manifold is the union of two invariant curves Ws(S) and Ws(T(S)) whose endpoints are repellers such that P≪ne Ws(S) ≪ne T(P). Continuing in this way, we obtain that T has inﬁnitely many minimal period-two solutions {Pi,T(Pi)}, which is in contradiction with the fact that T has at most eleven minimal period-two solutions. Hence ( ,) ≪ne E ≪ne T( ,). □ □ Corollary Assume that a> Ac, (¯y) < and D(p) ̸= . Then there exists one equi- librium point E which is a repeller. Further, the set int(Q(E)) ∪int(Q(E)) contains an odd number of minimal period-two solutions {( i,i),( ˜ i, ˜i)}, i = ,...,n + , such that ( i+,i+) ≪ne ( i,i) ≪ne E and E ≪ne ( ˜ i, ˜i) ≪ne ( ˜ i+, ˜i+), where ( ˜ i, ˜i) = T( i,i). Furthermore, odd indexed period-two points are saddles and even indexed period-two points are repellers. Proof By Theorem we have that T has one equilibrium point E(¯x, ¯y), which is a re- peller. By Theorem all minimal period-two solutions are hyperbolic and the number of minimal period-two solutions is ﬁnite. 5 Global behavior In this section we present global dynamics of system () in diﬀerent parametric regions. We have ﬁve parametric regions with diﬀerent dynamics which will be characterized by the following ﬁve theorems. 4.1 Period-two solution In view of Claim , T has at least one min- imal period-two solution which is a saddle point. Let {P,T(P)} be a minimal period- two solution which is a saddle point such that P≪ne E ≪ne T(P) and T has no minimal period-two solutions in JE,T(P)K and JP,EK. Such a minimal period-two solution exists in view of Theorem . The map Tsatisﬁes all conditions of Theorems , and , which yields the existence of the global stable manifolds Ws({P, ˜P}) which are the union of two curves Ws(P) and Ws(˜P) that have a common endpoint E. If T has minimal period-two solutions in int(Q(T(P))) ∪int(Q(P)), let {P, ˜P} (P≪ne ˜P) denote minimal period- two solutions such that T has no other minimal period-two solutions in JT(P),T(P)K and JP,PK. Then Ws(P) has the second endpoint at Pand Ws(T(P)) has the second endpoint at T(P) and P≪ne P≪ne E ≪ne T(P) ≪ne T(P). Furthermore, a minimal period-two solution {P,T(P)} is a repeller. Similarly as in Theorem , one can prove that int(Q(T(P))) ∪int(Q(P)) contains at least one minimal period-two solution which Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 21 of 32 http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 21 of 32 http://www.advancesindifferenceequations.com/content/2014/1/301 ﬀerence Equations 2014, 2014:301 Page 21 of 32 nceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 21 of 32 is a saddle point. Since the number of minimal period-two solutions is ﬁnite, continuing in this way, we will end with a minimal period-two solution which is a saddle point, from which the proof follows. □ Corollary If a> Acand (¯y) > , then there exists one equilibrium point E which is a saddle point. If D(p) ̸= , then int(Q(E)) ∪int(Q(E)) contains an even number of minimal period-two solutions {( i,i),( ˜ i, ˜i)}, i = ,...,n, such that ( i+,i+) ≪ne ( i,i) ≪ne E and E ≪ne ( ˜ i, ˜i) ≪ne ( ˜ i+, ˜i+) and ( ˜ i, ˜i) = T( i,i). Furthermore, even indexed period-two points are saddles and odd indexed period-two points are repellers. □ Proof The proof is similar as the proof of Corollary and it will be omitted. Based on a series of numerical simulations, we propose the following conjecture. 5.1 The case where the equilibrium points and period-two solutions are hyperbolic points (1 ̸= 0 and D(p) ̸= 0) Theorem Assume that < . Then system () has three equilibrium solutions E≪ne E≪ne E, where Eand Eare saddle points and Eis a repeller. In this case there exist four invariant continuous curves Ws(E), Ws(E), Wu(E), Wu(E), where Ws(E), Ws(E) have end points at E, and are graphs of increasing functions. The curves Wu(E), Wu(E) are the graphs of decreasing functions. Every solution {(xn,yn)} which starts below Ws(E)∪ Ws(E) in the south-east ordering is asymptotic to (,∞), and every solution {(xn,yn)} which starts above Ws(E) ∪Ws(E) in the south-east ordering is asymptotic to (∞,). The ﬁrst quadrant of initial condition Q= {(x,y) : x> ,y≥} is the union of ﬁve disjoint basins of attraction, i.e., Q= B(,∞) ∪B(∞,) ∪B(E) ∪B(E) ∪B(E), Q= B(,∞) ∪B(∞,) ∪B(E) ∪B(E) ∪B(E), where B(E) = {E}, B(E) = Ws(E), B(E) = Ws(E) and B(,∞) = (x,y)\|(x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈Ws(E) ∪Ws(E) , B(∞,) = (x,y)\|(˜x, ˜y) ⪯se (x,y) for some (˜x, ˜y) ∈Ws(E) ∪Ws(E) . Proof Theorem implies that there exist three equilibrium points, namely E, Eand E, such that E≪ne E≪ne E. In this case, Eand Eare saddle points and Eis a repeller. In view of (), the map T is competitive on R and strongly competitive on int(R). It follows from the Perron-Frobenius theorem and a change of variables [] that at each point the Jacobian matrix of a strongly competitive map has two real and distinct eigenvalues, the larger one in absolute value being positive, and that corresponding eigenvectors may be Proof Theorem implies that there exist three equilibrium points, namely E, Eand E, such that E≪ne E≪ne E. In this case, Eand Eare saddle points and Eis a repeller. In view of (), the map T is competitive on R and strongly competitive on int(R). It follows from the Perron-Frobenius theorem and a change of variables [] that at each point the Jacobian matrix of a strongly competitive map has two real and distinct eigenvalues, the larger one in absolute value being positive, and that corresponding eigenvectors may be Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 22 of 32 chosen to point in the direction of the second and ﬁrst quadrant, respectively. 5.1 The case where the equilibrium points and period-two solutions are hyperbolic points (1 ̸= 0 and D(p) ̸= 0) Also, one can show that if the map is strongly competitive, then no eigenvector is aligned with a coordinate axis. Since < implies a< Ac, we have that detJT(Ei) > , i = ,. Hence, all condi- tions of Theorems , and are satisﬁed, which yields the existence of the global sta- ble manifolds Ws(E), Ws(E) and the global unstable manifolds Wu(E), Wu(E), where Ws(E), Ws(E) are passing through the point Eand are graphs of increasing functions. The curves Wu(E), Wu(E) are the graphs of decreasing functions. Let W– = (x,y)\|(x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈Ws(E) ∪Ws(E) , W+ = (x,y)\|(˜x, ˜y) ⪯se (x,y) for some (˜x, ˜y) ∈Ws(E) ∪Ws(E) . Take (x,y) ∈W– ∩R. By Theorem we have that there exists n> such that Tn(x,y) ∈int((Q(E) ∪Q(E)) ∩R), n > n. In view of Theorem , since Eand Eare saddle points, we obtain that for all (x,y) ∈int((Q(E)∪Q(E))∩R), there exists a sub- solution (u,v) such that (x,y) ⪯se (u,v). By monotonicity we have that (,∞) ⪯se Tn(x,y) ⪯se Tn(u,v) ≪Eif (x,y) ∈int(Q(E) ∩R) and (,∞) ⪯se Tn(x,y) ⪯se Tn(u,v) ≪Eif (x,y) ∈int(Q(E) ∩R). For the sequence (un,vn) = Tn(u,v), we have (un+,vn+) ⪯se (un,vn) ⪯se (u,v). The sequence {un} is monotone decreasing and bounded from below and {vn} is monotone increasing. Since T has no other equilibrium point except E, Eand E, this implies that the sequence {un} has ﬁnite limit and vn →+∞ as n →∞. From un+= (bu n)/(A+ v n) we obtain that un →as n →∞. This implies Tn(x,y) →(,∞) as n →∞. If (x,y) ∈W+ ∩R, the proof is similar and we skip it. Another way of completing the proof is by using () of Lemma . □ □ Another way of completing the proof is by using () of Lemma . Theorem Assume that > . If Theorem Assume that > . If or a> Acand (¯y) > and T has no minimal period-two solution, then system () has one equilibrium solution E(¯x, ¯y), which is a saddle point. There exists the global stable manifold Ws(E) which is the graph of a continuous increasing function and the global unstable manifold Wu(E), which is the graph of a continuous decreasing function. Every solution {(xn,yn)} which starts below Ws(E) in the south-east ordering is asymptotic to (,∞), and every solution {(xn,yn)} which starts above Ws(E) in the south-east ordering is asymptotic to (∞,). The ﬁrst quadrant of initial condition Q= {(x,y) : x> ,y≥} is the union of three disjoint basins of attraction, Q= B(,∞) ∪B(∞,) ∪B(E), where B(E) = Ws(E) and B(,∞) = (x,y)\|(x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈Ws(E) ; B(∞,) = (x,y)\|(˜x, ˜y) ⪯se (x,y) for some (˜x, ˜y) ∈Ws(E) . Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 23 of 32 q http://www.advancesindifferenceequations.com/content/2014/1/301 q http://www.advancesindifferenceequations.com/content/2014/1/301 http://www.advancesindifferenceequations.com/content/2014/1/301 Figure 1 (a) Visual illustration of Theorem 17. (b) Visual illustration of Theorem 18. Figure 1 (a) Visual illustration of Theorem 17. (b) Visual illustration of Theorem 18. Figure 1 (a) Visual illustration of Theorem 17. (b) Visual illustration of Theorem 18. Proof The conditions of this theorem and a̸= Acimply all assumptions of Theorems , and for R. The proof of this theorem in this case is similar to the proof of Theorem  and will be skipped. Now, we assume that a= Ac. In view of Lemma , the set I = x, bx– A¯x ¯x : x ≥ A¯x b I = x, bx– A¯x ¯x : x ≥ A¯x b is invariant and contains equilibrium E, and T(x,y) = E for (x,y) ∈I. In view of the unique- ness of the global stable manifold, we conclude that Ws(E) = I. Let W– = (x,y)\|(x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈Ws(E) ; W+ = (x,y)\|(˜x, ˜y) ⪯se (x,y) for some (˜x, ˜y) ∈Ws(E) . Take any point (x,y) ∈W+(E). Then there exists the point (xl,yl) ∈I such that (xl,yl) ≪se (x,y). Since the map T is strongly competitive, then E = T(xl,yl) ≪se T(x,y). This implies T(x,y) ∈int(Q(E) ∩R). If (x,y) ∈W–(E), then there exists the point (xr,yr) ∈I such that (x,y) ≪se (xr,yr). global stable manifolds are given by Ws ( k+,k+),( ˜ k+, ˜k+) = Ws( k+,k+) ∪Ws( ˜ k+, ˜k+), k = ,...,n, where Ws( k+,k+) and Ws( ˜ k+, ˜k+) are the graphs of a continuous strictly in- creasing function such that Ws( ˜ k+, ˜k+) = T(Ws( k+,k+)) with endpoints at ( ˜ k, ˜k), ( ˜ k+, ˜k+) and ( k+,k+), ( k,k), k = ,...,n – , respectively, where ( ˜ , ˜) = ( ,) = E. The curve C = n k= Ws( k+,k+) ∪Ws( ˜ k+, ˜k+) ∪ ( k,k),( ˜ k, ˜k) separates R into two components W– and W+, which are basins of attraction of (,∞) and (∞,), respectively, where separates R into two components W– and W+, which are basins of attraction of (,∞) and (∞,), respectively, where W– = (x,y)\|(x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈C ; W+ = (x,y)\|(˜x, ˜y) ⪯se (x,y) for some (˜x, ˜y) ∈C . Further, for k = ,...,n, we have Further, for k = ,...,n, we have Further, for k = ,...,n, we have B ( k+,k+),( ˜ k+, ˜k+) = Ws( k+,k+) ∪Ws( ˜ k+, ˜k+); B ( k,k),( ˜ k, ˜k) = ( k,k),( ˜ k, ˜k) . The global unstable manifolds are given by The global unstable manifolds are given by Theorem Assume that > . If Since the map T is strongly competitive, then T(x,y) ≪se E = T(xr,yr). This implies T(x,y) ∈int(Q(E) ∩R). Similarly as in Theo- rem , one can prove that Tn(x,y) →(,∞) as n →∞if (x,y) ∈int(Q(E) ∩R) and Tn(x,y) →(∞,) as n →∞if (x,y) ∈int(Q(E) ∩R), from which the proof follows in this case. □ □ See Figure for visual illustration of Theorems and . Theorem If a> Acand (¯y) < , then there exists one equilibrium point E which is a repeller. If D(p) ̸= , then int(Q(E))∪int(Q(E)) contains an odd number of minimal period- two solutions {( i,i),( ˜ i, ˜i)}, i = ,...,n + , such that ( i+,i+) ≪ne ( i,i) ≪ne E and E ≪ne ( ˜ i, ˜i) ≪ne ( ˜ i+, ˜i+) and ( ˜ i, ˜i) = T( i,i). Furthermore, the odd indexed period-two points are saddles and the even indexed period-two points are repellers. The Page 24 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 global stable manifolds are given by 5.2 The case where at least one of the equilibrium points is non-hyperbolic and all minimal period-two solutions are hyperbolic (1 = 0 and D(p) ̸= 0) 5.2 The case where at least one of the equilibrium points is non-hyperbolic and all minimal period-two solutions are hyperbolic (1 = 0 and D(p) ̸= 0) Theorem Assume that = , ̸= . Then there exist two equilibrium points E(¯x, ¯y) and E(¯x, ¯y) such that E≪ne E, and the following hold: (a) If Eis non-hyperbolic, then there exist two invariant curves C+ and C+ that are con- tained in Q(E) with endpoint in ∂Q(E), which are the graphs of continuous strictly in- creasing functions. Further, there exists the global stable manifold Ws(E) with endpoint at E, which is a graph of a continuous increasing function and the global unstable mani- fold Wu(E), which is a graph of a continuous decreasing function. Every solution {(xn,yn)} which starts below Ws(E) ∪C+ in the south-east ordering is asymptotic to (,∞), and ev- ery solution {(xn,yn)} which starts above Ws(E) ∪C+ in the south-east ordering is asymp- totic to (∞,). The ﬁrst quadrant of initial condition Q= {(x,y) : x> ,y≥} is the union of four disjoint basins of attraction, Q= B(,∞) ∪B(∞,) ∪B(E) ∪B(E), where The global unstable manifolds are given by Wu ( k+,k+),( ˜ k+, ˜k+) = Wu( k+,k+) ∪Wu( ˜ k+, ˜k+), k = ,...,n, Wu ( k+,k+),( ˜ k+, ˜k+) = Wu( k+,k+) ∪Wu( ˜ k+, ˜k+), where Wu( k+,k+) and Wu( ˜ k+, ˜k+) are the graphs of continuous strictly decreas- ing functions such that Wu( ˜ k+, ˜k+) = T(Wu( k+,k+)) with endpoints at (,∞) and (∞,). where Wu( k+,k+) and Wu( ˜ k+, ˜k+) are the graphs of continuous strictly decreas- ing functions such that Wu( ˜ k+, ˜k+) = T(Wu( k+,k+)) with endpoints at (,∞) and (∞,). Proof In view of Corollary , the set int(Q(E)) ∪int(Q(E)) contains an odd number of minimal period-two solutions {( i,i),( ˜ i, ˜i)}, i = ,...,n+, such that ( i+,i+) ≪ne ( i,i) ≪ne E and E ≪ne ( ˜ i, ˜i) ≪ne ( ˜ i+, ˜i+) and ( ˜ i, ˜i) = T( i,i). Furthermore, the odd indexed period-two points are saddles and the even indexed period-two points are repellers. The map Tsatisﬁes all conditions of Theorems , and , which yields the existence of the global stable and unstable manifolds with the above properties. In view of Theorem , for (x,y) ∈W– ∩R, there exists n> such that Tn(x,y) ∈ n k= int Q( k+,k+) ∪int Q( ˜ k+, ˜k+) ∩R, n > n, Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 25 of 32 q http://www.advancesindifferenceequations.com/content/2014/1/301 Figure 2 Visual illustration of Theorem 19. Figure 2 Visual illustration of Theorem 19. Figure 2 Visual illustration of Theorem 19. and for (x,y) ∈W+ ∩R, there exists n> such that Tn(x,y) ∈ n k= int Q( k+,k+) ∪int Q( ˜ k+, ˜k+) ∩R, n > n. The global unstable manifolds are given by Similarly as in the proof of Theorem , one can prove that Similarly as in the proof of Theorem , one can prove that Similarly as in the proof of Theorem , one can prove that n k= int Q( k+,k+) ∪int Q( ˜ k+, ˜k+) ∩R ⊂B(,∞) and n k= int Q( k+,k+) ∪int Q( ˜ k+, ˜k+) ∩R ⊂B(∞,), □ which completes the proof. See Figure for visual illustration of Theorem . See Figure for visual illustration of Theorem . See Figure for visual illustration of Theorem . all minimal period-two solutions are hyperbolic (1 = 0 and D(p) ̸= 0) Theorem Assume that = , ̸= . Then there exist two equilibrium points E(¯x, ¯y) and E(¯x, ¯y) such that E≪ne E, and the following hold: E(x,y) and E(x,y) such that E≪ne E, and the following hold: (a) If Eis non-hyperbolic, then there exist two invariant curves C+ and C+ that are con- tained in Q(E) with endpoint in ∂Q(E), which are the graphs of continuous strictly in- creasing functions. Further, there exists the global stable manifold Ws(E) with endpoint at E, which is a graph of a continuous increasing function and the global unstable mani- fold Wu(E), which is a graph of a continuous decreasing function. Every solution {(xn,yn)} which starts below Ws(E) ∪C+ in the south-east ordering is asymptotic to (,∞), and ev- ery solution {(xn,yn)} which starts above Ws(E) ∪C+ in the south-east ordering is asymp- totic to (∞,). The ﬁrst quadrant of initial condition Q= {(x,y) : x> ,y≥} is the union of four disjoint basins of attraction, Q= B(,∞) ∪B(∞,) ∪B(E) ∪B(E), where (a) If Eis non-hyperbolic, then there exist two invariant curves C+ and C+ that are con- tained in Q(E) with endpoint in ∂Q(E), which are the graphs of continuous strictly in- creasing functions. Further, there exists the global stable manifold Ws(E) with endpoint at E, which is a graph of a continuous increasing function and the global unstable mani- fold Wu(E), which is a graph of a continuous decreasing function. Every solution {(xn,yn)} which starts below Ws(E) ∪C+ in the south-east ordering is asymptotic to (,∞), and ev- ery solution {(xn,yn)} which starts above Ws(E) ∪C+ in the south-east ordering is asymp- totic to (∞,). The ﬁrst quadrant of initial condition Q= {(x,y) : x> ,y≥} is the union of four disjoint basins of attraction, Q= B(,∞) ∪B(∞,) ∪B(E) ∪B(E), where Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 26 of 32 B(E) = Ws(E) and B(E) = (x,y) ∈R : (˜x, ˜y) ⪯se (x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈C+ and (˜x, ˜y) ∈C+  , B(,∞) = (x,y)\|(x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈Ws(E) ∪C+  , B(∞,) = (x,y)\|(˜x, ˜y) ⪯se (x,y) for some (˜x, ˜y) ∈Ws(E) ∪C+  . all minimal period-two solutions are hyperbolic (1 = 0 and D(p) ̸= 0) B(E) = (x,y) ∈R : (˜x, ˜y) ⪯se (x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈C+ and (˜x, ˜y) ∈C+  , (b) If Eis non-hyperbolic, then there exist two invariant curves C– and C– that are con- tained in Q(E) with endpoint in ∂Q(E), which are the graphs of continuous strictly in- creasing functions. Further, there exists the global stable manifold Ws(E) with endpoint at E, which is a graph of a continuous increasing function, and the global unstable mani- fold Wu(E), which is a graph of a continuous decreasing function. Every solution {(xn,yn)} which starts below Ws(E) ∪C– in the south-east ordering is asymptotic to (,∞), and ev- ery solution {(xn,yn)} which starts above Ws(E) ∪C– in the south-east ordering is asymp- totic to (∞,). The ﬁrst quadrant of initial condition Q= {(x,y) : x> ,y≥} is the union of four disjoint basins of attraction, Q= B(,∞) ∪B(∞,) ∪B(E) ∪B(E), where B(E) = Ws(E) and B(E) = (x,y) ∈R : (˜x, ˜y) ⪯se (x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈C– and (˜x, ˜y) ∈C–  , B(,∞) = (x,y)\|(x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈Ws(E) ∪C–  , B(∞,) = (x,y)\|(˜x, ˜y) ⪯se (x,y) for some (˜x, ˜y) ∈Ws(E) ∪C–  . (E) = (x,y) ∈R : (˜x, ˜y) ⪯se (x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈C– and (˜x, ˜y) ∈C–  , Proof We will prove statement (a). The proof of statement (b) is similar. By Theorem  we have that T has two equilibrium points Eand Esuch that E≪ne E. Then either E is non-hyperbolic and Eis a saddle point or Eis non-hyperbolic and Eis a saddle point. Assume that Eis non-hyperbolic. From Descartes’ rule of signs applied to () we have that a< Ac. Lemma implies that Tn(x,y) is asymptotic to either (,∞) or (∞,) or to an equilibrium point for all (x,y) ∈R. Let B(,∞) be the basin of attraction of (,∞), and let B(∞,) be the basin of attraction of (∞,). By using Theorem one can prove that int(Q(E)) ⊂B(,∞) and int(Q(E)) ⊂B(∞,). Let C+ denote the boundary of B(,∞) considered as a subset of Q(E) and C+ denote the boundary of B(∞,) considered as a subset of Q(E). It is easy to see that E ∈C+ and E ∈C+ . Similarly as in Claim , one can prove the following claim. all minimal period-two solutions are hyperbolic (1 = 0 and D(p) ̸= 0) Now Theorems , and yield the existence of the global stable manifold Ws(E) and the global unstable manifold Wu(E), where Ws(E) has an endpoint at Eand it is a graph of an increasing function. Similarly as in the proof of Theorem , one can see that W– = (x,y)\|(x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈Ws(E) ⊂B(,∞), W+ = (x,y)\|(˜x, ˜y) ⪯se (x,y) for some (˜x, ˜y) ∈Ws(E) ⊂B(∞,), and B(E) = Ws(E). and B(E) = Ws(E). Similarly as in Claim , one can prove the following claim. Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 27 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Claim Let C+ and C+ be the sets deﬁned as above. Then we have the following: (a) If (x,y) ∈B(∞,), then (x,y) ∈B(∞,) for all (x,y) ⪯se (x,y); (b) If (x,y) ∈B(,∞), then (x,y) ∈B(,∞) for all (x,y) ⪯se (x,y); (c) If (x,y) ∈C+ , then (x,y) ∈int(B(,∞)) for all (x,y) ≪se (x,y) and (x,y) /∈B(,∞) for all (x,y) ≪se (x,y); (d) If (x,y) ∈C+ , then (x,y) ∈int(B(∞,)) for all (x,y) ≪se (x,y) and (x,y) /∈B(∞,) for all (x,y) ≪se (x,y); (e) C+ ∩int(Q(E)) ̸= and C+ ∩int(Q(E)) ̸= ; (f) T(C+ ) ⊆C+ and T(C+ ) ⊆C+ ; (g) (x,y),(x,y) ∈C+ ⇒(x,y) ≪ne (x,y) or (x,y) ≪ne (x,y); (h) (x,y),(x,y) ∈C+ ⇒(x,y) ≪ne (x,y) or (x,y) ≪ne (x,y). (h) (x,y),(x,y) ∈C+ ⇒(x,y) ≪ne (x,y) or (x,y) ≪ne (x,y). Proof We prove statement (f). Since Ws(E) ∪C+ is the boundary of B(,∞) similar as in the proof of statement (d) of Claim , we have that T(Ws(E) ∪C+ ) ⊆Ws(E) ∪C+ . Take (x,y) ∈C+ \ E. If T(x,y) ∈Ws(E), then Tn(x,y) →Eas n →∞, which is in con- tradiction with B(E) = Ws(E) and B(E) ∩C+ = {E}. Hence, T(x,y) ∈C+ which implies that T(C+ ) ⊆C+ . The corresponding assertion for C+ is proved in a similar fashion. □ Now, we prove that C+ and C+ are the graphs of continuous strictly increasing functions. Let J be the projection of C+ onto the ﬁrst coordinate. From (g), no two distinct elements of C+ can be weakly related nor can they have the same projection onto the ﬁrst coordinate. Further, J contains [x,x] whenever x≤xbelong to J. Indeed, if x≤xbelong to J, then there exist yand ysuch that (x,y),(x,y) ∈C+ . q http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 all minimal period-two solutions are hyperbolic (1 = 0 and D(p) ̸= 0) From (c), we have that int(Q(x,y)) ⊂ int(B(,∞)) and int(Q(x,y)) ⊂int(B(∞,)). By using this it is easy to see that (x, ˆy) ∈C+  if and only if ˆy = inf{y : y ∈[y,y] and (x,y) ∈int(B(,∞))}, where x ∈[x,x]. This implies that J contains [x,x]. By (e), C+ ∩int(Q(E)) ̸= . From this it follows that J is an interval such that int(J) ̸= ∅and C+ is a connected set. Since points on C+ are non-comparable, C+  is the graph of a strictly increasing function f (x) of x ∈J. If there is a jump discontinuity at x∈J, let y– and y+ respectively be the left and right (distinct) limits of f (x) as x approaches x, respectively. The points (x,y–) and (x,y+) are comparable in ⪯se-ordering, which is in contradiction with (g). Thus f (x) is a continuous function. Since T has no equilibrium points in Q(E), similarly as in the proof of Theorem [], one can prove that C+ has its endpoints in ∂Q(E). The proof that considers C+ is similar and will be skipped. Take (x,y) ∈C+ and (x,y) ∈C+ . Since Eis the only equilibrium point in Q(E) and (,∞),(∞,) /∈Q(E) we have that Tn(x,y) and Tn(x,y) converge to Eas n →∞. If (˜x, ˜y) ⪯se (x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈C+ and (˜x, ˜y) ∈C+ , since T is competitive, we get Tn(˜x, ˜y) ⪯se Tn(x,y) ⪯se Tn(˜x, ˜y), which implies Tn(x,y) →Eas n →∞. □ Based on a series of numerical simulations, we propose the following conjec See Figure for visual illustration of Theorem . See Figure for visual illustration of Theorem . Conjecture Suppose that all assumptions of Theorem are satisﬁed, then the following hold: (a) B(E) = C+ = C+ ; (a) B(E) = C+ = C+ ; (b) B(E) = C– = C– . (b) B(E) = C– = C– . Theorem Assume that = , = and a< Ac. Then there exist the unique non- hyperbolic equilibrium point E(¯x, ¯y) and two invariant curves C– and C+ with endpoint in Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 Page 28 of 32 http://www.advancesindifferenceequations.com/content/2014/1/301 Figure 3 Visual illustration of statements (a) and (b) of Theorem 20. Figure 3 Visual illustration of statements (a) and (b) of Theorem 20. all minimal period-two solutions are hyperbolic (1 = 0 and D(p) ̸= 0) Figure 3 Visual illustration of statements (a) and (b) of Theorem 20. Figure 4 Visual illustration of Theorem 21. ∂R, which are the graphs of continuous strictly increasing functions. Every solution {(xn,yn)} which starts below Cin the south-east ordering is asymptotic to (,∞) and every solution {(xn,yn)} which starts above Cin the south-east ordering is asymptotic to (∞,). The ﬁrst quadrant of initial condition Q= {(x,y) : x> ,y≥} is the union of three disjoint basins of attraction, Q= B(,∞) ∪B(∞,) ∪B(E), where Figure 3 Visual illustration of statements (a) and (b) of Theorem 20. Figure 4 Visual illustration of Theorem 21. ∂R, which are the graphs of continuous strictly increasing functions. Every solution {(xn,yn)} which starts below Cin the south-east ordering is asymptotic to (,∞) and every solution {(xn,yn)} which starts above Cin the south-east ordering is asymptotic to (∞,). The ﬁrst quadrant of initial condition Q= {(x,y) : x> ,y≥} is the union of three disjoint basins of attraction, Q= B(,∞) ∪B(∞,) ∪B(E), where Figure 4 Visual illustration of Theorem 21. Figure 4 Visual illustration of Theorem 21. ∂R, which are the graphs of continuous strictly increasing functions. Every solution {(xn,yn)} which starts below Cin the south-east ordering is asymptotic to (,∞) and every solution {(xn,yn)} which starts above Cin the south-east ordering is asymptotic to (∞,). The ﬁrst quadrant of initial condition Q= {(x,y) : x> ,y≥} is the union of three disjoint basins of attraction, Q= B(,∞) ∪B(∞,) ∪B(E), where ∂R, which are the graphs of continuous strictly increasing functions. Every solution {(xn,yn)} which starts below Cin the south-east ordering is asymptotic to (,∞) and every solution {(xn,yn)} which starts above Cin the south-east ordering is asymptotic to (∞,). The ﬁrst quadrant of initial condition Q= {(x,y) : x> ,y≥} is the union of three disjoint basins of attraction, Q= B(,∞) ∪B(∞,) ∪B(E), where B(E) = (x,y) ∈R : (˜x, ˜y) ⪯se (x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈Cand (˜x, ˜y) ∈C ; B(,∞) = (x,y)\|(x,y) ⪯se (˜x, ˜y) for some (˜x, ˜y) ∈C ; B(∞,) = (x,y)\|(˜x, ˜y) ⪯se (x,y) for some (˜x, ˜y) ∈C . Proof The proof is similar to the proof of Theorem and will be skipped. Proof The proof is similar to the proof of Theorem and will be skipped. □ Based on a series of numerical simulations, we propose the following conjecture. Based on a series of numerical simulations, we propose the following conjecture. See Figure for visual illustration of Theorem . Based on a series of numerical simulations, we propose the following conjecture. See Figure for visual illustration of Theorem . See Figure for visual illustration of Theorem . See Figure for visual illustration of Theorem . Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 29 of 32 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 q http://www.advancesindifferenceequations.com/content/2014/1/301 Conjecture Suppose that all assumptions of Theorem are satisﬁed, then the following holds: B(E) = C= C. Appendix: Values of coefﬁcients pi for i = 0,...,22 p= a b  c A – ac A + a c A + a A + a  b c – A c , p= aA b  c A + ac  b c – A  A + a c  A – b c  A + a b cA  + a  A – A b c  + a  c b + A c b – A c , p= a b  c A – ac A + a c A + a A + a  b c – A c , p= aA b  c A + ac  b c – A  A + a c  A – b c  A + a b cA  + a  A – A b c  + a  c b + A c b – A c , p= c A + ac  b c – A  A + a c c b – A c b – A c b – A  A  + a  A c b + A b  – a  b cA + b c A  + a  A c – A b c  + a  c b + A c b + A c b + A  , p= c A + ac  b c – A  A + a c c b – A c b – A c b – A  A  + a  A c b + A b  – a  b cA + b c A  + a  A c – A b c  + a  c b + A c b + A c b + A  , p= Ab  c A + ac  c b + A  A + a c  c b – A c b – A  A  + a c  c b – A c b + A  A+ a  Ac b + A cb  + a  A – b c A – b c A  + a  c b + A c b – A c , p= c  c b + A  A + ac  c b + A c b – A  A  + a  c b + A c b + A  A  + a c  c b – A c b – A c b + A  A + a b  c b + A  A  – a  A c b + A cb  + a c  c b – A c b – A c b + A  A + a b  c b + A  A  – a  A c b + A cb  Hadžiabdi´c et al. Author details 1Division of Mathematics, Faculty of Mechanical Engineering, University of Sarajevo, Sarajevo, Bosnia and Herzegovina. 2Department of Mathematics, University of Rhode Island, Kingston, Rhode Island 02881-0816, USA. 3Department of Mathematics, University of Sarajevo, Sarajevo, Bosnia and Herzegovina. Competing interests Competing interests Competing interests The authors declare that they have no competing interests. g The authors declare that they have no competing interests. Authors’ contributions Each of the authors, VH, MRSK and EP, contributed to each part of this work equally and read and approved the ﬁnal version of the manuscript. Appendix: Values of coefﬁcients pi for i = 0,...,22 Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 31 of 32 + a c  c b + A  b c – A  + a  A + b c A + a  cA + b c A  + a  –Ac b – A c b + A c  + a c A + b c A – b c A  , p= A b ca +  c b + A c b + A  a  –  –Ac b + A c b + A c a  +  c A – b c A – b c A  a  + c b – A c b + A c b – A c  a + Ac  c b + A c b + A c b + A  , p= b  –A b c + A b c – A c + a A + a  cA + b c A + a  A b c – A c  – a  –c b + A c b + A c  + a –Ab c – A b c + A c  , p=  A + b c A  a + c c b + A c b – A  a  +  Ac b – A c b + A c  a  –  c A – b c A + b c A  a+ c  c b + A c b + A  , p= b c –Ab c + A b c – A c + a Ab c + a A  – a  c A + b c A  + a c b – A c b + A c  , p= a A – a c A – b c  A + c  –c b + A c b + A  A  – ac  –c b + A c b + A  A+ a  c b – A c b + A c  , p=b c  c b – A c b + a A – A c – a Ac A – b c  + a A c – A b c  , p= b c  c A + a A + a b c – A c , p= Ab c (a– Ac), + a c  c b + A  b c – A  + a  A + b c A + a  cA + b c A  + a  –Ac b – A c b + A c  + a c A + b c A – b c A  , p= A b ca +  c b + A c b + A  a  –  –Ac b + A c b + A c a  +  c A – b c A – b c A  a  + c b – A c b + A c b – A c  a + Ac  c b + A c b + A c b + A  , p= b  –A b c + A b c – A c + a A + a  cA + b c A p= b  –A b c + A b c – A c + a A + a  cA + b c A – a  –c b + A c b + A c  + a –Ab c – A b c + A c  , p=  A + b c A  a + c c b + A c b – A  a  –  c A – b c A + b c A  a+ c  c b + A c b + A  , p= b c –Ab c + A b c – A c + a Ab c + a A  – a  c A + b c A  + a c b – A c b + A c  , p= a A – a c A – b c  A + c  –c b + A c b + A  A  – ac  –c b + A c b + A  A+ a  c b – A c b + A c  , p=b c  c b – A c b + a A – A c – a Ac A – b c  + a A c – A b c  , Appendix: Values of coefﬁcients pi for i = 0,...,22 Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Hadžiabdi´c et al. Advances in Diﬀerence Equations 2014, 2014:301 http://www.advancesindifferenceequations.com/content/2014/1/301 Page 30 of 32 + Ac  c b + A c b + A  a  + A c  c b + A c b – A c b – A  a + A c  c b + A c b + A  , p= Ac b + A b  a +  A c b + A cb  a  +  A – b c A – b c A  a  + Ac c b + A c b + A c b – A  a  + A c  c b + A c b + A c b + A  a  + c  c b + A c b – A c b – A c b – A  a + A c  c b + A c b + A  , p= Ac b + A b  a +  A c b + A cb  a  +  A – b c A – b c A  a  + Ac c b + A c b + A c b – A  a  + A c  c b + A c b + A c b + A  a  + c  c b + A c b – A c b – A c b – A  a + A c  c b + A c b + A  , + Ac  c b + A c b + A c b – A  a p= b  c b + A c b + A c b – A c  Hadžiabdi´c et al. 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https://openalex.org/W2886150863	https://europepmc.org/articles/pmc6341410?pdf=render	English	null	A review of the effects of unilateral hearing loss on spatial hearing	Hearing research	2,019	cc-by	14,385	a r t i c l e i n f o . . . . . . . . . . . 26 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Contents lists available at ScienceDirect Hearing Research * Corresponding author. E-mail address: andrew.king@dpag.ox.ac.uk (A.J. King). Hearing Research 372 (2019) 17e28 Hearing Research 372 (2019) 17e28 a r t i c l e i n f o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2. The importance and limitations of binaural processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3. Prevalence of unilateral hearing loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a r t i c l e i n f o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4. Effects of unilateral hearing loss on the developing auditory system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 5. Adaptation to unilateral hearing loss in the mature auditory system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 6. Perceptual training for hearing-impaired listeners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a r t i c l e i n f o Article history: Received 26 March 2018 Received in revised form 5 July 2018 Accepted 9 August 2018 Available online 11 August 2018 Keywords: Sound localization Spatial release from masking Plasticity Binaural Monaural spectral cue Auditory cortex Article history: Received 26 March 2018 Received in revised form 5 July 2018 Accepted 9 August 2018 Available online 11 August 2018 Keywords: Sound localization Spatial release from masking Plasticity Binaural Monaural spectral cue Auditory cortex Article history: Received 26 March 2018 Received in revised form 5 July 2018 Accepted 9 August 2018 Available online 11 August 2018 The capacity of the auditory system to extract spatial information relies principally on the detection and interpretation of binaural cues, i.e., differences in the time of arrival or level of the sound between the two ears. In this review, we consider the effects of unilateral or asymmetric hearing loss on spatial hearing, with a focus on the adaptive changes in the brain that may help to compensate for an imbalance in input between the ears. Unilateral hearing loss during development weakens the brain's represen- tation of the deprived ear, and this may outlast the restoration of function in that ear and therefore impair performance on tasks such as sound localization and spatial release from masking that rely on binaural processing. However, loss of hearing in one ear also triggers a reweighting of the cues used for sound localization, resulting in increased dependence on the spectral cues provided by the other ear for localization in azimuth, as well as adjustments in binaural sensitivity that help to offset the imbalance in inputs between the two ears. These adaptive strategies enable the developing auditory system to compensate to a large degree for asymmetric hearing loss, thereby maintaining accurate sound locali- zation. They can also be leveraged by training following hearing loss in adulthood. Although further research is needed to determine whether this plasticity can generalize to more realistic listening con- ditions and to other tasks, such as spatial unmasking, the capacity of the auditory system to undergo these adaptive changes has important implications for rehabilitation strategies in the hearing impaired. © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Keywords: Sound localization Spatial release from masking Plasticity Binaural Monaural spectral cue Auditory cortex Contents 1. Introduction . . . . . . . . . . . . a r t i c l e i n f o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2. The importance and limitations of binaural processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3. Prevalence of unilateral hearing loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4. Effects of unilateral hearing loss on the developing auditory system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 5. Adaptation to unilateral hearing loss in the mature auditory system . . . . a r t i c l e i n f o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 6. Perceptual training for hearing-impaired listeners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 References . . . . . https://doi.org/10.1016/j.heares.2018.08.003 0378-5955/© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction potential mates or prey or avoiding and escaping from approaching predators. This is particularly the case if the source lies beyond the detection range of the other senses, either because it is located outside the visual ﬁeld or is too far away to be registered by other sensory receptors. The basis for directional hearing relies princi- pally on the fact that animals have two ears that are physically separated on either side of the head, or in the case of some insects, on other parts of the body. This means that, depending on the location of the sound source, the signals reaching each ear may An ability to localize and segregate different sound sources is extremely important for most species that can hear, often playing a crucial role in guiding behavioral responses, such as seeking out D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 18 i 1 i l d l i (A) l l l diff Research 372 (2019) 17 28 Fig. 1. Binaural cues to sound source location. (A) Interaural level differences as a function of sound azimuth and frequency. (B) Interaural time differences as a function of sound azimuth and frequency. Negative values indicate azimuths and corresponding binaural cue values on the left of the midline. Data for both cues are derived from head-related transfer function measurements (0 elevation) published in the CIPIC database by Algazi et al. (2001). (Copyright (c) 2001 The Regents of the University of California. All Rights Reserved). g ( ) differ in their time of arrival or intensity, giving rise to binaural spatial cues (reviewed by Blauert, 1997; Schnupp et al., 2011). A large number of studies have demonstrated the importance of binaural cues for sound-source localization, as well as for improving the perception of target sounds in the presence of other, interfering sounds (Bronkhorst, 2000), and a great deal is known about how these cues are processed in the brain (reviewed by Grothe et al., 2010). By eliminating binaural cues, or at least altering the relationship between the interaural acoustic differences and directions in space, unilateral or asymmetric hearing loss can have very disruptive effects on spatial hearing. Furthermore, monaural deprivation in infancy can induce maladaptive changes in the brain that may persist even if hearing in the affected ear is restored, resulting in longer-term deﬁcits in spatial hearing (Kaplan et al., 2016). 2. The importance and limitations of binaural processing Because cochlear hair cells are tuned to different sound fre- quencies, with their topographically organized outputs producing tonotopic maps throughout the core or lemniscal regions of the central auditory pathway, sound-source location has to be computed through the sensitivity of neurons to the physical cues generated by the geometry of the head and external ears. For sound sources located to one side of the midline, frequency-dependent interaural level differences (ILDs) may be generated by a combi- nation of the spectral ﬁltering effects produced by the external ears and the attenuation at the far ear due to the acoustic shadow cast by the head (Fig. 1A). In addition, the difference in path length from the sound source to each ear produces an interaural time difference (ITD) whose magnitude is determined by both the distance be- tween the ears and the angle subtended by the source relative to the head (Fig. 1B). Fig. 1. Binaural cues to sound source location. (A) Interaural level differences as a function of sound azimuth and frequency. (B) Interaural time differences as a function of sound azimuth and frequency. Negative values indicate azimuths and corresponding binaural cue values on the left of the midline. Data for both cues are derived from head-related transfer function measurements (0 elevation) published in the CIPIC database by Algazi et al. (2001). (Copyright (c) 2001 The Regents of the University of California. All Rights Reserved). Because of the tonotopic organization of the auditory system, these binaural comparisons mostly take place within frequency- speciﬁc channels. For simple periodic sounds, such as pure tones, the temporal ﬁne structure is represented by the phase-locked discharges of auditory neurons at relatively low frequencies only (e.g., Sumner and Palmer, 2012). Moreover, interaural phase dif- ferences become spatially ambiguous as the sound frequency is increased (Mills, 1972; Blauert, 1997). Conversely, the ILDs gener- ated by the shadowing effect of the head are the dominant locali- zation cue when the wavelength of the sound is less than the distance between the two ears and therefore for adult humans at frequencies above ~1700 Hz (Fig. 1A). This provides the basis for the duplex theory of sound localization (Strutt, 1907), whereby ITDs and ILDs are utilized for localizing low-frequency and high- frequency sounds, respectively. the source is varied (Blauert,1997; Carlile et al., 2005). 1. Introduction However, as described in the following sections, there is growing evidence that the plasticity of central auditory processing can help to partially compensate for loss of hearing in one ear, leading to some recovery in the ability to localize sound (e.g. Knudsen et al., 1984; Keating et al., 2013, 2015, 2016). In this article, we review the effects of asymmetric hearing loss on spatial pro- cessing, both during development and in later life, and consider the factors that may promote adaptive changes in the brain and their potential clinical relevance. 4. Effects of unilateral hearing loss on the developing auditory system Experimental studies of the effects of unilateral hearing loss on spatial hearing have focussed primarily on the consequences of conductive loss (reviewed in Keating and King, 2013). It is impor- tant to note that sensorineural hearing loss can produce spec- trotemporal processing deﬁcits that would be expected to affect neural sensitivity to spatial localization cues (Moore, 1996; Felix and Portfors, 2007; Trujillo and Razak, 2013). Nevertheless, inducing a conductive hearing loss in one ear has the great advantage from an experimental perspective that it is, in principle, fully reversible. For example, monaural occlusion can be used in both humans and animals to produce a temporary imbalance in input between the two ears. From a clinical standpoint, under- standing how the brain responds to conductive hearing loss can provide insight into the consequences of otitis media with effusion and other disorders that affect sound transmission through the external or middle ear. g ( g ) Although spatial release from masking can occur in the absence of subjective sound localization, two key processes that support this phenomenon, better-ear listening and binaural unmasking, are explicitly dependent upon the interaural disparities that arise when a sound source is located to one side of the head. Better-ear listening can improve the signal-to-noise-ratio (SNR) at one ear for a target sound if the masker is attenuated due to the shadowing effect of the head. In realistic speech-in-noise scenarios, such as when multiple spatially-separated maskers are present, the better ear may ﬂuctuate over time and frequency. Consequently, better- ear effects are thought to result from the auditory system's ability to “glimpse” these short-term changes in SNR (e.g., Brungart and Iyer, 2012; Schoenmaker and van de Par, 2017). In contrast to better-ear effects, binaural unmasking involves a comparison of information at the two ears (Licklider, 1948; Durlach, 1963). Detection thresholds for bilaterally presented pure tones in noise can be up to 15 dB lower when the phase of either signal is fully inverted in one ear, a measure known as the binaural masking level difference. Similar manipulations are known to improve the intel- ligibility of masked speech (measured using the binaural intelligi- bility level difference; Levitt and Rabiner, 1967). 3. Prevalence of unilateral hearing loss Estimates of the prevalence of unilateral hearing loss, in which the impairment is restricted to one ear, vary with numerous factors, including the age of the subjects and, of course, the type and extent of the hearing loss (e.g. Bess et al., 1998; Lee et al., 2011; Berninger and Westling, 2011). For example, minimal sensorineural hearing loss in one ear (15e40 dB HL) has been reported in 3% of sampled school-age children (Bess et al., 1998). In terms of the potential impact on spatial hearing, it is just as important to consider asymmetric hearing loss, where both ears might be affected but to differing degrees. This is particularly the case in young children, where the changing incidence of either unilateral or bilateral otitis media with effusion with age is likely to provide highly variable experience of spatial cues for the majority of individuals (Hogan et al., 1997; Whitton and Polley, 2011). Furthermore, treatments for hearing loss may actually exacerbate asymmetric hearing, such as when individuals with severe to profound bilateral deafness receive a cochlear implant in one ear only or sequentially in the two ears. This may also be the case if there is a delay in providing a device to the affected ear when hearing loss is unilateral. Fig. 2. Monaural spectral cues to sound source elevation. Pinna gain for the right ear is shown as a function of sound frequency and elevation for sounds presented at 0 az- imuth. Data are derived from head-related transfer function measurements published in the CIPIC database by Algazi et al. (2001). (Copyright (c) 2001 The Regents of the University of California. All Rights Reserved). (or target detection) thresholds in the presence of interfering sounds when the target and masking sounds are spatially sepa- rated. Spatial release from masking is one process that can support auditory stream segregation, including the capacity to perceive a particular speaker's voice in “cocktail-party” situations, where other, interfering, sounds are simultaneously present (Cherry,1953; Middlebrooks, 2017). The improvement in speech intelligibility that results from the spatial separation of the sound sources varies in magnitude with the nature of the masker, with a greater beneﬁt being obtained with informational masking than with energetic masking (Arbogast et al., 2002). 2. The importance and limitations of binaural processing Spectral cues are also important in the horizontal plane as they provide the basis for determining whether sound sources are located in front or behind the listener, and therefore for resolving the cones of confusion that are inherent in the way binaural cues vary with spatial location (Blauert, 1997; Schnupp et al., 2011). Although these cues otherwise appear to contribute little to localization in the horizontal plane, which instead relies principally on ITDs and ILDs (Macpherson and Middlebrooks, 2002), we shall show in the following that the relative weighting of the cues used to localize sound sources in azimuth can change with experience, particularly following hearing loss in one ear. Because of their unusual ear asymmetry, barn owls are able to use the two binaural cues for localizing sounds at any angle relative to the head, relying on ILDs in the vertical plane while ITDs provide the principal basis for localization in the horizontal plane (Knudsen and Konishi, 1979). In most other species, however, the skull is symmetrical, with the values of both binaural cues varying pre- dominantly in the horizontal plane. Vertical localization relies instead on spectral localization cues (Fig. 2), i.e., frequency- dependent changes in the level of the sound as the location of In addition to providing a basis for localizing sounds, the ability to extract interaural information facilitates target detection in noisy environments (Darwin, 2006; Eramudugolla et al., 2008; Maddox and Shinn-Cunningham, 2012), a phenomenon known as spatial release from masking. This refers to the change in speech reception D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 19 Fig. 2. Monaural spectral cues to sound source elevation. Pinna gain for the right ear is shown as a function of sound frequency and elevation for sounds presented at 0 az- imuth. Data are derived from head-related transfer function measurements published in the CIPIC database by Algazi et al. (2001). (Copyright (c) 2001 The Regents of the University of California. All Rights Reserved). localization and spatial release from masking depend on binaural processing and will therefore be impaired, particularly if the target sound is located on that side, if hearing is lost in one ear. 4. Effects of unilateral hearing loss on the developing auditory system For example, Slattery and Middlebrooks (1994) compared the localization ability of monaural subjects who had congenital deaf- ness in one ear with that of normal-hearing controls wearing a monaural earplug to simulate asymmetric hearing loss. Monaural occlusion in the controls severely disrupted sound localization in the horizontal plane, with the subjects displaying a large lateral response bias towards the open ear, and also affected vertical localization, particularly on the side of the plugged ear. In contrast, The capacity of the developing auditory system to compensate for asymmetric hearing loss by becoming more dependent on the spectral cues generated by the intact ear has been demonstrated most clearly by rearing ferrets with one ear occluded with earplugs that attenuated acoustical inputs by 15e45 dB in a frequency- dependent fashion and delayed them by ~110 ms (Keating et al., 2013, 2016). The use of an animal model affords more control over the age of onset and duration of the hearing loss than is possible in studies in people. In these experiments, monaural oc- clusion began at around 4 weeks of age, corresponding to the onset of auditory function in this altricial species, and continued for several months until the animals were fully grown. During this time, brief intermittent periods of normal hearing were provided by removing the earplug, in order to more closely mimic the ﬂuc- tuating periods of hearing loss associated with otitis media with effusion. Fig. 3. Binaural masking level difference (BMLD) in 19 patients before and after sur- gery to correct congenital unilateral hearing loss resulting from an abnormal external and/or middle ear on one side. The BMLD (N0S0 minus N0Sp) is the difference in detection threshold of a tone presented either in phase or with the phase reversed between the ears in the presence of broadband noise, which was always presented in phase at the two ears. Some subjects had post-operative MLDs in the normal range, whereas others showed a persistent deﬁcit in binaural processing. Modiﬁed with permission from Wilmington et al. (1994). The animals were trained to perform a free-ﬁeld sound locali- zation task in which noise bursts were presented from one of 12 loudspeakers positioned at 30 intervals around the perimeter of the testing chamber (Fig. 5A). 4. Effects of unilateral hearing loss on the developing auditory system The physiological changes induced by unilateral or asymmetric stimu- lation can be interpreted in terms of competitive interactions tak- ing place in the developing brain between each ear. From a clinical perspective, they likely underpin the condition of amblyaudia or “lazy ear”, the persistent deﬁcit in binaural processing experienced by people with a developmental history of asymmetric hearing loss (Snik et al., 1994; Kaplan et al., 2016). The consequences have been found to include impairments in sound localization and binaural unmasking, which can outlast restoration of function to the pre- viously deprived ear (Clements and Kelly, 1978; Beggs and Foreman, 1980; Pillsbury et al., 1991; Wilmington et al., 1994; Moore et al., 1999; Gray et al., 2009) (Fig. 3). electrophysiological recordings, and observed impaired binaural integration, with greater reorganization in the primary auditory cortex (A1) than in the inferior colliculus (IC). Furthermore, they found that this plasticity is more pronounced in infancy than in older animals. Other electrophysiological studies have also re- ported that abnormal binaural processing is present after correc- tion of the unilateral hearing loss (Clopton and Silverman, 1977; Silverman and Clopton, 1977; Brugge et al., 1985) or following stimulation via bilateral cochlear implants (Tillein et al., 2016). The physiological changes induced by unilateral or asymmetric stimu- lation can be interpreted in terms of competitive interactions tak- ing place in the developing brain between each ear. From a clinical perspective, they likely underpin the condition of amblyaudia or “lazy ear”, the persistent deﬁcit in binaural processing experienced by people with a developmental history of asymmetric hearing loss (Snik et al., 1994; Kaplan et al., 2016). The consequences have been found to include impairments in sound localization and binaural unmasking, which can outlast restoration of function to the pre- viously deprived ear (Clements and Kelly, 1978; Beggs and Foreman, 1980; Pillsbury et al., 1991; Wilmington et al., 1994; Moore et al., 1999; Gray et al., 2009) (Fig. 3). although two of the monaural patients tested gave similar results to the controls, the other three showed little or no lateral response bias and localized sounds on their deaf and hearing sides equally well. Slattery and Middlebrooks (1994) proposed that these lis- teners had learned to use the spectral cues of their intact ear to judge the lateral angle of a sound source, but also noted that the head-shadow effect may have inﬂuenced their performance. 4. Effects of unilateral hearing loss on the developing auditory system Subsequent work in monaural listeners has conﬁrmed this (Van Wanrooij and Van Opstal, 2004; Agterberg et al., 2014). Thus, relative to binaural controls, the horizontal localization judgments of monaural humans are much more affected by stimulus level, suggestive of a dependence on the attenuating effects of the head. Furthermore, in some cases, performance was found to be impaired by degrading spectral cues either by ﬁlling the concha of the intact ear with wax or by using low-frequency sounds where those cues provide little directional information. Monaural subjects appear to be quite variable, however, in their capacity to use spectral cues to localize in azimuth (Van Wanrooij and Van Opstal, 2004; Agterberg et al., 2014). Apart from individuals with total deafness in one ear, an important consideration is whether plasticity in the processing of spectral cues can enhance the localization accuracy of subjects with partial hearing loss and who may therefore have access to binaural cues that provide conﬂicting spatial information. Human listeners with a normal history of binaural hearing during childhood who then experience impaired hearing in one ear, due either to acquired conductive hearing loss (Agterberg et al., 2012) or the presence of an earplug (Van Wanrooij and Van Opstal, 2007), can use spectral cues to localize low-level broadband sounds that are insufﬁciently loud to reach the affected ear. However, based on the degradation in performance observed at higher sound levels when the input to the impaired ear is further reduced by covering it with a muff, Agterberg et al. (2012) concluded that listeners with acquired unilateral conductive hearing loss are also able to use their abnormal binaural cues to localize sounds in azimuth (Fig. 4). Although these ﬁndings are indicative of maladaptive plasticity in binaural processing following unilateral hearing loss, other changes can take place that help to compensate for the impaired spatial hearing that would otherwise be observed. As previously mentioned, monaural spectral cues normally appear to contribute little to lateral location judgments (Macpherson and Middlebrooks, 2002). However, several studies have reported that some human listeners with single-sided deafness or severe-to-profound hearing loss in one ear can localize broadband or high-pass noise stimuli accurately in the horizontal plane (Newton, 1983; Slattery and Middlebrooks, 1994; Van Wanrooij and Van Opstal, 2004; Rothpletz et al., 2012; Agterberg et al., 2014; Firszt et al., 2017). 4. Effects of unilateral hearing loss on the developing auditory system A number of studies in animals have examined the effects of unilateral hearing loss during development on the morphology (Coleman and O'Connor, 1979; Webster and Webster, 1979; Moore et al., 1989), connectivity (Moore et al., 1989) and response prop- erties (Clopton and Silverman, 1977; Silverman and Clopton, 1977; Moore and Irvine, 1981; Brugge et al., 1985; Popescu and Polley, 2010; Polley et al., 2013; Keating et al., 2013, 2015) of neurons at different levels of the auditory system. The results of many (though not all) of these studies are consistent with unilateral hearing loss causing a weakening of the representation of the deprived ear and a strengthening of the representation of the intact ear. Similarly, chronic stimulation of one ear via a cochlear implant during early life has been shown to result in a pronounced reorganization of cortical responses in humans (Gordon et al., 2013) and cats (Kral et al., 2013) in favor of the stimulated ear. Much less attention has been given to the role of spectral cues in spatial release from masking. Because the spectral ﬁltering pro- vided by the head, and particularly the external ears, is direction dependent, this will contribute to the better-ear effect at high sound frequencies. Indeed, there is some evidence that speech intelligibility in the presence of spatially-separated masking noise improves if natural spectral cues are available than when they are not (Rychtarikova et al., 2011). Nevertheless, both sound In terms of the consequences of unilateral or asymmetric hearing loss on spatial hearing, it is important to ask what effect this shift in aural preference has on neural sensitivity to binaural cues. Popescu and Polley (2010) addressed this by rearing rats with one ear canal ligated, which was reversed prior to carrying out D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 20 electrophysiological recordings, and observed impaired binaural integration, with greater reorganization in the primary auditory cortex (A1) than in the inferior colliculus (IC). Furthermore, they found that this plasticity is more pronounced in infancy than in older animals. Other electrophysiological studies have also re- ported that abnormal binaural processing is present after correc- tion of the unilateral hearing loss (Clopton and Silverman, 1977; Silverman and Clopton, 1977; Brugge et al., 1985) or following stimulation via bilateral cochlear implants (Tillein et al., 2016). 4. Effects of unilateral hearing loss on the developing auditory system These data were obtained without using the subject's bone-anchored device (BCD off) (A), and in the BCD off condition with an additional muff over the impaired ear to further alter binaural cues (B). The gains of responses (obtained from the slopes of the regression lines ﬁtted to the data) to stimuli with levels of 55 dB SPL (solid gray regression lines) and 65 dB SPL (solid black regression lines) decreased signiﬁcantly when the impaired ear was covered with the muff, indicating that the subjects were relying on binaural cues for localization, whereas this was not the case at the lower level of 45 dB SPL (gray dashed regression lines), which was unlikely to be audible at the deprived ear. g ¼ response gain. Reproduced with permission from Agterberg et al. (2012). Fig. 4. Unaided sound-localization responses for one subject with a unilateral conductive hearing loss in the left ear. The stimulus was broadband noise (0.5e20 kHz). These data were obtained without using the subject's bone-anchored device (BCD off) (A), and in the BCD off condition with an additional muff over the impaired ear to further alter binaural cues (B). The gains of responses (obtained from the slopes of the regression lines ﬁtted to the data) to stimuli with levels of 55 dB SPL (solid gray regression lines) and 65 dB SPL (solid black regression lines) decreased signiﬁcantly when the impaired ear was covered with the muff, indicating that the subjects were relying on binaural cues for localization, whereas this was not the case at the lower level of 45 dB SPL (gray dashed regression lines), which was unlikely to be audible at the deprived ear. g ¼ response gain. Reproduced with permission from Agterberg et al. (2012). substantial degree to the asymmetric hearing loss (King et al., 2000; Keating et al., 2013, 2015) (Fig. 5B and C). achieved via a partial compensatory adjustment in ILD sensitivity (Keating et al., 2015) (Fig. 6). Both forms of adaptation can be observed in the same animals, with largely separate populations of A1 neurons showing adaptive plasticity in the processing of monaural spectral cues and binaural cues (Keating et al., 2016). Following removal of the earplug, these animals were able to localize sounds as accurately as the controls (Fig. 5C). This lack of an after-effect argues against the basis for adaptation being a sys- tematic remapping of sensitivity to the altered binaural cues. 4. Effects of unilateral hearing loss on the developing auditory system To examine the role of pinna cues at the non-occluded ear, Keating et al. (2013) randomized the spectrum of the broadband noise bursts across trials so that it was not possible to determine whether spectral features were due to the ﬁltering effects of the head and ears or were instead properties of the stimulus itself. When tested with the ear plugged, sound localization performance in ferrets raised with one ear occluded declined as the amount of spectral randomization was increased, but this effect largely disappeared once the earplug was removed (Fig. 5D). In other words, the ani- mals' horizontal localization behavior was guided by spectral cues in the asymmetric hearing loss condition, but not when normal binaural inputs were available. This was conﬁrmed by calculating the mean stimulus spectrum preceding responses to each of the 12 loudspeaker locations, which revealed high-frequency spectral features that matched the directional transfer function of the intact ear. Electrophysiological recordings from these animals showed that A1 neurons carried more information about the spectral cues available at the non-occluded ear, but again only when a conductive hearing loss was applied to the previously occluded ear (Keating et al., 2013) (Fig. 5E and F). It is unclear whether providing these animals with intermittent episodes of normal hearing while they were being raised with one ear occluded is required for the observed adaptation in their spatial hearing abilities. However, it has been shown that providing cats with brief periods of binocular vision during development can reduce the amblyopia, or loss of visual acuity, that would otherwise result from monocular deprivation (Mitchell et al., 2003, 2011). It is therefore possible that some experience of normal hearing during development may be necessary if spatial hearing abilities are to be preserved following asymmetric hearing loss, which has implica- tions for the timing of treatment in children with hearing disorders (Gordon et al., 2013; Keating and King, 2013). 4. Effects of unilateral hearing loss on the developing auditory system The performance of the animals was assessed as the duration, level and spectral composition of the stimulus were varied, by measuring both the accuracy and latency of the initial head orienting response made following sound pre- sentation and the loudspeaker/reward spout subsequently approached. As expected, acute monaural occlusion in normally- reared control animals resulted in an immediate decline in locali- zation accuracy (Fig. 5C). However, ferrets raised with an earplug placed in one ear and tested with that ear still occluded were able to localize broadband sounds reasonably well at all locations tested, indicating that the developing auditory system had adapted to a Fig. 3. Binaural masking level difference (BMLD) in 19 patients before and after sur- gery to correct congenital unilateral hearing loss resulting from an abnormal external and/or middle ear on one side. The BMLD (N0S0 minus N0Sp) is the difference in detection threshold of a tone presented either in phase or with the phase reversed between the ears in the presence of broadband noise, which was always presented in phase at the two ears. Some subjects had post-operative MLDs in the normal range, whereas others showed a persistent deﬁcit in binaural processing. Modiﬁed with permission from Wilmington et al. (1994). D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 21 Fig. 4. Unaided sound-localization responses for one subject with a unilateral conductive hearing loss in the left ear. The stimulus was broadband noise (0.5e20 kHz). These data were obtained without using the subject's bone-anchored device (BCD off) (A), and in the BCD off condition with an additional muff over the impaired ear to further alter binaural cues (B). The gains of responses (obtained from the slopes of the regression lines ﬁtted to the data) to stimuli with levels of 55 dB SPL (solid gray regression lines) and 65 dB SPL (solid black regression lines) decreased signiﬁcantly when the impaired ear was covered with the muff, indicating that the subjects were relying on binaural cues for localization, whereas this was not the case at the lower level of 45 dB SPL (gray dashed regression lines), which was unlikely to be audible at the deprived ear. g ¼ response gain. Reproduced with permission from Agterberg et al. (2012). Fig. 4. Unaided sound-localization responses for one subject with a unilateral conductive hearing loss in the left ear. The stimulus was broadband noise (0.5e20 kHz). 5. Adaptation to unilateral hearing loss in the mature auditory system Reversible manipulation of acoustic localization cues has been widely used to probe the adaptive capabilities of the auditory system in adulthood. Plasticity during development is clearly important for calibrating neural circuits during the period when these cues are naturally changing in value as the head and ears grow (Schnupp et al., 2003). It also provides a potential means of adjusting to recurring periods of hearing loss that may be experi- enced during infancy (Hogan et al.,1997; Whitton and Polley, 2011). Although studies in barn owls (Knudsen et al., 1984; Knudsen, 1985) and rodents (Popescu and Polley, 2010; Polley et al., 2013) have shown that changes in binaural processing in response to asymmetric hearing loss are restricted to, or at least most pro- nounced during, a sensitive period of development, there is now overwhelming evidence in mammals that the adult brain can also This strategy of up-weighting spectral cues in a context- dependent fashion following a history of asymmetric hearing loss enables accurate sound localization to be maintained irrespective of whether the hearing loss is present or not in the other ear. In fact, the auditory system possesses an even greater capacity for ac- commodating abnormal spatial cues. If access to spectral localiza- tion cues is minimized by using narrowband noise bursts as stimuli, ferrets raised with one ear occluded still exhibit adaptive plasticity in both their behavioral and cortical responses, but this is now D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 22 p , J g / g ( ) 5. Adaptation to asymmetric hearing loss during infancy can be achieved by reweighting auditory spatial cues. (A) Schematic of setup used for measuring sound localization in horizontal plane by adult ferrets. Twelve loudspeakers were located at 30 intervals around the perimeter of the apparatus. 0 is straight ahead, with negative numbers ating locations to the animal's left. A trial was initiated by the animal licking a spout at the center of the chamber. This triggered the presentation of a burst of broadband noise a ﬂat spectrum from one of the loudspeakers; the animal received a water reward for making a correct approach-to-target response. (B) Average joint distributions of stimulus response location for ferrets raised wearing an earplug in the left ear (interspersed with brief periods of normal hearing); the size of the circles represents the proportion o for each stimulus-response combination. 5. Adaptation to unilateral hearing loss in the mature auditory system Thus, different strategies for recovering sound localization accuracy in the presence of asymmetric hearing loss are also present in adulthood, with individual subjects varying in the extent to which they adapted by cue reweighting or cue remapping (Keating et al., 2016). Several studies have shown that horizontal localization by adult humans can adapt to varying degrees to asymmetric hearing loss induced by occluding one ear, resulting in a partial recovery in their ability to localize sound (Bauer et al., 1966; Butler, 1987; Kumpik et al., 2010; Irving and Moore, 2011; Keating et al., 2016). An important question is whether this plasticity is driven solely by training on the localization task or whether other factors contribute. Although listeners with normal hearing can learn within a few hours to reinterpret the relationship between auditory localization cues and directions in space (e.g., Mendonça et al., 2013; Shinn-Cunningham et al., 1998; Zahorik et al., 2006), the spacing of the trials seems to be important for adaptation to hearing loss in one ear (Musicant and Butler, 1980; Kumpik et al., 2010). For example, Kumpik et al. (2010) observed steady im- provements in performance in subjects who wore an earplug all day (except during showering or sleep) if the sound localization training was distributed across several days, but not in a second group who completed a similar number of trials compressed into one day. This implies that a period of memory consolidation may be required for adaptation to asymmetric hearing loss. The behavioral plasticity observed in ferrets raised with a uni- lateral conductive hearing loss is mirrored by changes in the pro- cessing of sound localization cues in A1 (Keating et al., 2013, 2015). However, adaptive changes in the auditory spatial tuning of neu- rons in the superior colliculus have also been described in monaurally-deprived animals (King et al., 1988, 2000), so the site of plasticity remains unclear. The ability of adult ferrets to compen- sate with training to temporary loss of hearing in one ear requires a functioning auditory cortex (Nodal et al., 2012), but is also impaired if the layer V neurons in A1 that project to the IC are selectively eliminated (Bajo et al., 2010). with error bars showing bootstrapped 95% conﬁdence intervals. Acutely plugging one ear (‘Plug’) in the normally-raised control ferrets caused a substantial drop in localization accuracy. Signiﬁcantly higher scores were achieved by the juvenile-plugged ferrets, and these animals localized just as accurately as the control group when the earplug was removed (‘No plug’). (D) Effect of disrupting spectral cues by increasing the degree of spectral randomization in the stimuli on localization accuracy by juvenile-plugged animals with and without an earplug in place. (E) Recordings were made bilaterally in the primary auditory cortex (A1) of these animals. (F) Neurons in juvenile-plugged animals were more sensitive to the monaural spatial cues provided to the intact ear and less sensitive to the other available cues; this is indicated by the higher weighting index (mean ± 95% conﬁdence intervals) in juvenile-plugged animals than in the control group (whose mean values are indicated by the horizontal dashed lines). Increased weighting of spectral cues in juvenile- plugged animals was observed only when a virtual earplug was introduced to the previously occluded ear during the recordings. Adapted with permission from Keating et al. (2013). 5. Adaptation to unilateral hearing loss in the mature auditory system Thus, although the auditory cortex plays a critical role in spatial hearing and in the experience- dependent plasticity that allows the brain to compensate for asymmetric reversible hearing loss, its descending projections appear to play a speciﬁc role in retraining the auditory system. Experiments in monaurally-plugged adult ferrets have shown that the extent of the recovery in localization accuracy is deter- mined by the frequency of training (Kacelnik et al., 2006). These animals adapted more quickly and more extensively when pro- vided with daily training than when the training sessions were more spread out, even though the same overall number of trials were included. The ferret experiments also demonstrated that the improvements in localization accuracy were speciﬁc to auditory training, and that neither vision nor feedback about the accuracy of the response were required for some adaptation to take place (Kacelnik et al., 2006). It is likely, however, that other sensory, motor and cognitive factors may promote learning (Strelnikov et al., 2011; Carlile et al., 2014) when abnormal auditory cues are expe- rienced. For example, a greater improvement in auditory localiza- tion accuracy has been observed in human listeners wearing an earplug if performance feedback is provided, and especially if the auditory stimuli are accompanied by spatially-congruent visual cues (Strelnikov et al., 2011). 5. Adaptation to unilateral hearing loss in the mature auditory system These data were obtained with the earplug in place; note the similarity in the accuracy of the localization responses on the plugged non-plugged sides. (C) Percentage correct scores for control and juvenile-plugged groups, with individual animals denoted by symbols. Horizontal lines indicate mean values Fig. 5. Adaptation to asymmetric hearing loss during infancy can be achieved by reweighting auditory spatial cues. (A) Schematic of setup used for measuring sound localization in the horizontal plane by adult ferrets. Twelve loudspeakers were located at 30 intervals around the perimeter of the apparatus. 0 is straight ahead, with negative numbers indicating locations to the animal's left. A trial was initiated by the animal licking a spout at the center of the chamber. This triggered the presentation of a burst of broadband noise with a ﬂat spectrum from one of the loudspeakers; the animal received a water reward for making a correct approach-to-target response. (B) Average joint distributions of stimulus and response location for ferrets raised wearing an earplug in the left ear (interspersed with brief periods of normal hearing); the size of the circles represents the proportion of trials for each stimulus-response combination. These data were obtained with the earplug in place; note the similarity in the accuracy of the localization responses on the plugged and non-plugged sides. (C) Percentage correct scores for control and juvenile-plugged groups, with individual animals denoted by symbols. Horizontal lines indicate mean values, D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 23 learn to utilize abnormal spatial cues (reviewed by Mendonça 2014). is occluded. Perceptual learning studies carried out in listeners with normal hearing have shown that sensitivity to binaural spatial cues can improve with training (Wright and Fitzgerald, 2001; Kumpik et al., 2009; Sand and Nilsson, 2014) and, as mentioned in the previous section, binaural plasticity represents part of the basis for adaptation to asymmetric hearing loss during infancy. Although Kumpik et al. (2010) found no changes in ITD or ILD sensitivity over a week long period of monaural occlusion in adult humans, during which performance on a free-ﬁeld localization task gradually improved, exposing subjects to altered cues only during training sessions did result in remapping of both binaural cues onto appropriate locations (Keating et al., 2016) (Fig. 7C and D). 6. Perceptual training for hearing-impaired listeners Nevertheless, reweighting of different spatial cues is not the only means of learning to localize sounds accurately when one ear th id bl id f l i t di th t l h d f i d i d d tili daptation to asymmetric hearing loss during infancy by remapping the altered binaural cues onto new locations in space. (AeC) Joint distributions o expressed as degrees (deg) azimuth, for a control ferret with normal hearing (A) and a control (B) and juvenile-plugged (JP) ferret (C) wearing an earplug represents the number of trials (n) corresponding to each stimulus-response combination. (D) Mean unsigned error for control and earplugged ferrets, norm orrespond to perfect and chance performance, respectively. Error bars show bootstrapped 95% conﬁdence intervals. Controls wearing an earplug (n ¼ 6 ferre an normal hearing controls (n ¼ 4; P < 0.001, bootstrap test). While wearing an earplug, juvenile-plugged ferrets (n ¼ 2) made smaller errors than acutely pl , bootstrap test). (E) Mean binaural interaction (±s. e.m.) as a function of ILD across neurons recorded in A1 of control ferrets under normal hearing cond eparately for left (n ¼ 142 units, black) and right (n ¼ 177 units, gray) A1. Best ILDs for each hemisphere are indicated by arrows. (F) Binaural interaction funct uvenile-plugged ferrets under normal hearing conditions, which are shifted, relative to controls, in the appropriate direction to compensate for the hea uring development. Adapted with permission from Keating et al. (2015). D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 24 Fig. 6. Adaptation to asymmetric hearing loss during infancy by remapping the altered binaural cues onto new locations in space. (AeC) Joint distributions of stimulus and response, expressed as degrees (deg) azimuth, for a control ferret with normal hearing (A) and a control (B) and juvenile-plugged (JP) ferret (C) wearing an earplug in the left ear. Grayscale represents the number of trials (n) corresponding to each stimulus-response combination. (D) Mean unsigned error for control and earplugged ferrets, normalized so that 0 and 1 correspond to perfect and chance performance, respectively. Error bars show bootstrapped 95% conﬁdence intervals. Controls wearing an earplug (n ¼ 6 ferrets) made larger errors than normal hearing controls (n ¼ 4; P < 0.001, bootstrap test). 6. Perceptual training for hearing-impaired listeners Compared with the large body of work that has examined the effects of sound localization training in normal-hearing listeners with unperturbed or perturbed hearing, attempts to translate such training to clinical populations have only recently gathered pace. This is perhaps because of a prior emphasis on providing speech recognition training (Henshaw and Ferguson, 2013; Fu et al., 2015), and also possibly because the potential beneﬁts of functional plasticity in bilateral and bimodal artiﬁcial or ampliﬁed hearing for spatial masking release and sound localization have only recently started to be recognized. Recently, Firszt et al. (2015) provided a rich free-ﬁeld sound localization training regime with visuospatial feedback and showed that adults with severe to profound unilateral hearing loss can be trained to more accurately localize broadband sounds with complex spectral and temporal structure in the hori- zontal plane, and that this improvement generalized to the locali- zation of monosyllabic words. This ﬁnding is somewhat analogous to those of the monaural ear-plugging studies described above with normal-hearing listeners, although the relative contribution of changes in the weights given to head shadow versus spectral cues was not quantiﬁed in that study, and so the extent to which the improvements might generalize to real-world spatial scenes is unclear. As with monaural deprivation during development, when broadband sounds are used as stimuli, adaptation of auditory localization behavior to asymmetric hearing loss in adulthood is based on subjects learning to rely more than before on the un- changed spectral localization cues provided by the normal ear (Kacelnik et al., 2006; Kumpik et al., 2010; Keating et al., 2016) (Fig. 7A and B). These ﬁndings therefore support the growing body of evidence from studies in which spectral localization cues are altered by mechanically reshaping the external ear (Hofman et al., 1998; Van Wanrooij and Van Opstal, 2007; Carlile et al., 2014; Trapeau et al., 2016; Watson et al., 2017) or by presenting virtual acoustic space stimuli using non-individualized head-related transfer functions (Zahorik et al., 2006; Parseihian and Katz, 2012) for considerable plasticity in the way these cues are processed in the brain. 6. Perceptual training for hearing-impaired listeners While wearing an earplug, juvenile-plugged ferrets (n ¼ 2) made smaller errors than acutely plugged controls (P < 0.001, bootstrap test). (E) Mean binaural interaction (±s. e.m.) as a function of ILD across neurons recorded in A1 of control ferrets under normal hearing conditions. Data are plotted separately for left (n ¼ 142 units, black) and right (n ¼ 177 units, gray) A1. Best ILDs for each hemisphere are indicated by arrows. (F) Binaural interaction functions (mean ± s. e.m.) in juvenile-plugged ferrets under normal hearing conditions, which are shifted, relative to controls, in the appropriate direction to compensate for the hearing loss expe- rienced during development. Adapted with permission from Keating et al. (2015). Given the considerable evidence from earplugging studies that a key step in compensating for an imbalance in inputs between the two ears is to change the weighting of different spatial cues, with the auditory brain becoming more dependent on the unchanged spectral cues available at the non-affected ear, it is important to ask how clinically relevant this might be. It is clear that at least some people who are deaf in one ear do indeed utilize monaural spectral cues for localization in the horizontal plane (e.g. Newton, 1983; Slattery and Middlebrooks, 1994; Van Wanrooij and Van Opstal, 2004). Furthermore, the improvement in sound localization sometimes reported in blind individuals has been attributed to their greater sensitivity to spectral cues corresponding to lateral D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 25 human listeners can relearn to localize sound after introducing an asymmetric hearing loss by occluding one ear. (A) Sound localization performan aining session for one subject who wore an earplug in the right ear during the localization tests. Scores for each session (dots) were ﬁtted using linear lope values, which quantiﬁed the rate of adaptation. Relative to ﬂat-spectrum noise (blue), much less adaptation occurred with random-spectrum n efulness of spectral cues to sound location. (B) Adaptation rate is shown for ﬂat- and random-spectrum stimuli for different subjects (gray lines; n ¼ 1 rovements in localization performance with training. Mean adaptation rates across subjects (±bootstrapped 95% conﬁdence intervals) are shown in ulus types. Dotted black lines indicate adaptation rates observed in a previous study (Kumpik et al., 2010). (C) Mean error magnitude plotted as a fu ne subject when pure tones were used as the stimuli. 6. Perceptual training for hearing-impaired listeners Data are plotted separately for low- (1 kHz, dark blue) and high-frequency (8 kHz, light blue was associated with a reduction in error magnitude, producing negative values for the change (D) in error magnitude. (D) D error for low- and hig ach subject (gray lines; n ¼ 11). Mean values for D error across subjects (±bootstrapped 95% conﬁdence intervals) are shown in blue. Although ther fferences for the adaptation observed at the two tone frequencies, almost all values are <0, indicating that error magnitude declined over the trainin ws D error values that would have been observed if human listeners had adapted as well as ferrets reared with a unilateral earplug (Keating et al., 20 (2016). Fig. 7. Adult human listeners can relearn to localize sound after introducing an asymmetric hearing loss by occluding one ear. (A) Sound localization performance (% correct) as a function of training session for one subject who wore an earplug in the right ear during the localization tests. Scores for each session (dots) were ﬁtted using linear regression (lines) to calculate slope values, which quantiﬁed the rate of adaptation. Relative to ﬂat-spectrum noise (blue), much less adaptation occurred with random-spectrum noise (pink), which limits the usefulness of spectral cues to sound location. (B) Adaptation rate is shown for ﬂat- and random-spectrum stimuli for different subjects (gray lines; n ¼ 11). Positive values indicate improvements in localization performance with training. Mean adaptation rates across subjects (±bootstrapped 95% conﬁdence intervals) are shown in blue and pink for the two stimulus types. Dotted black lines indicate adaptation rates observed in a previous study (Kumpik et al., 2010). (C) Mean error magnitude plotted as a function of training session for one subject when pure tones were used as the stimuli. Data are plotted separately for low- (1 kHz, dark blue) and high-frequency (8 kHz, light blue) tones. Improved performance was associated with a reduction in error magnitude, producing negative values for the change (D) in error magnitude. (D) D error for low- and high-frequency tones plotted for each subject (gray lines; n ¼ 11). Mean values for D error across subjects (±bootstrapped 95% conﬁdence intervals) are shown in blue. Although there are pronounced individual differences for the adaptation observed at the two tone frequencies, almost all values are <0, indicating that error magnitude declined over the training sessions. 6. Perceptual training for hearing-impaired listeners Dotted red line shows D error values that would have been observed if human listeners had adapted as well as ferrets reared with a unilateral earplug (Keating et al., 2015). Adapted from Keating et al. (2016). sound locations (Doucet et al., 2005; Voss et al., 2011), providing further evidence for compensatory plasticity in the use of spectral localization cues. can help hearing-impaired subjects to understand target speech in the presence of spatially-separated masking speech (Carr Levy et al., 2015), spectral cues are seriously distorted by the use of microphones that do not sit inside the auditory canal, such as in behind-the-ear hearing aids (Moore and Popelka, 2016). There is some indication that inclusion of algorithms that preserve pinna cues can improve horizontal localization and speech perception in noise in hearing aid users (Kuk et al., 2013; Xu and Han, 2014; Korhonen et al., 2015; Gomez and Seeber, 2017). However, this However, whether listeners provided with hearing devices can beneﬁt in the affected ear in a similar fashion is more questionable. However, whether listeners provided with hearing devices can beneﬁt in the affected ear in a similar fashion is more questionable. For one thing, the progressive loss of high-frequency hearing in age-related sensorineural hearing loss will restrict the availability of spectral cues. Although modern hearing aids can have band- widths of up to 10 kHz or more (Kuk and Baekgaard, 2009), which For one thing, the progressive loss of high-frequency hearing in age-related sensorineural hearing loss will restrict the availability of spectral cues. Although modern hearing aids can have band- widths of up to 10 kHz or more (Kuk and Baekgaard, 2009), which D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 26 will depend on whether the hearing aids provide sufﬁcient ampliﬁcation for individual listeners at the high frequencies where most of the directional information is available in these cues. with acquired unilateral conductive hearing loss: improved directional hearing with a bone-conduction device. Hear. Res. 286, 9e18. with acquired unilateral conductive hearing loss: improved directional hearing with a bone-conduction device. Hear. Res. 286, 9e18. Agterberg, M.J., Hol, M.K., Van Wanrooij, M.M., Van Opstal, A.J., Snik, A.F., 2014. Single-sided deafness and directional hearing: contribution of spectral cues and high-frequency hearing loss in the hearing ear. Front. Neurosci. 8, 188. 6. Perceptual training for hearing-impaired listeners The ﬁnding that listeners with one ear plugged can be trained to partially recover their sound localization accuracy by learning to remap the distorted ILDs and ITDs onto appropriate spatial loca- tions (Keating et al., 2016) potentially offers much greater scope for utilizing adaptive plasticity to promote improvements in spatial hearing in the hearing impaired. Indeed, the results of this study raise the possibility of adopting targeted training strategies based on the residual hearing abilities of individual patients and therefore the localization cues they have available. This is also relevant to patients with cochlear implants whose limited spatial hearing abilities can be improved if they adapt their ILD sensitivity to the range of values provided by the output of the implants (Dorman et al., 2014; Dorman et al., 2015) and by enhancing the availabil- ity of ILDs at low frequencies (Brown, 2014). Recent studies have started to examine the effects of training on cochlear implant users who have either been implanted bilaterally or retain access to binaural information as a result of having one good ear. There is some indication that sound localization training can promote binaural hearing, both with unilateral implantation when the other ear is preserved (Nawaz et al., 2014) and with bilateral cochlear implants (Tyler et al., 2010). Furthermore, a training paradigm in which auditory and visual stimuli were randomly interleaved has been shown to improve the auditory localization accuracy and cortical coding of ILDs in adult ferrets ﬁtted with bilateral cochlear implants following deafening in infancy (Isaiah et al., 2014). Algazi, V.R., Duda, R.O., Thompson, D.M., Avendano, C., 2001. The CIPIC HRTF database. In: Proceedings of the IEEE Workshop on Applications of Signal Processing to Audio and Electroacoustics. Mohonk Mountain House, New Paltz, NY, pp. 99e102. pp Arbogast, T.L., Mason Jr., C.R., G.K, 2002. The effect of spatial separation on infor- mational and energetic masking of speech. J. Acoust. Soc. Am. 112, 2086e2098. B j V M N d l F R M D R Ki A J 2010 Th d di i lli l Bajo, V.M., Nodal, F.R., Moore, D.R., King, A.J., 2010. The descending corticocollicular pathway mediates learning-induced auditory plasticity. Nat. Neurosci. 13, 253e260. Bauer, R.W., Matuzsa, J.L., Blackmer, F., Glucksberg, S., 1966. Noise localization after unilateral attenuation. J. Acoust. Soc. Am. 40, 441e444. Beggs, W.D., Foreman, D.L., 1980. Sound localization and early binaural experience in the deaf. Br. J. Audiol. 14, 41e48. 7. Conclusions Carlile, S., Martin, R., McAnally, K., 2005. Spectral information in sound localization. Int. Rev. Neurobiol. 70, 399e434. The studies discussed in this review have demonstrated that the auditory system can adjust to changes in the available sound localization cues in ways that can help to preserve spatial hearing abilities. This can be achieved either by reweighting different cues, as demonstrated by the evidence for greater reliance on spectral cues when ITDs and ILDs are compromised by unilateral hearing loss, or by learning a new relationship between altered binaural cues and sound source location. Utilizing the remarkable plasticity of auditory localization mechanisms in the treatment of clinical populations will require the development of training protocols that are practical to use outside the laboratory and which confer maximum generalization to regions of space and stimulus types other than those used for training. This includes identifying the type of feedback most likely to promote learning, with recent studies suggesting that sensorimotor feedback can improve the rate and extent of adaptation to altered spatial cues (Carlile et al., 2014; Keating et al., 2016). Most importantly, if plasticity in the neural processing of auditory spatial cues is to have therapeutic value, it will be necessary to show that it extends to more realistic and challenging listening situations than those typically used in the laboratory, and that the beneﬁts include not only a recovery in sound localization accuracy, but also improved speech-in-noise perception. Carr Levy, S., Freed, D.J., Nilsson, M., Moore, B.C.J., Puria, S., 2015. Extended high- frequency bandwidth improves speech reception in the presence of spatially separated masking speech. Ear Hear. 36, e214ee224. p g p Cherry, E.C., 1953. Some experiments on the recognition of speech, with one and with two ears. J. Acoust. Soc. Am. 25, 975e979. 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Relevance of spectral cues for auditory spatial processing in the occipital cortex of the blind. Front. Psychol. 2, 48. basic and clinical studies. J. Assoc. Res. Otolaryngol. Xu, J., Han, W., 2014. Improvement of adult BTE hearing aid wearers' front/back localization performance using digital pinna-cue preserving technologies: an evidence-based review. Kor.J. Audiol. 18, 97e104. Whitton, J.P., Polley, D.B., 2011. Evaluating the perceptual and pathophysiological consequences of auditory deprivation in early postnatal life: a comparison of y y g , Wilmington, D., Gray, L., Jahrsdoerfer, R., 1994. Binaural processing after corrected congenital unilateral conductive hearing loss. Hear. Res. 74, 99e114. Webster, D.B., Webster, M., 1979. Effects of neonatal conductive hearing loss on brain stem auditory nuclei. Ann. Otol. Rhinol. Laryngol. 88, 684e688. D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 12, 535e547. W i h B A Fi ld M B 2001 Diff f h d Wright, B.A., Fitzgerald, M.B., 2001. Different patterns of human discrimination learning for two interaural cues to sound-source location. Proc. Natl. Acad. Sci. U.S.A. 98, 12307e12312. Watson, C.J.G., Carlile, S., Kelly, H., Balachandar, K., 2017. The generalization of auditory accommodation to altered spectral cues. Sci. Rep. 7, 11588. Xu, J., Han, W., 2014. Improvement of adult BTE hearing aid wearers' front/back localization performance using digital pinna-cue preserving technologies: an evidence-based review. Kor.J. Audiol. 18, 97e104. Webster, D.B., Webster, M., 1979. Effects of neonatal conductive hearing loss on b i di l i A O l Rhi l L l 88 684 688 Webster, D.B., Webster, M., 1979. Effects of neonatal conductive hearing loss on brain stem auditory nuclei. Ann. Otol. Rhinol. Laryngol. 88, 684e688. brain stem auditory nuclei. Ann. Otol. Rhinol. Laryngol. 88, 684e688. Wilmington, D., Gray, L., Jahrsdoerfer, R., 1994. Binaural processing after corrected congenital unilateral conductive hearing loss. Hear. Res. 74 99e114. y y g Wilmington, D., Gray, L., Jahrsdoerfer, R., 1994. Binaural processing after corrected congenital unilateral conductive hearing loss. Hear. Res. 74, 99e114. Zahorik, P., Bangayan, P., Sundareswaran, V., Wang, K., Tam, C., 2006. Perceptual recalibration in human sound localization: learning to remediate front-back reversals. J. Acoust. Soc. Am. 120, 343e359. Whitton, J.P., Polley, D.B., 2011. Evaluating the perceptual and pathophysiological consequences of auditory deprivation in early postnatal life: a comparison of
https://openalex.org/W4236866509	https://indexlaw.org/index.php/conpedireview/article/download/3601/3106	Portuguese	null	Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica	Conpedi Law Review	2,016	cc-by	11,393	RESUMO A interceptação das comunicações telefônicas é meio de obtenção de prova que vem sendo aplicada em casos de grande repercussão pública e gerando muitas polêmicas, como a realização de interpretações das conversas por parte dos agentes policiais, que terminam por conduzir o convencimento judicial com a atribuição de significados ocultos aos termos usados pelos interlocutores. O objeto deste trabalho é perquirir a legitimidade para valorar o conteúdo das conversas monitoradas e a validade das decisões proferidas com base nas interpretações prejudiciais diante do princípio da imparcialidade e do modelo epistêmico de conhecimento que é o processo. Palavras-chave: Interceptação telefônica, Imparcialidade, Modelo epistêmico, Verdade, Valoração da prova OS "HERMENEUTAS DOS GRAMPOS": UMA DISFUNCIONALIDADE EPISTÊMICA 1Antonio Eduardo Ramires Santoro 2Francisco Ramalho Ortigão Farias e-ISSN: 2448-3931 DOI: 10.21902/clr.v2i1.271 Received on January 07, 2016 Approved on April 27, 2016 Responsible Editor: Raymundo Juliano Feitosa Associate Editor: Fernando Galindo Ayuda Evaluation Process: Double Blind Review pelo SEER/OJS e-ISSN: 2448-3931 DOI: 10.21902/clr.v2i1.271 Received on January 07, 2016 Approved on April 27, 2016 Responsible Editor: Raymundo Juliano Feitosa Associate Editor: Fernando Galindo Ayuda Evaluation Process: Double Blind Review pelo SEER/OJS 1 Pelo que se tem notícia, há no Brasil basicamente três sistemas de TI utilizados para recepção e armazenamento Dados colhidos do Processo no 0.00.000.001328/2012-95, que tramitou junto ao Conselho Nacional do Ministério Público e se tratava de um Pedido de Providência formulado pelo Conselho Federal da Ordem dos Advogados do Brasil, consistente no requerimento de auditoria e inspeção nos sistemas de escuta e monitoramento de interceptações telefônicas utilizados pelas unidades do ministério público brasileiro1, mostram que, a partir das consultas feitas às 30 unidades do ministério público brasileiro, 8 (oito) adquiriram o Sistema Guardião (o ministério público federal e o ministério público dos estados de Goiás, Mato Grosso, Rio Grande do Norte, Rio Grande do Sul, São Paulo, Santa Catarina e o Distrito Federal); 6 (seis) adquiriram o Sistema Wytron (o ministério público dos estados de Alagoas, Amapá, Ceará, Maranhão, Pará e Rondônia); 3 (três) adquiriram o Sistema Sombra (o ministério público dos estados da Bahia, Mato Grosso do Sul e Paraíba); 4 (quatro) utilizam o Sistema Guardião disponibilizado ou cedido por órgãos do Poder Executivo (o ministério público dos estados do Espírito Santo, Minas Gerais, Amazonas e Tocantins); 9 (nove) não possuem ou não têm acesso a qualquer um desses sistemas (o ministério público militar, o ministério público do trabalho e o ministério público dos estados de Sergipe, Pernambuco, Acre, Paraná, Piauí, Roraima e Rio de Janeiro). RESUMEN La intervención de las comunicaciones telefónicas es una medida de investigación que se ha aplicado en casos de gran atención pública y con muchas controversias, como, por ejemplo, las interpretaciones policiales de las conversaciones, que en última instancia conduce la convicción judicial asignando significados ocultos a los términos utilizados por las personas investigadas. El objeto de este trabajo es investigar la legitimidad para hacer la valoración del contenido de las conversaciones y la validez de los juicios basados en las interpretaciones policiales preliminares bajo el principio de imparcialidad y el modelo epistémico del conocimiento que es el proceso. Palabras-claves: Intervención telefónica, Imparcialidad, Modelo epistémico, Verdad, Valoración de la prueba Palabras-claves: Intervención telefónica, Imparcialidad, Modelo epistémico, Verdad, Valoración de la prueba Palabras-claves: Intervención telefónica, Imparcialidad, Modelo epistémico, Verdad, Valoración de la prueba 1 Professor Adjunto de Direito Processual Penal da Universidade Federal do Rio de Janeiro - UFRJ, Rio de Janeiro (Brasil). Pós-doutor pela Universidad Nacional de La Matanza – UNLaM, Buenos Aires (Argentina). Doutor e Mestre pela Universidade Federal do Rio de Janeiro - UFRJ, Rio de Janeiro (Brasil). E-mail: antoniosantoro@direito.ufrj.br 2 Professor de Prática Penal e Coordenador do Núcleo de Prática Jurídica da Universidade Federal do Rio de Janeiro- FND/UFRJ, Rio de Janeiro (Brasil) ; Doutorando pela Universidade Federal Fluminense - UFF, Rio de Janeiro (Brasil). E-mail: franciscoortigao@uol.com.br CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 163 Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica 1 INTRODUÇÃO 1 INTRODUÇÃO Em 1996 entrou em vigor a Lei no 9.296 que regulou a parte final do artigo 5o, inciso XII da Constituição, dispondo regras sobre a interceptação das comunicações telefônicas e telemáticas, medida que se tornou, 10 (anos) depois e sobretudo com o advento dos avanços tecnológicos comunicacionais, em uma das maiores panaceias para o suposto conhecimento da verdade no processo penal. Essas medidas têm por característica alijar a defesa da participação dialética na produção da prova, vez que são determinadas pelo magistrado, de ofício ou a requerimento da autoridade policial ou do ministério público, são executadas pelos investigadores com participação direta e efetiva das operadoras de telefonia com a utilização de sistemas de tecnologia da informação especialmente desenvolvidos para realizar a escuta e o armazenamento de áudios1, implicam em um monitoramento comumente por longo tempo (meses ou até anos) em que o cidadão é vigiado à sorrelfa, sem qualquer oportunidade de exercer o comezinho (porém fundamental) direito de não se autoincriminar, com a submissão de todos os dados ao conhecimento do juiz que deferiu e prorrogou a medida, lamentavelmente o mesmo juiz que irá julgar a ação penal condenatória eventualmente proposta em face do investigado, que não tem oportunidade de exercer o contraditório direto. Assim, em que pese ter essa medida nascido da relativização constitucionalmente admitida do direito fundamental ao sigilo das comunicações, da intimidade e da privacidade, g p Portanto, das 30 (trinta) unidades do ministério público, 21 (vinte e uma) adquiriram ou utilizam sistemas de TI que se destinam a receber e armazenar dados obtidos de interceptações telefônicas ou de dados. Destas 21 (vinte e uma) unidades que operam sistemas de monitoramento de comunicações, 12 (doze) “não dispõem de ato normativo versando sobre procedimentos e rotinas adotadas”1 e 18 (dezoito) recorrem a policiais civis e/ou militares na operação. Quanto à aquisição desses sistemas pelos Departamentos de Polícia Federal dos Estados não há dados tão precisos quanto esses constantes do processo que tramitou no Conselho Nacional do Ministério Público, mas dados do Portal da Transparência do governo federal demonstram que as empresas Dígitro Tecnologia Ltda., Federal Tecnologia de Software Ltda.-EPP e Wytron Technology Corp. Ltda. comercializaram com o Departamento de Polícia Federal, sendo, ademais, amplamente divulgada a contratação do Sistema Guardião pelas Superintendências da Polícia Federal de Santa Catarina, Paraná, São Paulo e Rio de Janeiro. CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 Pelo que se tem notícia, há no Brasil basicamente três sistemas de TI utilizados para recepção e armazenamento Dados colhidos do Processo no 0.00.000.001328/2012-95, que tramitou junto ao Conselho Nacional do Ministério Público e se tratava de um Pedido de Providência formulado pelo Conselho Federal da Ordem dos Advogados do Brasil, consistente no requerimento de auditoria e inspeção nos sistemas de escuta e monitoramento de interceptações telefônicas utilizados pelas unidades do ministério público brasileiro1, mostram que, a partir das consultas feitas às 30 unidades do ministério público brasileiro, 8 (oito) adquiriram o Sistema Guardião (o ministério público federal e o ministério público dos estados de Goiás, Mato Grosso, Rio Grande do Norte, Rio Grande do Sul, São Paulo, Santa Catarina e o Distrito Federal); 6 (seis) adquiriram o Sistema Wytron (o ministério público dos estados de Alagoas, Amapá, Ceará, Maranhão, Pará e Rondônia); 3 (três) adquiriram o Sistema Sombra (o ministério público dos estados da Bahia, Mato Grosso do Sul e Paraíba); 4 (quatro) utilizam o Sistema Guardião disponibilizado ou cedido por órgãos do Poder Executivo (o ministério público dos estados do Espírito Santo, Minas Gerais, Amazonas e Tocantins); 9 (nove) não possuem ou não têm acesso a qualquer um desses sistemas (o ministério público militar, o ministério público do trabalho e o ministério público dos estados de Sergipe, Pernambuco, Acre, Paraná, Piauí, Roraima e Rio de Janeiro). Portanto, das 30 (trinta) unidades do ministério público, 21 (vinte e uma) adquiriram ou utilizam sistemas de TI que se destinam a receber e armazenar dados obtidos de interceptações telefônicas ou de dados. Destas 21 (vinte e uma) unidades que operam sistemas de monitoramento de comunicações, 12 (doze) “não dispõem de ato normativo versando sobre procedimentos e rotinas adotadas”1 e 18 (dezoito) recorrem a policiais civis e/ou militares na operação. Quanto à aquisição desses sistemas pelos Departamentos de Polícia Federal dos Estados não há dados tão 2 PRADO, Geraldo. Prova penal e sistema de controles epistêmicos: a quebra da cadeia de custódia das provas obtidas por métodos ocultos. São Paulo: Marcial Pons, 2014, p. 78. 3 MAYA, André Machado. Imparcialidade e processo penal: da prevenção da competência ao juiz das garantias. Rio de Janeiro: Lumen Juris, 2011, trata da questão da prevenção como critério positivo de competência, em clara oposição ao direito à imparcialidade, realizando uma ampla leitura das decisões do Tribunal Europeu de Direitos Humanos; GUERRERO PALOMARES, Salvador. La Imparcialidad Objetiva del Juez Penal: Análisis jurisprudencial y valoración crítica. Pamplona: Aranzadi, 2009, faz uma ampla análise das decisões dos Tribunais Espanhóis e do Tribunal Europeu de Direitos Humanos sobre a violação da imparcialidade objetiva, verificável conforme o caso concreto conforme o conteúdo das decisões proferidas antes da propositura da ação penal. Ambos os trabalhos são extremamente valiosos para o contexto deste trabalho. 4 O legislador ordinário foi gramaticalmente infeliz na redação do artigo 5o da Lei 9.296/96. Redigiu o dispositivo sem uma vírgula esclarecedora. Veja-se a redação da Lei: 2 PRADO, Geraldo. Prova penal e sistema de controles epistêmicos: a quebra da cadeia de custódia das provas obtidas por métodos ocultos São Paulo: Marcial Pons 2014 p 78 1 INTRODUÇÃO 1 \| p. 163-180 \| JAN/JUN. 2016 164 164 Antonio Eduardo Ramires Santoro e Francisco Ramalho Ortigão Farias não se deve ignorar a existência de conflitos com os direitos de estatura constitucional ao contraditório e à ampla defesa, já que se trata do que Geraldo Prado chamou de método oculto de investigação2, inviabilizando a participação de quem está sendo monitorado, bem como do direito ao silêncio e, mais amplamente, o direito de não produzir provas contra si mesmo, posto não ser dada opção ao investigado de falar ou calar-se enquanto conversa (e eventualmente confessa) sobre o ato que possa ter praticado preteritamente. Essas incompatibilidades são geralmente ignoradas nos casos concretos pela jurisprudência brasileira. Há que se ter em vista que a prática da interceptação telefônica nesses quase 20 (vinte) anos de vigência da Lei no 9.296/96 desvelou usos conflituosos com os direitos fundamentais, que vão além das vulnerações ínsitas à própria natureza da medida anteriormente citadas. Isso pode se dar por uma regulação legal equivocada, omissa ou dúbia. É uma regulação legal equivocada, por exemplo, a regra de determinação da competência pela prevenção inserta no artigo 1o da Lei no 9.296/96, em que o magistrado que defere e acompanha a execução da medida e, portanto, toma conhecimento do conteúdo das conversas interceptadas sem qualquer participação da defesa, vai julgar a ação penal proposta, violando o princípio da imparcialidade da jurisdição3. Há clara omissão proposital na Lei no 9.296/96, precisamente no seu artigo 5o, ao deixar para o juiz definir “a forma de execução da diligência” em sua decisão que a defere, em vez de dispor expressamente sobre o assunto. O mesmo artigo 5o contém uma dubiedade que até hoje cobra o seu preço. Trata-se da péssima definição do prazo máximo de duração da medida que, na falta de uma vírgula esclarecedora4, deixou para a jurisprudência estabelecer o tempo durante o qual o cidadão pode ficar sendo vigiado pelas agências de persecução penal. CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. “Art. 5o A decisão será fundamentada, sob pena de nulidade, indicando também a forma de execução da diligência que não poderá exceder o prazo de 15 (quinze) dias, renovável por igual tempo uma vez comprovada a indispensabilidade do meio de prova.” Observe que uma vírgula colocada antes da expressão “uma vez”, tornaria essa expressão uma locução conjuntiva subordinativa condicional, com o mesmo sentido de “se” ou “caso” e não limitaria expressamente a prorrogação a apenas uma única vez. Veja-se que o trecho em comento teria a seguinte redação “...não poderá exceder o prazo de 15 (quinze) dias, renovável por igual tempo, uma vez comprovada a indispensabilidade do meio de prova.” “Art. 5o A decisão será fundamentada, sob pena de nulidade, indicando também a forma de execução da diligência que não poderá exceder o prazo de 15 (quinze) dias, renovável por igual tempo uma vez comprovada a indispensabilidade do meio de prova.” “Art. 5o A decisão será fundamentada, sob pena de nulidade, indicando também a forma de execução da diligência que não poderá exceder o prazo de 15 (quinze) dias, renovável por igual tempo uma vez comprovada a indispensabilidade do meio de prova.” Observe que uma vírgula colocada antes da expressão “uma vez”, tornaria essa expressão uma locução conjuntiva subordinativa condicional com o mesmo sentido de “se” ou “caso” e não limitaria expressamente a “Art. 5o A decisão será fundamentada, sob pena de nulidade, indicando também a forma de execução da diligência que não poderá exceder o prazo de 15 (quinze) dias, renovável por igual tempo uma vez comprovada a indispensabilidade do meio de prova.” Observe que uma vírgula colocada antes da expressão “uma vez”, tornaria essa expressão uma locução conjuntiva subordinativa condicional, com o mesmo sentido de “se” ou “caso” e não limitaria expressamente a prorrogação a apenas uma única vez. Veja-se que o trecho em comento teria a seguinte redação “...não poderá exceder o prazo de 15 (quinze) dias, renovável por igual tempo, uma vez comprovada a indispensabilidade do meio de prova.” De outro lado, se existisse uma vírgula depois da expressão “uma vez” estaríamos diante de um adjunto adverbial de intensidade que modifica o adjetivo “renovável” e, portanto, indicaria que só é possível renovar a diligência uma única vez, como se pode ver pela redação modificada do trecho “...não poderá exceder o prazo de 15 (quinze) dias, renovável por igual tempo uma vez, comprovada a indispensabilidade do meio de prova.” Todavia essa vírgula não existe Cezar Peluso. Por maioria e algum desconforto com alguns votos5, foi decidido que as prorrogações eram válidas, mas o Ministro Gilmar Mendes afirmou expressamente: Cezar Peluso. Por maioria e algum desconforto com alguns votos5, foi decidido que as prorrogações eram válidas, mas o Ministro Gilmar Mendes afirmou expressamente: Sem me comprometer – tenho impressão de que vamos nos deparar com outros casos e já me abalanço a pensar em proporcionalidade para, eventualmente, fixar limites temporais em relação a isso –, é preciso deixar bem claro que até mesmo as prorrogações precisam ser especificamente fundamentadas.6 Naturalmente a oportunidade para rever sua posição está no citado Recurso Extraordinário 625.263, cuja Repercussão Geral foi admitida pelo Plenário Virtual do Supremo Tribunal Federal em 13 de junho de 2013. No entanto, é importante ressaltar que antes do julgamento do recebimento da denúncia no Inq 2424 (“Operação Furacão”), o Ministro Gilmar não expunha em suas decisões qualquer dúvida em acolher a tese da possibilidade das prorrogações sucessivas das medidas de interceptação telefônica, cabendo perquirir o que o teria feito admitir poder mudar de opinião. Ao assumir a Presidência do Supremo Tribunal Federal em abril de 2008, o Ministro Gilmar Mendes concedeu uma entrevista à Revista Veja (edição de 19 de abril de 2008) em que, ao ser perguntado sobre a apuração da “CPI dos Grampos” de que existiriam naquele momento mais de 500.000 (quinhentas mil) “escutas telefônicas” autorizadas pela Justiça no país, respondeu o seguinte: Os juízes devem ter mais cuidado em relação a isso. A lei prevê que o prazo para uma interceptação telefônica é de quinze dias. Mas o entendimento dos juízes é que esses quinze dias podem ser renovados de maneira ilimitada. O resultado é que hoje 5 O Ministro Marco Aurélio votou pela invalidade das prorrogações reiteradas, como já havia feito nas outras oportunidades. A Ministra Carmen Lúcia acompanhou o voto condutor, mas ressalvou que não entendia ser possível a prorrogação das autorizações, e sim uma “nova ordem de interceptação” com base em fatos novos. O Ministro Lewandowski entendeu ser possível a renovação em caso de “crimes de natureza permanente (...) ou crimes complexos”. O Ministro Eros Grau afirmou que “se (...) pensar nas garantias que esta Corte deve tornar positivas em relação a cada indivíduo (...) acompanho a exposição do Ministro Marco Aurélio”, mas “neste caso (...) não tenho dúvidas em acompanhar o relator”. O Ministro Carlos Britto votou na mesma linha da Ministra Carmen Lúcia. A Ministra Ellen Gracie votou com o relator. 1 INTRODUÇÃO 2016 165 Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica Diante da subversão prática da interceptação telefônica de meio de obtenção de prova em estado de vigilância (sendo mesmo chamado em casos concretos de monitoramento ou acompanhamento telefônico e lacanianamente revelando a natureza do seu uso), a jurisprudência dos tribunais superiores foi obrigada a se pronunciar sobre medidas que duraram meses ou anos. O Supremo Tribunal Federal definiu, no julgamento do Habeas Corpus 83.515, de relatoria do Ministro Nelson Jobim, no dia 16 de setembro de 2004, por maioria, que eram válidas as diligências de interceptação telefônica que, com as renovações, duraram 7 (sete) meses. Ocorre que em 09 de setembro de 2008, a Sexta Turma do Superior Tribunal de Justiça, sob a relatoria do Ministro Nilson Naves, adotou posição diversa ao julgar o Habeas Corpus 76.686 e definir que não era possível renovar ilimitadamente as autorizações de interceptação das comunicações. Dessa decisão foi interposto o Recurso Extraordinário 625.263, o qual foi distribuído para relatoria do Ministro Gilmar Mendes. A importância da distribuição deste recurso para o Ministro Gilmar Mendes é mais profunda do que se poderia, a princípio, imaginar. Isso porque no dia 20 de novembro de 2008 (onze dias depois do julgamento proferido pela Sexta Turma do Superior Tribunal de Justiça em sentido oposto ao entendimento dominante no Supremo Tribunal Federal), a sessão Plenária do Supremo decidiu sobre o recebimento da denúncia no Inq 2424, conhecida como “Operação Furacão”, que terminou por levar preventivamente à prisão empresários, bicheiros, desembargadores federais e até um Ministro do Superior Tribunal de Justiça. Um dos espinhosos temas que foram tratados nesse julgamento era sobre a legalidade das diversas prorrogações da medida de interceptação telefônica proferidas pelo Ministro De outro lado, se existisse uma vírgula depois da expressão “uma vez” estaríamos diante de um adjunto adverbial de intensidade que modifica o adjetivo “renovável” e, portanto, indicaria que só é possível renovar a diligência uma única vez, como se pode ver pela redação modificada do trecho “...não poderá exceder o prazo de 15 (quinze) dias, renovável por igual tempo uma vez, comprovada a indispensabilidade do meio de prova.” Todavia, essa vírgula não existe. CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 \| \| 166 166 Antonio Eduardo Ramires Santoro e Francisco Ramalho Ortigão Farias Cezar Peluso. 1 INTRODUÇÃO Por maioria e algum desconforto com alguns votos5, foi decidido que as prorrogações eram válidas, mas o Ministro Gilmar Mendes afirmou expressamente: 5 O Ministro Marco Aurélio votou pela invalidade das prorrogações reiteradas, como já havia feito nas outras oportunidades. A Ministra Carmen Lúcia acompanhou o voto condutor, mas ressalvou que não entendia ser possível a prorrogação das autorizações, e sim uma “nova ordem de interceptação” com base em fatos novos. O Ministro Lewandowski entendeu ser possível a renovação em caso de “crimes de natureza permanente (...) ou crimes complexos”. O Ministro Eros Grau afirmou que “se (...) pensar nas garantias que esta Corte deve tornar positivas em relação a cada indivíduo (...) acompanho a exposição do Ministro Marco Aurélio”, mas “neste caso (...) não tenho dúvidas em acompanhar o relator”. O Ministro Carlos Britto votou na mesma linha da Ministra Carmen Lúcia. A Ministra Ellen Gracie votou com o relator. O Ministro Celso de Mello votou com o relator, ressalvando sua preocupação na mesma forma do entendimento do Ministro Marco Aurélio, que ironizou o voto proferido: “O SENHOR MINISTRO CELSO DE MELLO: Peço vênia, Senhor Presidente, na linha de precedente firmado pelo Plenário do Supremo Tribunal Federal (HC 83.515/RS, Rel. Min. NELSON JOBIM), notadamente sobre a questão pertinente à prorrogação do prazo de autorização para interceptação telefônica (Lei n o 9.296/96, art. 5o), para acompanhar o eminente Relator, não obstante partilhe – como o fazem os demais Juízes desta Suprema Corte – das preocupações reveladas pelo Ministro MARCO AURÉLIO. O SENHOR MINISTRO MARCO AURÉLIO – Mas, para este caso, as preocupações não valem.” (grifos no original) 6 Inteiro teor do Acórdão. Inq 2424/RJ. STF. Pleno. Disponível em <http://redir.stf.jus.br/paginadorpub/paginador.jsp?docTP=AC&docID=609608> . Acesso em 13 de marco de 2015. “O SENHOR MINISTRO CELSO DE MELLO: Peço vênia, Senhor Presidente, na linha de precedente firmado pelo Plenário do Supremo Tribunal Federal (HC 83.515/RS, Rel. Min. NELSON JOBIM), notadamente sobre a questão pertinente à prorrogação do prazo de autorização para interceptação telefônica (Lei n o 9.296/96, art. 5o), para acompanhar o eminente Relator, não obstante partilhe – como o fazem os demais Juízes desta Suprema Corte – das preocupações reveladas pelo Ministro MARCO AURÉLIO. O S O S O A CO A É O l ” ( if 6 Inteiro teor do Acórdão. Inq 2424/RJ. STF. Pleno. Disponível em <http://redir.stf.jus.br/paginadorpub/paginador.jsp?docTP=AC&docID=609608> . Acesso em 13 de marco de 2015. Cezar Peluso. Por maioria e algum desconforto com alguns votos5, foi decidido que as prorrogações eram válidas, mas o Ministro Gilmar Mendes afirmou expressamente: O Ministro Celso de Mello votou com o relator, ressalvando sua preocupação na mesma forma do entendimento do Ministro Marco Aurélio, que ironizou o voto proferido: “O SENHOR MINISTRO CELSO DE MELLO: Peço vênia, Senhor Presidente, na linha de precedente firmado pelo Plenário do Supremo Tribunal Federal (HC 83.515/RS, Rel. Min. NELSON JOBIM), notadamente sobre a questão pertinente à prorrogação do prazo de autorização para interceptação telefônica (Lei n o 9.296/96, art. 5o), para acompanhar o eminente Relator, não obstante partilhe – como o fazem os demais Juízes desta Suprema Corte – das preocupações reveladas pelo Ministro MARCO AURÉLIO. É 6 Inteiro teor do Acórdão. Inq 2424/RJ. STF. Pleno. Disponível em <http://redir.stf.jus.br/paginadorpub/paginador.jsp?docTP=AC&docID=609608> . Acesso em 13 de marco de 2015. CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 167 Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica existem escutas instaladas há dois ou três anos em um mesmo telefone. Esses procedimentos precisam ser revistos. Outra questão delicada é a divulgação desse conteúdo por agentes policiais antes mesmo de o juiz ser informado sobre ele. Não temos hoje mecanismos para coibir isso. É notória a participação dos agentes policiais na divulgação, às vezes até em consórcio com órgãos de imprensa. Acostumamo-nos a isso de maneira equivocada. O Judiciário, que autoriza as escutas, tem responsabilidade por isso.7 De se observar que o Ministro Gilmar Mendes já externava incômodo não apenas com a questão do prazo de duração das medidas, mas também com a divulgação dos conteúdos das conversas interceptadas feita pelos agentes policiais junto aos órgão de imprensa antes mesmo de chegar ao conhecimento do juiz, muito embora não tenha proferido decisão no sentido de limitar temporalmente o prazo de duração ou a quantidade admitia de prorrogações. 7 Disponível em http://www.conjur.com.br/2008-abr-19/ministro_gilmar_mendes_entrevista_veja?pagina=3. Acessado em 22.01.2016. 8 Disponível em < http://www.migalhas.com.br/Quentes/17,MI68048,11049- Revista+Veja+A+Abin+gravou+o+ministro+Gilmar+Mendes>. Acessado em 22.01.2016. As histórias sobre esse tema são de todos conhecidas. De fato, hoje verificamos certa disfuncionalidade no modelo... Hoje, então estamos diante de situações bastante delicadas, espacialmente diante dos abusos noticiados e cometidos. Recentemente, em uma visita a São Paulo, Senhores Ministros, eu conversava com uma, talvez, das mais importantes editoras de jornais Cezar Peluso. Por maioria e algum desconforto com alguns votos5, foi decidido que as prorrogações eram válidas, mas o Ministro Gilmar Mendes afirmou expressamente: Foi a própria Revista Veja (em edição de 3 de setembro de 2008), a qual o Ministro Gilmar Mendes havia concedido a citada entrevista, que divulgou o conteúdo de uma conversa telefônica havida entre o próprio Ministro e o então Senador Demóstenes Torres e acrescentou que a Abin o estava monitorando juntamente com a Polícia Federal para investigar as circunstâncias nas quais o Ministro Gilmar Mendes havia concedido liberdade ao banqueiro Daniel Dantas, pois, segundo a referida revista, “a Polícia Federal e a Abin interpretaram a decisão como uma confirmação de que alguma coisa errada se passava no gabinete do ministro...”.8 Tendo essa reportagem sido publicada em 03 de setembro de 2008, surgiu 17 (dezessete) dias depois, quando do já mencionado julgamento do recebimento da denúncia no Inq 2424 (“Operação Furacão”) em 20 de setembro de 2008, a oportunidade para que o Ministro Gilmar Mendes se pronunciasse sobre as interceptações telefônicas. Nesse contexto fático e político, torna-se mais claro não apenas o porquê do Ministro ter deixado aberta a possibilidade de rever sua posição sobre o período máximo de duração da medida, mas, sobretudo, sua posição contra os agentes policiais que de fato realizam a interceptação, chamando-os de “hermeneutas dos grampos”: As histórias sobre esse tema são de todos conhecidas. De fato, hoje verificamos certa disfuncionalidade no modelo... CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 168 Antonio Eduardo Ramires Santoro e Francisco Ramalho Ortigão Farias do Brasil, que fora atingida, numa dessas operações, porque teria aceito um encontro, um almoço, um jantar, com uma das pessoas investigadas. Essa pessoa, aparentemente, buscava no jornal uma retratação por uma notícia publicada, por isso, tentava, então marcar um almoço, e mandaram entregar um envelope. Esse envelope virou conteúdo de propina – porque, claro, nós temos, aí, os hermeneutas dos grampos. Então, essa é uma seara em que temos abusos de toda índole. ( ) Quase exagero ao dizer que uma Corte como esta cumpre uma função muito mais importante – e essa função não é perceptível –, não pelo que ela faz – e ela faz muito, como temos demonstrado aqui, ao longo do tempo –, mas pelo que ela evita que se faça. Quando ela inibe que, desde o primeiro grau, desde o agente policial, o gendarme tentado a virar ditador comece a dar devaneios aos seus sonhos. 9 Disponível em < http://redir.stf.jus.br/paginadorpub/paginador.jsp?docTP=AC&docID=609608>. Acessado em 22.01.2016. Cezar Peluso. Por maioria e algum desconforto com alguns votos5, foi decidido que as prorrogações eram válidas, mas o Ministro Gilmar Mendes afirmou expressamente: É exatamente isso que temos que fazer neste tipo de matéria.9 (sem grifo no original) O objeto do presente trabalho é precisamente perquirir a legitimidade para realizar a interpretação das conversas gravadas durante uma medida de interceptação telefônica judicialmente autorizada na forma da Lei no 9.296/96. Os problemas a serem aqui tratados são: a autoridade ou os agentes policiais estão legitimados a realizar interpretações sobre as conversas gravadas durante uma medida de interceptação telefônica que executaram por ordem judicial? Quais os efeitos de eventuais interpretações? Há vedação no ordenamento a um tal procedimento? A hipótese considerada é que somente o juiz está autorizado a interpretar os fatos, não havendo qualquer validade em atos dessa natureza praticados pela autoridade ou pelos agentes policiais, os quais sequer devem ser admitidos aos autos do processo. Para tanto se realizou uma pesquisa bibliográfica de natureza descritiva e explicativa sobre a natureza da prova no processo penal e sua relação com a verdade e, com base em análise qualitativa dos princípios informadores e dispositivos legais aplicáveis, verificar a hipótese de manipulação da formação da convicção judicial como um elemento nulificador dos atos praticados por juiz cuja imparcialidade tenha sido comprometida. 2 QUESTÕES TERMINOLÓGICAS RELATIVAS À PROVA E APLICADAS À INTERCEPTAÇÃO DAS COMUNICAÇÕES TELEFÔNICAS Ponto fundamental à compreensão do problema diz respeito à compreensão terminológica referente ao tema probatório. Sabe-se que o termo prova é usado de forma indiscriminada para designar uma variada gama de significados, daí a sua natureza polissêmica tanto no trato comum, como no discurso jurídico. CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 169 Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica Entretanto, faz-se necessário realizar algumas distinções para efetiva compreensão da proposta de pesquisa realizada neste trabalho. A primeira distinção diz respeito à compreensão do que vem a ser elemento de prova e resultado da prova. Elementos de prova, no inglês evidence, são os “...dados objetivos que confirmam ou negam uma asserção a respeito de um fato que interessa à causa”10 e sobre os quais o juiz vai realizar um procedimento inferencial para chegar a alguma conclusão sobre os fatos. Já o resultado da prova, no inglês proof, é a própria conclusão que o julgador extrai dos diversos elementos de prova existentes, por meio de um procedimento intelectual para estabelecer a veracidade ou não dos fatos alegados. Estes fatos alegados são chamados de objeto de prova. Há que se distinguir, ainda, fonte de prova, meio de prova e meio de obtenção ou investigação de prova. Fonte de prova são as pessoas ou coisas que podem fornecer uma informação apreciável sobre o objeto de prova, ou seja, os fatos alegados. Daí porque as fonte podem ser reais (documentos lato sensu) ou pessoais (testemunhas, acusado, vítima, perito, assistentes técnicos). Meios de prova são instrumentos ou atividades endoprocessuais que se desenvolvem perante o juiz, com conhecimento e participação das partes, pelos quais as fontes de prova introduzem elementos de prova no processo. Diferenciam-se dos meios de investigação de prova, também chamados meios de pesquisa da prova ou meios de obtenção de prova, que são atividades extraprocessuais, que podem ser produzidos na fase investigatória, sem a participação do investigado, baseado no fator “surpresa”11 e não podem ser repetidos. Nosso Código de Processo Penal não distingue entre meios de prova e meios de investigação de prova. O Codice di Procedura Penale12 italiano distingue no Livro III, Titulo II os meios de prova (testemunhal, confronto ou acareação, reconhecimento, reprodução judicial, pericial e documental) e no Título III os meios de pesquisa da prova (inspeções, buscas, sequestros e interceptações das conversas ou comunicações). 10 GOMES FILHO, Antonio Magalhães. “Notas sobre a terminologia da prova (reflexos no processo penal brasileiro)” In YARSHELL, Flávio Luiz e ZANOIDE DE MORAES, Maurício. Estudos em homenagem à Professora Ada Pellegrini Grinover. São Paulo: DPJ Editora, 2005, p. 307. 11 11 TONINI, Paolo. A prova no processo penal italiano. Tradução Alexandra Martins e Daniela Mróz. São Paulo: Revista dos Tribunais, 2002, p. 242. 12 , , p 12 Disponível em <http://www.polpenuil.it/attachments/048_codice_di_procedura_penale.pdf>. Acesso mar.2015. 10 GOMES FILHO, Antonio Magalhães. “Notas sobre a terminologia da prova (reflexos no processo penal brasileiro)” In YARSHELL, Flávio Luiz e ZANOIDE DE MORAES, Maurício. Estudos em homenagem à Professora Ada Pellegrini Grinover. São Paulo: DPJ Editora, 2005, p. 307. 11 TONINI, Paolo. A prova no processo penal italiano. Tradução Alexandra Martins e Daniela Mróz. São Paulo: Revista dos Tribunais, 2002, p. 242. 12 Disponível em <http://www.polpenuil.it/attachments/048_codice_di_procedura_penale.pdf>. Acesso em 25. Professora Ada Pellegrini Grinover. São Paulo: DPJ Editora, 2005, p. 307. 11 TONINI, Paolo. A prova no processo penal italiano. Tradução Alexandra Martins e Daniela Mróz. São Paulo: Revista dos Tribunais, 2002, p. 242. 12 Di í l <h // l il i / h /048 di di d l df> A 25 13 Art. 155, CPP: “O juiz formará sua convicção pela livre apreciação da prova produzida em contraditório judicial, não podendo fundamentar sua decisão exclusivamente nos elementos informativos colhidos na investigação, ressalvadas as provas cautelares, não repetíveis e antecipadas.” 2 QUESTÕES TERMINOLÓGICAS RELATIVAS À PROVA E APLICADAS À INTERCEPTAÇÃO DAS COMUNICAÇÕES TELEFÔNICAS A interceptação das comunicações telefônicas no Brasil, que como já visto está regulada pela Lei no 9.296 de 1996, constitui um meio de investigação ou de obtenção ou de CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 170 Antonio Eduardo Ramires Santoro e Francisco Ramalho Ortigão Farias pesquisa da prova, cuja aptidão para levar ao processo elementos probatórios deve ser analisada de maneira cuidadosa. CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 3 O CONTRADITÓRIO NA INTERCEPTAÇÃO TELEFÔNICA OU A FALTA DELE É muito comum tratar o direito ao contraditório no processo penal apenas no que respeita ao aspecto argumentativo, como a possibilidade de falar sobre o que for aduzido pela parte oposta. Porém essa é uma visão simplista que ignora dever o contraditório ir muito além da aparência formal para se consubstanciar em poder de informação, força de confronto e paridade de armas. O exercício pleno desse direito no processo penal deve passar por seis momentos: (1) a oportunidade de postular a prova em igualdade de oportunidades e condições; (2) a possibilidade de impugnar a prova postulada pela parte adversa; (3) a possibilidade de impugnar a decisão que admite a prova; (4) a participação e assistência na produção da prova; (5) a possibilidade aportar considerações sobre a validade do elemento gerado e razões para influenciar o magistrado na valoração dos elementos válidos antes que se convertam em resultados probatórios; (6) a possibilidade de controlar a racionalidade da decisão do julgador pela impugnação dos fundamentos por via recursal. Assim se conclui que o contraditório é um direito constitucionalmente assegurado, que concede às partes de um processo judicial a faculdade de tomar conhecimento (informação) e de discutir todos os elementos apresentados ao julgador (poder de reação) em igualdade de condições (paridade de armas) com a finalidade de influenciar sua decisão (poder de influência) , que é responsável por torná-lo eficaz, e controlar sua racionalidade (direito ao recurso). Em outras palavras, o contraditório se torna palpável na expressão do poder de influência que a parte é capaz de exercer sobre a formação cognitiva do juiz, fazendo entender que além de apresentar os elementos obrigatórios, formais (ou aparentes) e materiais (ou substanciais), o contraditório precisa se mostrar eficaz. Contudo, o legislador no artigo 15513 do Código de Processo Penal (CPP) aparentemente trouxe uma exceção à exigência do contraditório judicial, relegando a um segundo plano o princípio do contraditório em prol da expansão de possibilidades de investigação. Essa opção legislativa obedece a uma lógica inquisitória intrínseca ao Código de CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 171 Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica Processo Penal, que muito já se modificou com a reforma acontecida em 2008, mas deixou neste artigo seus resquícios. Processo Penal, que muito já se modificou com a reforma acontecida em 2008, mas deixou neste artigo seus resquícios. 14 SUPREMO TRIBUNAL FEDERAL. INQUÉRITO Nº 2.266/AMAPÁ. RELATOR MINISTRO GILMAR MENDES. TRIBUNAL PLENO. Julgamento em 26.5.2011, DJe de 13.3.2012. Disponível em: <http://redir.stf.jus.br/paginadorpub/paginador.jsp?docTP=TP&docID=1812853>. Acessado em 10/02/2015. 15“Quanto al contraddittorio impossibile, esso andrebbe rigorosamente inteso come legittimante l'impiego processuale di strumenti gnoseologici dei quali sia "accertata" (cioè indubbia, sicura, innegabile; quindi, non soltanto presunta o convenzionamente stabilita dal legislatore) l'inconciliabilità con il contraddittorio perché intrinsecamente incompatibili con quest'ultimo (come avviene per le intercettazione di comunicazioni, la cui attendibilità sarebbe inevitabilmente compromessa da un preavviso de loro comprimento alla persona 3 O CONTRADITÓRIO NA INTERCEPTAÇÃO TELEFÔNICA OU A FALTA DELE 2016 172 Antonio Eduardo Ramires Santoro e Francisco Ramalho Ortigão Farias interceptação de comunicações como exemplo, uma vez que um aviso de seu cumprimento à pessoa sob investigação não é possível por motivos óbvios, fazendo faltar o elemento informação/conhecimento intrínseco ao exercício pleno do contraditório. Ora, como seria concebível um contraditório verdadeiro exercido a posteriori diante dos já citados fatores de composição inegociáveis desse direito, a saber: acesso irrestrito às informações pertinentes ao caso, instrumentos de insurgência contra a pretensão acusatória, paridade de armas e, em especial, poder de influência na decisão judicial? O que está por trás dessa prática que se instaurou no processo penal brasileiro? A facilitação a que as agências de persecução introduzam elementos para formação do convencimento do juiz no processo penal é, naturalmente, o principal objetivo. Ocorre que sua utilização indiscriminada denota certa ideologia repressiva, tendente a fazer prevalecer no embate axiológico à defesa social sobre a liberdade. Sobre esse tema, Geraldo Prado escreveu: Em um cenário de tensão entre liberdade e segurança e sob a inspiração da retórica do risco, os Estados produzem normativas que acossam os direitos fundamentais visando ampliar os recursos à disposição da repressão penal. As pesquisas policias são incrementadas com o emprego de métodos ocultos de investigação autorizados judicialmente, como a interceptação telefônica e de e- mails, as escutas domiciliares e a infiltração de agentes, convertendo-se a prática em modelo de atuação preliminar. 16 Em um cenário de tensão entre liberdade e segurança e sob a inspiração da retórica do risco, os Estados produzem normativas que acossam os direitos fundamentais visando ampliar os recursos à disposição da repressão penal. As pesquisas policias são incrementadas com o emprego de métodos ocultos de investigação autorizados judicialmente, como a interceptação telefônica e de e- mails, as escutas domiciliares e a infiltração de agentes, convertendo-se a prática em modelo de atuação preliminar. 16 Muitos são os prejuízos desse proceder, desde a contaminação das provas até a drástica redução da possibilidade de defesa pela restrição de informações. Todavia, uma questão sobre a qual doutrina e jurisprudência dificilmente se debruçam é o comprometimento cognitivo do julgador que teve contato unilateralmente com a acusação (e sua tese) a fim de decidir sobre a autorização da cautelar, ficando prevento para julgar a causa. 3 O CONTRADITÓRIO NA INTERCEPTAÇÃO TELEFÔNICA OU A FALTA DELE Todavia, numa leitura processual-constitucional, é inconcebível que uma escolha de política criminal possa preterir um princípio constitucional, ponto em que a doutrina converge com os tribunais superiores, reconhecendo como indispensável o contraditório, sob pena de nulidade do processo. Desse entendimento nasceu o contraditório diferido, que consiste no exercício do contraditório extemporâneo à obtenção da prova, na tentativa de salvar a utilização de provas cautelares, não repetíveis e antecipadas. O comportamento de muitos doutrinadores – que cunharam os termos “contraditório diferido”, “contraditório postergado” e “contraditório retardado” – e dos tribunais indica que essa versão do contraditório tem sido amplamente aceita. Exemplo disso é a decisão do Supremo Tribunal Federal abaixo colacionada. O inquérito não possui contraditório, mas as medidas invasivas deferidas judicialmente devem se submeter a esse princípio [do contraditório], e a sua subtração acarreta nulidade. Obviamente não é possível falar-se em contraditório absoluto quando se trata de medidas invasivas e redutoras da privacidade. Ao investigado não é dado conhecer previamente - sequer de forma concomitante - os fundamentos da medida que lhe restringe a privacidade. Intimar o investigado da decisão de quebra de sigilo telefônico tornaria inócua a decisão. Contudo, isso não significa a ineficácia do princípio do contraditório. Com efeito, cessada a medida, e reunidas as provas colhidas por esse meio, o investigado deve ter acesso ao que foi produzido, nos termos da Súmula Vinculante nº 14. Os fundamentos da decisão que deferiu a escuta telefônica, além das decisões posteriores que mantiveram o monitoramento devem estar acessíveis à parte investigada no momento de análise da denúncia e não podem ser subtraídas da Corte, que se vê tolhida na sua função de apreciar a existência de justa causa da ação penal. Trata-se de um contraditório diferido, que permite ao cidadão exercer um controle sobre as invasões de privacidade operadas pelo Estado.14 Entretanto, apesar da conveniência da aplicação desta pretensa modalidade de contraditório, doutrinadores mais críticos caminham no sentido de refutar esse entendimento, como Giulio Ubertis, para quem, se faltar algum dos elementos essenciais do contraditório, estaremos diante de um contraditório impossível. O próprio Ubertis15 usa o caso da CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 16 PRADO, Geraldo. “Prova penal e sistema de controles epistêmicos: a quebra da cadeia de custódia das provas obtidas por métodos ocultos”. 1ª ed. São Paulo: Marcial Pons, 2014, p. 59. sottoposta alle indagini) oppure perché recanti elementi di prova contenustiticamente o strutturalmente diversi da quelli che sarebbero generabili esso (...).” UBERTIS, Giulio. op. cit., p. 338. p p 17 Nesse sentido vale a leitura de FESTINGER, Leon. Teoria da dissonância cognitiva. Tradução Eduardo Almeida. Rio de Janeiro: Zahar editores, 1975. Sobre a influência que os elementos cognitivos da investigação exercem sobre a formação da convicção judicial SCHÜNEMANN, Bernd. O juiz como um terceiro manipulado Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica Esse é o típico caso da interceptação telefônica, na qual esse contato prévio com as informações provavelmente se repetirá com a prorrogação (ou as prorrogações) do tempo de escuta, que dependem de decisão fundamentada18, ou seja, o magistrado disporá sobre os motivos que o levaram ao convencimento de que os indícios apresentados são suficientes para suspeitar do investigado, denotando já haver uma aproximação da cognição do julgador com a tese (ou futura tese) acusatória. Tal fenômeno foi descrito por Paulo Biskup de Aquino como a transformação de meros indícios em elementos definidores da figura delitiva. Ele explica que “...cada vez mais se utiliza fatores indiciários para incriminar sujeitos, principalmente para a decretação de medidas cautelares em busca de provas, como é o caso da interceptação telefônica e a lei do crime organizado”.19 Porém o problema mais desafiador gerado pela prova produzida cautelarmente decorre dos casos em que o sigilo é indissociável do procedimento de obtenção do conteúdo da informação, como ocorre na interceptação e também na infiltração de agentes. Em situações como essas, de provas ocultas20, é possível identificar implicações que tornam atualmente impraticável o pleno exercício do contraditório. Basicamente, no caso das provas cautelares em geral, o exercício do contraditório deveria se dar ainda na fase investigatória com uma rigorosa cadeia de custódia do material probatório, como pensada por Prado21, e a prevenção negativa do julgador – como previsto, e.g., nas legislações francesa22 e portuguesa23, onde existe a figura do juiz de instrução – se p p 21 “No direito brasileiro praticamente não há referências doutrinárias à cadeia de custódia, designação pela qual é conhecido o dispositivo que pretende assegurar a integridade dos elementos probatórios, não obstante o seu significado em termos de redução de complexidade de garantia constitucional contra a prova ilícita.” PRADO, Geraldo. op. cit., p. 80. p , p 21 “No direito brasileiro praticamente não há referências doutrinárias à cadeia de custódia, designação pela qual é conhecido o dispositivo que pretende assegurar a integridade dos elementos probatórios, não obstante o seu significado em termos de redução de complexidade de garantia constitucional contra a prova ilícita.” PRADO, Geraldo. op. cit., p. 80. p p ç 18 Art. 5º, lei 9.296/96: “A decisão será fundamentada, sob pena de nulidade, indicando também a forma de execução da diligência, que não poderá exceder o prazo de quinze dias, renovável por igual tempo uma vez comprovada a indispensabilidade do meio de prova. ”. (grifo nosso) q a) Puníveis com pena de prisão superior, no seu máximo, a 3 anos; no processo penal? Uma confirmação empírica dos efeitos perseverança e aliança in Estudos de direito penal, direito processual penal e filosofia do direito. Tradução Luís Greco. São Paulo: Marcial Pons, 2013. 18 no processo penal? Uma confirmação empírica dos efeitos perseverança e aliança in Estudos de direito penal, direito processual penal e filosofia do direito. Tradução Luís Greco. São Paulo: Marcial Pons, 2013. 18 Art. 5º, lei 9.296/96: “A decisão será fundamentada, sob pena de nulidade, indicando também a forma de execução da diligência, que não poderá exceder o prazo de quinze dias, renovável por igual tempo uma vez comprovada a indispensabilidade do meio de prova. ”. (grifo nosso) comprovada a indispensabilidade do meio de prova. . (grifo nosso) 19 AQUINO, Paulo Biskup de., ROLAND, Claudia Symone Dias. “As interceptações telefônicas e o processo penal brasileiro: uma reflexão”. 1ª ed. Curitiba: Editora Prismas, 2015, p. 63-64. 20 PRADO op cit p 59 e ss no processo penal? Uma confirmação empírica dos efeitos perseverança e aliança in Estudos de direito penal, direito processual penal e filosofia do direito. Tradução Luís Greco. São Paulo: Marcial Pons, 2013. 18 Art. 5º, lei 9.296/96: “A decisão será fundamentada, sob pena de nulidade, indicando também a forma de execução da diligência, que não poderá exceder o prazo de quinze dias, renovável por igual tempo uma vez comprovada a indispensabilidade do meio de prova. ”. (grifo nosso) 19 AQUINO, Paulo Biskup de., ROLAND, Claudia Symone Dias. “As interceptações telefônicas e o processo penal brasileiro: uma reflexão” 1ª ed Curitiba: Editora Prismas 2015 p 63 64 AQUINO, Paulo Biskup de., ROLAND, Claudia Symone Dias. “As interceptações telefônicas e o processo penal brasileiro: uma reflexão”. 1ª ed. Curitiba: Editora Prismas, 2015, p. 63-64. 20 PRADO. op. cit., p. 59 e ss. 3 O CONTRADITÓRIO NA INTERCEPTAÇÃO TELEFÔNICA OU A FALTA DELE Neste caso o contraditório diferido termina por servir apenas para legitimar a prova unilateral, na medida em que a cognição do julgador já se formou pelo contato inicial com as informações, de tal sorte que novos conjuntos de elementos cognitivos acabam por se submeter a procedimentos psicológicos de afastamento ou redução da dissonância cognitiva com a prevalência dos elementos conhecidos previamente17. CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 173 Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica 22 22 Article 100-3, Códe de Procédure Pénale: “Le juge d'instruction ou l'officier de police judiciaire commis par lui peut requérir tout agent qualifié d'un service ou organisme placé sous l'autorité ou la tutelle du ministre chargé des télécommunications ou tout agent qualifié d'un exploitant de réseau ou fournisseur de services de télécommunications autorisé, en vue de procéder à l'installation d'un dispositif d'interception.” 23 23Artigo 187, Código de Processo Penal português: “1 - A intercepção e a gravação de conversações ou comunicações telefónicas só podem ser autorizadas durante o inquérito, se houver razões para crer que a diligência é indispensável para a descoberta da verdade ou que a prova seria, de outra forma, impossível ou muito difícil de obter, por despacho fundamentado do juiz de instrução e mediante requerimento do Ministério Público, quanto a crimes: q a) Puníveis com pena de prisão superior, no seu máximo, a 3 anos; a) Puníveis com pena de prisão superior, no seu máximo, a 3 anos; b) Relativos ao tráfico de estupefacientes; b) Relativos ao tráfico de estupefacientes; CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 174 Antonio Eduardo Ramires Santoro e Francisco Ramalho Ortigão Farias apresentam em conjunto como possível solução, que é acompanhada da possibilidade de a defesa exercer atos de investigação24 tais quais os praticados pela polícia e pelo Ministério Público, conferindo equidistância às partes. Contudo, quando se trata da interceptação das comunicações telefônicas, devido ao segredo que lhe é imanente, nenhuma dessas propostas parece apta a salvar a essência do contraditório, que resta perdida pela impossibilidade de igualdade de acesso a informação e de contra-argumentação gerados pela quebra de paridade, culminando também em perda de força de influência no convencimento do juiz. Pontue-se que, pela essência da interceptação das comunicações telefônicas, não é dado às partes, em igualdade de condições, as oportunidades para: (1) postular a prova; (2) discutir a admissão da prova antes da decisão que o faz e (3) participar da produção da prova. c) De detenção de arma proibida e de tráfico de armas; d) De contrabando; d) De contrabando; e) De injúria, de ameaça, de coacção, de devassa da vida privada e perturbação da paz e do sossego, quando cometidos através de telefone; f) De ameaça com prática de crime ou de abuso e simulação de sinais de perigo; ou c) De detenção de arma proibida e de tráfico de armas; c) De detenção de arma proibida e de tráfico de armas; d) De contrabando; e) De injúria, de ameaça, de coacção, de devassa da vida privada e perturbação da paz e do sossego, quando cometidos através de telefone; f) De ameaça com prática de crime ou de abuso e simulação de sinais de perigo; ou g) De evasão, quando o arguido haja sido condenado por algum dos crimes previstos nas alíneas anteriores.” 24 A reforma sofrida pelo sistema processual italiano é um exemplo interessante sobre a necessidade de, em sistema acusatório, permitir-se, ao lado da investigação estatal, outra realizada pela defesa. SCARANCE FERNANDES, Antonio. “O Equilíbrio na Investigação Criminal” in YARSHELL, Flávio Luis; MORAES, Maurício Zanoide (org.). Estudos em homenagem à professora Ada Pellegrini Grinover. São Paulo: DPJ, 2005, p. 327. 25 FERRAJOLI, Luigi. Direito e razão: teoria do garantismo penal 4a ed. Tradutores Ana Paula Zomer Sica, Fauzi Hassan Choukr, Juarez Tavares e Luiz Flávio Gomes. São Paulo: Revista dos Tribunais, 2014, p. 70. 4 CONCLUSÃO - O PROCESSO COMO MODELO EPISTEMOLÓGICO DE CONHECIMENTO CONDICIONANTE DA VALIDADE DA DECISÃO A formação do convencimento judicial não pode se dar ao arrepio da verdade. Entretanto, não esta se adotando um conceito de verdade como equivalência, pois que cada parte constrói sua narrativa sobre os fatos e a narrativa construída pelo juiz deve ser completa e coerente para que seja reputada racional. Luigi Ferrajoli esclarece que tanto do ponto de vista epistemológico, como político, como jurídico, o que se exige é “que a legitimidade das decisões penais se condicione à verdade empírica de suas motivações”25. Todavia, essa racionalidade é obtida a posteriori da efetiva formação da convicção judicial, pois que verificada pela motivação exposta. ) ; e) De injúria, de ameaça, de coacção, de devassa da vida privada e perturbação da paz e do sossego, quando cometidos através de telefone; De ameaça com prática de crime ou de abuso e simulação de sinais de perigo; ou evasão, quando o arguido haja sido condenado por algum dos crimes previstos nas alíneas anteriores.” 24 A reforma sofrida pelo sistema processual italiano é um exemplo interessante sobre a necessidade de, em sistema acusatório, permitir-se, ao lado da investigação estatal, outra realizada pela defesa. SCARANCE FERNANDES, Antonio. “O Equilíbrio na Investigação Criminal” in YARSHELL, Flávio Luis; MORAES, Maurício Zanoide (org.). Estudos em homenagem à professora Ada Pellegrini Grinover. São Paulo: DPJ, 2005, p. 327. p 25 FERRAJOLI, Luigi. Direito e razão: teoria do garantismo penal 4a ed. Tradutores Ana Paula Zomer Sica, Fauzi Hassan Choukr, Juarez Tavares e Luiz Flávio Gomes. São Paulo: Revista dos Tribunais, 2014, p. 70. p 25 FERRAJOLI, Luigi. Direito e razão: teoria do garantismo penal 4a ed. Tradutores Ana Paula Zomer Sica, Fauzi Hassan Choukr, Juarez Tavares e Luiz Flávio Gomes. São Paulo: Revista dos Tribunais, 2014, p. 70. CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 175 Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica O processo, segundo Michele Taruffo, sob uma perspectiva metodológica, pode ser analisado “como um ‘modelo epistemológico’ do conhecimento dos fatos com base nas provas”26. E ressalta: Em todo e qualquer procedimento de caráter epistêmico tem importância decisiva o método, ou seja, o conjunto das modalidades com que são selecionadas, controladas e utilizadas as informações que servem para demonstrar a veracidade das conclusões. 29 SCARANCE FERNANDES, Antonio. “Tipicidade e sucedâneos de prova” in: SCARANCE FERNANDES, Antonio, GAVIÃO DE ALMEIDA, José Raul e ZANOIDE DE MORAES, Maurício (coordenadores) Provas no Processo Penal: estudo comparado. São Paulo: RT, 2012, p. 15. 26 TARUFFO, Michele. Uma simples verdade: o juiz e a construção dos fatos. Tradução Vitor de Paula Ramos. São Paulo: Marcial Pons, 2012, p. 160. 27 Ibid, p. 164. , p 28 ANDRÉS IBÁÑEZ, Perfecto. Prueba y convicción judicial en el proceso penal. Buenos Aires: Hammurabi, 2009, p 49. 29 26 TARUFFO, Michele. Uma simples verdade: o juiz e a construção dos fatos. Tradução Vitor de Paula Ramos. São Paulo: Marcial Pons, 2012, p. 160. 27 Ibid, p. 164. 28 ANDRÉS IBÁÑEZ, Perfecto. Prueba y convicción judicial en el proceso penal. Buenos Aires: Hammurabi, 2009, p 49. 29 SCARANCE FERNANDES, Antonio. “Tipicidade e sucedâneos de prova” in: SCARANCE FERNANDES, Antonio, GAVIÃO DE ALMEIDA, José Raul e ZANOIDE DE MORAES, Maurício (coordenadores) Provas no Processo Penal: estudo comparado. São Paulo: RT, 2012, p. 15. 33 De forma muito resumida, a interceptação das comunicações telefônicas não geram elementos de prova e todos os dados obtidos da interceptação que o juiz valora são sucedâneos de prova posto que (1) não cabe ao juiz analisar o fonograma ou as transcrições das conversas telefônicas interceptadas como se elementos de prova fossem, pois que não se deve confundir meio de obtenção de prova (a interceptação) com o suporte em que se registram os dados colhidos (documento), de sorte que tal procedimento implicaria em substituir um meio de (obtenção de) prova por outro; e (2) não cabe ao juiz usar na audiência ou mesmo na sentença o que foi colhido na fase anterior à propositura da ação penal durante a interceptação das comunicações telefônicas porque esta descobre fontes pessoais (e não reais), às quais obrigatoriamente só podem gerar dados a serem introduzidos ao processo e aptos a serem valorados (elementos de prova) se submetidos ao meio de prova oral, com seu sistema específico de exercício do contraditório (direto e cruzado), não se podendo aplicar-lhe o contraditório diferido que é próprio do meio de prova documental cujo elemento é pré-existente. 30 PRADO, Geraldo. op. cit., p. 78. 31 4 CONCLUSÃO - O PROCESSO COMO MODELO EPISTEMOLÓGICO DE CONHECIMENTO CONDICIONANTE DA VALIDADE DA DECISÃO No âmbito do processo isso equivale a fazer referência sobretudo às regras que disciplinam a produção das provas e sua utilização, ou seja, ao “direito das provas” e à equivalente noção anglo-americana da law of evidence.27 Portanto, resta claro que o processo penal se legitima pela busca do conhecimento da verdade com base nas provas. Certo de que os fatos estão no passado, as provas nada mais são do que signos transmitidos, são materiais semióticos que representam a única via de acesso ao conhecimento28 e que, como em todo procedimento de caráter epistêmico, devem ser obtidas com estrita observância do método de produção e utilização. Há, portanto, que se definir se a interceptação das comunicações telefônicas é um meio de investigação de prova típico ou atípico. Neste ponto é importante pontuar que meios típicos não se caracterizam meramente por estarem previstos em lei, pois, como pontua Scarance, apoiado na lição de Antonio Laronga, “a prova típica é aquela prevista e dotada de procedimento próprio para sua efetivação; a prova atípica, por conseguinte, é aquela que, prevista ou não, é destituída de procedimento para sua produção.”29 Nesse sentido seriam típicos aqueles cuja previsão e procedimento estão regulamentadas, seja o procedimento próprio ou por remissão. De outro lado, não estando previsto o meio ou, ainda que previsto, se o procedimento não está regulamentado ou é objeto de remissão, está-se diante de um meio atípico. Ora, o art. 5o da Lei no 9.296 de 1996 prevê não apenas que a decisão que defere a medida deve ser fundamentada, mas que o juiz deve indicar “a forma de execução da diligência”, omitindo-se na regulamentação do procedimento aplicável. Ademais, o §2o do art. 6o da mesma Lei determina que “cumprida a diligência, a autoridade policial encaminhará o resultado da interceptação ao juiz, acompanhado de auto Ademais, o §2o do art. 6o da mesma Lei determina que “cumprida a diligência, a autoridade policial encaminhará o resultado da interceptação ao juiz, acompanhado de auto CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 176 Antonio Eduardo Ramires Santoro e Francisco Ramalho Ortigão Farias circunstanciado, que deverá conter o resumo das operações realizadas”, mas não define o que se entende por “resultado da interceptação” e, de forma exauriente, o que deve conter “o auto circunstanciado”, apenas referindo-se ao resumo das operações. Ibid. 32 Assim compreendido o fenômeno processual que decorre de dois fatores: (1) o uso, na audiência de julgamento, como elementos probatórios de elementos colhidos em fases anteriores e (2) a substituição de um meio de prova por outro. (SCARANCE, op. cit., p. 30) 33 30 PRADO, Geraldo. op. cit., p. 78. 31 Ibid. 32 31 Ibid. 32 30 PRADO, Geraldo. op. cit., p. 78. 31 Ibid. 32 Assim compreendido o fenômeno processual que decorre de dois fatores: (1) o uso, na audiência de julgamento, como elementos probatórios de elementos colhidos em fases anteriores e (2) a substituição de um meio de prova por outro. (SCARANCE, op. cit., p. 30) 33 De forma muito resumida, a interceptação das comunicações telefônicas não geram elementos de prova e todos os dados obtidos da interceptação que o juiz valora são sucedâneos de prova posto que (1) não cabe ao juiz analisar o fonograma ou as transcrições das conversas telefônicas interceptadas como se elementos de prova fossem, pois que não se deve confundir meio de obtenção de prova (a interceptação) com o suporte em que se registram os dados colhidos (documento), de sorte que tal procedimento implicaria em substituir um meio de (obtenção de) prova por outro; e (2) não cabe ao juiz usar na audiência ou mesmo na sentença o que foi colhido na fase anterior à propositura da ação penal durante a interceptação das comunicações telefônicas porque esta descobre fontes pessoais (e não reais), às quais obrigatoriamente só podem gerar dados a serem introduzidos ao processo e aptos a serem valorados (elementos de prova) se submetidos ao meio de prova oral, com seu sistema específico de exercício do contraditório (direto e cruzado), não se podendo aplicar-lhe o contraditório diferido que é próprio do meio de prova documental cujo elemento é pré-existente. 4 CONCLUSÃO - O PROCESSO COMO MODELO EPISTEMOLÓGICO DE CONHECIMENTO CONDICIONANTE DA VALIDADE DA DECISÃO circunstanciado, que deverá conter o resumo das operações realizadas”, mas não define o que se entende por “resultado da interceptação” e, de forma exauriente, o que deve conter “o auto circunstanciado”, apenas referindo-se ao resumo das operações. Dessa forma, como muito bem observou Geraldo Prado, quando a legislação silencia sobre o procedimento probatório, há exigência de motivação da “decisão que defere o emprego de métodos ocultos de investigação importa”30 não apenas na indicação dos elementos que convencem acerca da sua adequação, mas “ainda, na definição dos meios de sua execução e fiscalização”31. Isso significa que o procedimento da interceptação das comunicações telefônicas não é regulamentado, sendo deixado ao juiz, no ato decisório, fazê-lo. Isso implica em que a interceptação das comunicações é um meio de investigação de prova atípico. Neste ponto faremos uma observação neste trabalho de pesquisa para ressalvar nosso entendimento pessoal sobre a possibilidade de que a interceptação telefônica gere elementos de prova valoráveis pelo juiz. Compreendemos tratar-se de um sucedâneo de prova32 que é apto apenas a descobrir fontes de prova que devem se submeter ao contraditório apropriado para que forneçam elementos valoráveis33. Todavia, a jurisprudência admite que o juiz valore os conteúdos de conversas obtidos durante as diligências de interceptação telefônica, portanto nos importa trabalhar com esse entendimento majoritário para analisar o problema da hermenêutica policial ou pré-judicial. A falta de densidade dos elementos constantes do §2o do art. 4 CONCLUSÃO - O PROCESSO COMO MODELO EPISTEMOLÓGICO DE CONHECIMENTO CONDICIONANTE DA VALIDADE DA DECISÃO 6o da Lei no 9.296/96, a saber, “resultado da interceptação” e o exato conteúdo do “o auto circunstanciado” implica em que não se saiba se os chamados resultados da interceptação são as transcrições (integrais ou parciais), se são os fonogramas ou o suporte magnético que os armazena (CD, DVD, HD, etc.), tampouco se sabe o que podem a autoridade policial e seus agentes fazer constar do 33 De forma muito resumida, a interceptação das comunicações telefônicas não geram elementos de prova e todos os dados obtidos da interceptação que o juiz valora são sucedâneos de prova posto que (1) não cabe ao juiz analisar o fonograma ou as transcrições das conversas telefônicas interceptadas como se elementos de prova fossem, pois que não se deve confundir meio de obtenção de prova (a interceptação) com o suporte em que se registram os dados colhidos (documento), de sorte que tal procedimento implicaria em substituir um meio de (obtenção de) prova por outro; e (2) não cabe ao juiz usar na audiência ou mesmo na sentença o que foi colhido na fase anterior à propositura da ação penal durante a interceptação das comunicações telefônicas porque esta descobre fontes pessoais (e não reais), às quais obrigatoriamente só podem gerar dados a serem introduzidos ao processo e aptos a serem valorados (elementos de prova) se submetidos ao meio de prova oral, com seu sistema específico de exercício do contraditório (direto e cruzado), não se podendo aplicar-lhe o contraditório diferido que é próprio do meio de prova documental cujo elemento é pré-existente. CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 177 177 Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica “auto circunstanciado”, que normalmente, na prática, é um conjunto de atos como relatórios, análises, transcrições parciais e, o pior de tudo, interpretações sobre o suposto significado das conversas ouvidas. Essa atividade interpretativa da autoridade e dos agentes policiais cria uma disfunção no sistema de interceptações telefônicas, porquanto diante da ausência de tempo para ouvir as milhares de horas de conversas captadas pelos sistemas de tecnologia da informação, o juiz decide sobre as prorrogações com base nas informações fornecidas pela autoridade policial, que já estão previamente valoradas e, portanto, conduzem a formação da convicção judicial de forma metodologicamente mais perniciosa que a mera inexistência de contraditório direto. p , p 35 Id. p. 208. 34 SCHUNEMANN. Op. cit., p. 213. 35 35 Id. p. 208. CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 4 CONCLUSÃO - O PROCESSO COMO MODELO EPISTEMOLÓGICO DE CONHECIMENTO CONDICIONANTE DA VALIDADE DA DECISÃO Isso porque mais grave do que não ser dado ao juiz ter contato direto com a informação bruta sem a participação das partes, a prática da interceptação telefônica no Brasil, demonstra que o primeiro contato que o julgador tem é com uma narrativa da verdade construída por quem sequer é parte do processo (ou do futuro processo) e que termina por macular sua imparcialidade pelos chamados efeito aliança, segundo o qual “o comportamento do juiz pode ser explicado também simplesmente pelo fato de que, diante de uma situação obscura, ele se orienta segundo uma prévia avaliação oriunda de uma pessoa por ele aceita como competente”34, e efeito perseverança, qual seja, “as informações que confirmam uma hipótese que, em algum momento anterior fora considerada correta, são sistematicamente superestimadas, enquanto as informações contrárias são sistematicamente menosprezadas”35. Neste sentido, para que o magistrado defira uma medida de interceptação telefônica, ele deve primeiramente aderir à construção narrativa da verdade de quem a requer e, com muito mais razão, deve densificar essa adesão para prorrogar sucessivamente as medidas. Ao contrário, qualquer versão narrativa que se apresente dissonante é submetida a um mecanismo de equilíbrio do sistema cognitivo, que faça desaparecer as contradições, restaurando a consonância. Dessa forma, se o sucesso de qualquer atividade da defesa já restava maculada com a inoperância do contraditório diferido, a admissão da atividade interpretativa da autoridade policial e seus agentes implica na impossibilidade de garantir o respeito ao princípio da imparcialidade judicial, do contraditório e da ampla defesa. Neste sentido, a inexistência de um rígido procedimento probatório para a interceptação telefônica, implica na necessidade de compatibilizá-la aos princípios constitucionais que norteiam o processo, de tal sorte que a omissão legislativa sobre a CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 178 Antonio Eduardo Ramires Santoro e Francisco Ramalho Ortigão Farias possibilidade de que os agentes executores da medida interpretem seu conteúdo significa na sua proibição. O processo é um modelo epistemológico de conhecimento cuja não observância macula a livre formação do convencimento e a existência de fundamentação da decisão não elide a vulneração dos direitos fundamentais, em especial a necessária imparcialidade. A atividade valorativa dos “hermeneutas dos grampos” invalida a decisão judicial proferida com base na interceptação telefônica prejudicialmente interpretada. Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica MARTINS, Rui Cunha. O ponto cego do direito: the brazilian lessons. 3ª ed. São Paulo: Atlas, 2013. ____. O mapeamento procesual da “verdade”. In: “Decisão Judicial”. São Paulo: Marcial Pons, 2013. MAYA, André Machado. Imparcialidade e proceso penal: da prevençào da competencia ao juiz das garantías. Rio de Janeiro: Lumen Juris, 2011. MUÑOZ CONDE, Francisco. De las prohibiciones probatorias al derecho procesal penal del enemigo. Buenos Aires: Hammurabi, 2008. ____. Valoración de las grabaciones audiovisuales en el proceso penal. 2ª ed. Buenos Aires: Hammurabi, 2007. ____. La búsqueda de la verdade en el processo penal. 3a edição. Buenos Aires: Hammurabi, 2007. NIEVA FENOLL, Jordi. La duda en el proceso penal. Barcelona: Marcial Pons, 2013. ____. La valoración de la prueba. Barcelona: Marcial Pons, 2010. PRADO, Geraldo. Prova penal e sistema de controles epistêmicos: a quebra da cadeia de custódia das provas obtidas por métodos ocultos. São Paulo: Marcial Pons, 2014. RODRIGUES, Benjamim Silva. A monitorização dos fluxos inormacionais e comunicacionais. Volume I. Coimbra: Coimbra, 2009. ROSA, Gabriela Porto. A construção da verdade no processo penal. Rio de Janeiro: Lumen Juris, 2015. ROXIN, Claus. La prohibición de autoincriminación y de las escuchas domiciliarias. Buenos Aires: Hammurabi, 2008. SCARANCE FERNANDES, Antonio. “O Equilíbrio na Investigação Criminal” in YARSHELL, Flávio Luis; MORAES, Maurício Zanoide (org.). Estudos em homenagem à professora Ada Pellegrini Grinover. São Paulo: DPJ, 2005. ____.“Tipicidade e sucedâneos de prova” in: SCARANCE FERNANDES, Antonio, GAVIÃO DE ALMEIDA, José Raul e ZANOIDE DE MORAES, Maurício (coordenadores) Provas no Processo Penal: estudo comparado. São Paulo: RT, 2012, p. 15. SCHÜNEMANN, Bernd. O juiz como um terceiro manipulado no processo penal? Uma confirmação empírica dos efeitos perseverança e aliança in Estudos de direito penal, direito processual penal e filosofia do direito. Tradução Luís Greco. São Paulo: Marcial Pons, 2013. STRECK, Lenio Luiz. Verdade e Consenso: Constituição, Hermenêutica e Teorias Discursivas. 4a ed. São Paulo: Saraivva, 2012. TARUFFO, Michele. Uma simples verdade: o juiz e a construção dos fatos. Tradução Vitor de Paula Ramos. São Paulo: Marcial Pons, 2012. TONINI, Paolo. A prova no processo penal italiano. Tradução Alexandra Martins e Daniela Mróz. São Paulo: Revista dos Tribunais, 2002. UBERTIS, Giulio. “Il contradittorio nella formazione dela prova penale.” In YARSHELL, Flávio Luiz e ZANOIDE DE MORAES, Maurício. Estudos em homenagem à Professora Ada Pellegrini Grinover. São Paulo: DPJ Editora, 2005. VALENTE, Manuel Monteiro Guedes. 5 REFERÊNCIAS ABEL LLUCH, Xavier e RICHARD GONZÁLEZ, Manuel. Estudios sobre prueba penal volumen III: Actos de investigación y medios de prueba en el proceso penal: diligencias de instrucción, entrada y registro, intervención de comunicaciones, valoración y revisión de la prueba en vía de recurso. Madri: La Ley Actualidad, 2013. AGUILAR, Francisco. Dos Conhecimentos Fortuitos Obtidos Através de Escutas Telefónicas. Coimbra: Almedina, 2004. AQUINO, Paulo Biskup de., ROLAND, Claudia Symone Dias. “As interceptações telefônicas e o processo penal brasileiro: uma reflexão”. 1ª ed. Curitiba: Editora Prismas, 2015. ANDRÉS IBÁÑEZ, Perfecto. Prueba y convicción judicial en el proceso penal. Buenos Aires: Hammurabi, 2009. BACIGALUPO, Enrique. El debido proceso penal. Buenos Aires: Hammurabi, 2007. CONTI, Carlotta e TONINI, Paolo. Il diritto delle prove penali. Milão: Giuffrè, 2012. COSTA ANDRADE, Manuel da. Sobre as Proibições de prova em processo penal. Coimbra: Coimbra, 2006. FERRAJOLI, Luigi. Direito e razão: teoria do garantismo penal 4a ed. Tradutores Ana Paula Zomer Sica, Fauzi Hassan Choukr, Juarez Tavares e Luiz Flávio Gomes. São Paulo: Revista dos Tribunais, 2014. FESTINGER, Leon. Teoria da dissonância cognitiva. Tradução Eduardo Almeida. Rio de Janeiro: Zahar editores, 1975. GOMES, Luiz Flávio e MACIEL, Silvio. Interceptação Telefônica: Comentários à Lei 9.296, de 24.07.1996. 3a edição. São Paulo: RT, 2014. GOMES FILHO, Antonio Magalhães. “Notas sobre a terminologia da prova (reflexos no processo penal brasileiro)” In YARSHELL, Flávio Luiz e ZANOIDE DE MORAES, Maurício. Estudos em homenagem à Professora Ada Pellegrini Grinover. São Paulo: DPJ Editora, 2005. ____. A motivacão das decisões penais. 2a edição. São Paulo: RT, 2013. GÖSSEL, Karl Heinz. El derecho procesal penal en el Estado de Derecho. Obras completas. Tomo I. Santa Fe: Rubinzal-Culzoni, 2007. GRINOVER, Ada Pellegrini. Provas Ilícitas, Interceptações e Escutas. 1a edição. Brasília: Gazeta Jurídica, 2013. GUERRERO PALOMARES, Salvador. La Imparcialidad Objetiva del Juez Penal: Análisis jurisprudencial y valoración crítica. Pamplona: Aranzadi, 2009. CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 179 179 Os "Hermeneutas dos Grampos": Uma Disfuncionalidade Epistêmica Escutas Telefónicas: da excepcionalidade à vulgaridade. 2a ed. Coimbra: Almedina, 2008. CONPEDI LAW REVIEW \| OÑATI, ESPANHA \| v. 2 \| n. 1 \| p. 163-180 \| JAN/JUN. 2016 180
https://openalex.org/W4396648691	https://journal.unismuh.ac.id/index.php/sigma/article/download/11571/pdf	Indonesian	null	SCAFFOLDING: UPAYA MENGATASI KESULITAN PESERTA DIDIK DALAM MENYELESAIKAN SOAL ARITMATIKA SOSIAL	Sigma /SIGMA	2,023	cc-by-sa	923	24 07 2023 09 08 2023 10/08/2023 145 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial SIGMA: JURNAL PENDIDIKAN MATEMATIKA Volume 15 Nomor 2, Halaman 145 - 157 p-ISSN: 2085-3610, e-ISSN: 2746-7503 https://journal.unismuh.ac.id/index.php/sigma SIGMA: JURNAL PENDIDIKAN MATEMATIKA Volume 15 Nomor 2, Halaman 145 - 157 p-ISSN: 2085-3610, e-ISSN: 2746-7503 https://journal.unismuh.ac.id/index.php/sigma SIGMA: JURNAL PENDIDIKAN MATEMATIKA Volume 15 Nomor 2, Halaman 145 - 157 p-ISSN: 2085-3610, e-ISSN: 2746-7503 https://journal.unismuh.ac.id/index.php/sigma SIGMA: JURNAL PENDIDIKAN MATEMATIKA Volume 15 Nomor 2, Halaman 145 - 157 p-ISSN: 2085-3610, e-ISSN: 2746-7503 https://journal.unismuh.ac.id/index.php/sigma SIGMA: JURNAL PENDIDIKAN MATEMATIKA Volume 15 Nomor 2, Halaman 145 - 157 p-ISSN: 2085-3610, e-ISSN: 2746-7503 https://journal.unismuh.ac.id/index.php/sigma ndonesia , Indonesia ABSTRACT ABSTRACT The existence of this research is aimed at explaining the kinds of mistakes that students often make when working on social arithmetic problems, as well as the application of the scaffolding method as an effort to overcome these errors. This study applies a research method in the form of a literature study, by collecting data, analyzing data, and drawing conclusions. The data used was taken from related articles about various types of errors in solving social arithmetic problems and about the scaffolding method published from 2015 to 2023. There were 10 articles that would be reviewed, all of which were indexed by Google Scholar, and 8 of them sinta accredited. The results of this study indicate that errors when solving social arithmetic problems consist of errors at the stages of reading the questions, understanding the questions, transforming the questions, processing skills, and writing the final results. The type of scaffolding that can be used to overcome errors at the stages of reading questions, transforming questions, processing skills, and writing the final results is level 2 scaffolding, namely explaining and reviewing. While the type of scaffolding that can be used to overcome errors at the stage of understanding the questions is level 2 scaffolding, namely explaining, reviewing, and restructuring. Keyword: Scaffolding; Student Difficulties; Solve Problems; Social Arithmetic SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 Cara Menulis Sitasi 15 (2) 145-157 https://doi.org/10.26618/sigma.v15i2.11571 146 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial 147 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial (Puspaningtyas, 2019) SIGMA: Jurnal Pendidikan Matematika, 15 (2), 2023 Hanik Setyawati1, Putri Nur Malasari2 SIGMA: Jurnal Pendidikan Matematika, 15 (2), 2023 Hanik Setyawati1, Putri Nur Malasari2 SIGMA: Jurnal Pendidikan Matematika, 15 (2), 2023 Hanik Setyawati1, Putri Nur Malasari2 SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 148 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial 149 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial SIGMA: Jurnal Pendidikan Matematika, 15 (2), 2023 Hanik Setyawati1, Putri Nur Malasari2 SIGMA: Jurnal Pendidikan Matematika, 15 (2), 2023 Hanik Setyawati1, Putri Nur Malasari2 SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 150 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial Soal k 151 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial Soal SIGMA: Jurnal Pendidikan Matematika, 15 (2), 2023 Hanik Setyawati1, Putri Nur Malasari2 152 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial Soal SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 Soal SIGMA: Jurnal Pendidikan Matematika, 15 (2), 2023 Hanik Setyawati1, Putri Nur Malasari2 SIGMA: Jurnal Pendidikan Matematika, 15 (2), 2023 Hanik Setyawati1, Putri Nur Malasari2 154 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial . 155 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial × 𝐻𝐽−𝐻𝐵 𝐻𝐵 × 100% × 𝐻𝐽−𝐻𝐵 𝐻𝐵 × 100% × 153 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 Rp.1.260.000,00−Rp.1.200.000,00 Rp.1.200.000,00 × 100% Rp. 60.000,00 Rp.1.200.000,00 × 100% 5% SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 Rp.1.260.000,00−Rp.1.200.000,00 Rp.1.200.000,00 × 100% Rp. 60.000,00 Rp.1.200.000,00 × 100% 5% SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 Rp.1.260.000,00−Rp.1.200.000,00 Rp.1.200.000,00 × 100% Rp. 60.000,00 Rp.1.200.000,00 × 100% 5% Rp.1.260.000,00−Rp.1.200.000,00 Rp 1 200 000 00 × 100% Rp.1.260.000,00−Rp.1.200.000,00 Rp 1 200 000 00 × 100% SIGMA: Jurnal Pendidikan Matematika, 15 (2), 2023 Hanik Setyawati1, Putri Nur Malasari2 SIGMA: Jurnal Pendidikan Matematika, 15 (2), 2023 Hanik Setyawati1, Putri Nur Malasari2 156 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 157 \| Scaffolding: Upaya Mengatasi Kesulitan Peserta Didik dalam Menyelesaikan Soal Aritmatika Sosial SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 SIGMA: Jurnal Pendidikan Matematika, 15 (2), Desember 2023 Hanik Setyawati 1, Putri Nur Malasari 2 Using the concrete-representational-abstract approach to enhance students’ interest in mathematics refers to the primer mathematical skills. Inquiry Co-operation model: An effort to enhance students’ mathematical literacy proficiency. SIGMA: Jurnal Pendidikan Matematika, 15 (2), 2023 Hanik Setyawati1, Putri Nur Malasari2
https://openalex.org/W3173495640	https://www.frontiersin.org/articles/10.3389/fmars.2021.669329/pdf	English	null	Heavy Metal, Rare Earth Element and Pb Isotope Dynamics in Mussels During a Depuration Experiment in the Gulf of Aqaba, Northern Red Sea	Frontiers in marine science	2,021	cc-by	14,625	Heavy Metal, Rare Earth Element and Pb Isotope Dynamics in Mussels During a Depuration Experiment in the Gulf of Aqaba, Northern Red Sea T l B lt b t1 2* Eld d G t H h2 3 d Adi T f t i 1 2 Tal Benaltabet1,2, Eldad Gutner-Hoch2,3 and Adi Torfstein1,2 1 The Fredy and Nadine Herrmann Institute of Earth Sciences, Hebrew University of Jerusalem, Jerusalem, Israel, 2 The Interuniversity Institute for Marine Sciences, Eilat, Israel, 3 School of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel 1 The Fredy and Nadine Herrmann Institute of Earth Sciences, Hebrew University of Jerusalem, Jerusalem, Israel, 2 The Interuniversity Institute for Marine Sciences, Eilat, Israel, 3 School of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Mussels are considered highly efﬁcient marine biomonitors, tracing anthropogenic and natural variations in heavy metals and various organic compounds. While heavy metals depuration processes in biomonitors are of growing interest, less knowledge is available regarding their Pb isotopes and rare earth elements (REEs) accumulation- release dynamics, and their response to short-term anthropogenic and terrigenous perturbations. Here, we report the results of a relocation experiment where a group of mussels (Brachidontes pharaonis) were extracted from a contaminated lagoon in the Gulf of Aqaba, northern Red Sea, and placed in water tanks that were ﬂushed continuously with fresh, uncontaminated seawater. Specimens were removed periodically from the water table over a period of 13 weeks and trace and REEs and Pb isotopic compositions were determined separately for mussel’s shells and soft tissues. The results display a clear decrease over time in the concentrations of various heavy metals and REEs in the soft tissue, in concert with a similar shift in the Pb isotopic compositions toward seawater values. By contrast, the elemental and Pb isotopic composition of the shell presents little change over time. Coupling between the Pb isotopic composition of corresponding soft tissue and shell samples allows back-calculation of the timing and magnitude of abrupt pollution events and presents a novel approach for monitoring short-term pollution events. Nevertheless, given the coastal setting of the studied samples, it is important to consider the effects of terrigenous material on the results. Accordingly, Al-normalized element concentrations, Pb isotopes and calculated Ce anomalies, are used to identify two distinct terrigenous end members controlling the contaminated lagoon and the pristine site. The study demonstrates the potential of using mussels as robust biomonitors of natural and anthropogenic environmental perturbations through the combination between elemental concentrations and the isotopic composition of Pb. Keywords: biomonitoring, mussels, depuration, heavy metals, Pb isotopes, rare earth elements, red sea, pollution Correspondence: Tal Benaltabet tal.benaltabet@mail.huji.ac.il Specialty section: This article was submitted to Marine Pollution, a section of the journal Frontiers in Marine Science Received: 18 February 2021 Accepted: 11 May 2021 Published: 28 June 2021 Edited by: Kenneth Mei Yee Leung, City University of Hong Kong, Hong Kong, SAR China Reviewed by: Chee Kong Yap, Putra Malaysia University, Malaysia Paula Sánchez Marín, Spanish Institute of Oceanography, Spain Correspondence: Tal Benaltabet tal.benaltabet@mail.huji.ac.il Correspondence: Tal Benaltabet tal.benaltabet@mail.huji.ac.il ORIGINAL RESEARCH published: 28 June 2021 doi: 10.3389/fmars.2021.669329 INTRODUCTION Various species have been recognized to serve as reliable biomonitors, including ascidians, polychaetes, copepods, mussels, clams, oysters, snails and ﬁsh (e.g., Hutchinson et al., 1995; Van der Oost et al., 2003; Horiguchi, 2006; Cebrian et al., 2007; Raisuddin et al., 2007; Zhou et al., 2008; Zega et al., 2009; Moloukhia and Sleem, 2011; Carmichael et al., 2012; Tzafriri-Milo et al., 2019). Of these, mussels have been recognized as particularly reliable biomonitors because they have the ability to ﬁlter high volumes of water, allowing for the relatively signiﬁcant accumulation of pollutants in their tissue (Phillips, 1976; Roditi et al., 2000). The use of mussels as biomonitors was introduced in the mid-1970s by Goldberg (1975), who proposed that specimens from coastal and open ocean sites could be beneﬁcial to assess the spatial and temporal trends of various compound concentrations, such as halogenated hydrocarbons, transuranics, heavy metals, and petroleum. This resulted in the establishment of the global program known as “Mussel Watch” (Goldberg et al., 1978; Farrington et al., 1983), which widely uses mussels from the genus Mytilus for marine biomonitoring. The intertidal mussel Brachidontes pharaonis from the family Mytilidae is present in the coasts of the western Paciﬁc Ocean, Indian Ocean, Red Sea, and Mediterranean Sea (Taylor, 1971; Sasekumar, 1974; Barash and Danin, 1986; Morton, 1988). Several studies have demonstrated the metal bio-accumulation capacity of B. pharaonis at polluted and pristine locations along the coasts of the Mediterranean Sea (Göksu et al., 2005; Karayakar et al., 2007; Dar et al., 2018; Hamed et al., 2020). However, to the best of our knowledge, none has presented the depuration dynamics of this species. Despite numerous studies of the accumulation of heavy metals p The Gulf of Aqaba (GoA) is a deep oligotrophic water body connected to the Red Sea across the shallow Straits of Tiran in its southern part (Figure 1). The regional climate is extremely arid (rainfall <30 mm/year), therefore freshwater input from precipitation and ﬂuvial run-oﬀare quantitatively insigniﬁcant, with only a few rain events and associated ﬂashﬂoods each winter, compared to high ﬂuxes of atmospheric dust deposition (Genin et al., 1995; Almogi-Labin et al., 2008; Genin, 2008; Lazar et al., 2008). Two major cities reside in the northern tip of the GoA: Eilat (Israel) and Aqaba (Jordan) (Figure 1). INTRODUCTION elemental retention and depuration dynamics are still of growing interest. In addition to anthropogenic inputs, terrigenous sources such as atmospheric deposition, rivers and sediments are a prominent source of heavy metals and other elements to the marine environment (Turekian, 1977; Bruland et al., 2013). Similar to anthropogenic pollution such as discrete events of oil pollution and industrial or sewage discharge, terrigenous inputs can also have an abrupt nature, with relatively short episodes of increased ﬂuxes such as dust storms (Mahowald et al., 2009; Ternon et al., 2010), ﬂash ﬂoods (Katz et al., 2015), and large-scale sediment resuspension events (Bruland et al., 2008; Torfstein et al., 2020). The terrigenous components may also contribute anthropogenic sourced metals, as these may adsorb heavy metals onto their surfaces during transportation (Nriagu and Pacyna, 1988; Cziczo et al., 2009). Due to the complexity of sampling these and other anthropogenic driven events, examples of the use of mussels as biomonitors for abrupt pulses of marine pollution are scarce. Concentrations of heavy metals in the oceans, perturbated by anthropogenic processes, are particularly high in coastal areas (e.g., Steding et al., 2000; Buck et al., 2005; Xu et al., 2014), especially those proximal to big cities, where the discharge of untreated industrial and human waste is most signiﬁcant (Van Geen et al., 1997; Boyle, 2019). Yet, evaluating the degree of contamination in coastal environments remains challenging because of the complexity in sampling and analyzing water samples and the short residence times of some contaminants in seawater following pollution events. Alternatively, it is possible to use the abundances of contaminants in solid phases within the marine environment as temporal and spatial tracers of pollution. These could be suspended or deposited sediment particles that may adsorb dissolved elements onto their surface from surrounding water, or living organisms that actively circulate seawater and hence incorporate dissolved elements internally, i.e., biomonitors (Rainbow, 2002). An advantage of using marine biomonitors is the ease of processing and analyses (relative to seawater) and their supposedly short response time to local changes in seawater heavy metal contents, whereas solid sediment particles tend to accumulate surrounding elements overtime but do not respond to short term perturbations in the seawater composition. Citation: Benaltabet T, Gutner-Hoch E and Torfstein A (2021) Heavy Metal, Rare Earth Element and Pb Isotope Dynamics in Mussels During a Depuration Experiment in the Gulf of Aqaba, Northern Red Sea. Front. Mar. Sci. 8:669329. doi: 10.3389/fmars.2021.669329 June 2021 \| Volume 8 \| Article 669329 1 Frontiers in Marine Science \| www.frontiersin.org Metals Depuration Dynamics in Mussels Benaltabet et al. Frontiers in Marine Science \| www.frontiersin.org INTRODUCTION The location of the Interuniversity Institute (IUI) for Marine Sciences, where the mussels were relocated to, is marked by an empty triangle in panel (B). The location of the Peace Lagoon is presented in panel (C), including the exact site of sampling (white triangle). Map taken from Google Maps. been used to trace the source of environmental pollution in coastal environments (Labonne et al., 2001, 1998; Richardson et al., 2001; Dang et al., 2015). (Herut et al., 1999; Chase et al., 2011, 2006; Chien et al., 2019). The shell and soft tissues were analyzed for their heavy metal and REEs concentrations and Pb isotopic composition over a period of 13 weeks in order to demonstrate the depuration trends from highly contaminated values to the low and natural baseline. The distribution of Rare Earth Elements (REEs) in seawater has been shown to be a useful proxy for quantifying and distinguishing between terrigenous and anthropogenic inputs to the oceans (de Baar et al., 1985; Elderﬁeld, 1988; Hatje et al., 2016). In recent years, the increasing use of REEs in industrial (La, Ce, Pr, Sm, Nd, and Tm) and medical (Gd) ﬁelds have resulted in their intrusion into the marine environment and subsequent accumulation in marine biota (Gwenzi et al., 2018; Squadrone et al., 2019). The low concentrations of REEs in seawater hamper their application as in-situ marine- environmental monitors. By contrast, elevated abundances in various marine organisms renders them useful biomonitor for REEs in the marine environment (Bonnail et al., 2017; Ma et al., 2019; Wang et al., 2019). INTRODUCTION Marine based commerce activity, together with the environmental impacts of big cities, are often in conﬂict with the well-being of natural resources, making the GoA marine habitats highly sensitive to anthropogenic contamination (Abelson et al., 1999; Wielgus et al., 2004). A man-made inland lagoon (the “Peace Lagoon”, Figure 1) was constructed to support the development of tourism along the seashore. Yet overestimates of the water exchange rates with the open sea had resulted in an almost isolated inland lagoon with very little water exchange with the open waters. Consequently, the lagoon accumulated contaminants and organic material over time, and its interstitial waters became anoxic. At present, the lagoon is considered contaminated and unﬁt for recreation activities. Several previous studies have provided information about metals in, or near, the GoA, ranging between analyses of seawater (Chase et al., 2011, 2006; Chien et al., 2019; Benaltabet et al., 2020), atmospheric dust (Chen et al., 2008; Torfstein et al., 2017; Chien et al., 2019) and studies of surface sediments (Al-Taani et al., 2014; Barakat et al., 2015). Despite these eﬀorts, we still lack a clear understanding of the sources of contamination, their spread range and their long and short-term impact on the GoA and its marine ecosystem. In addition to the use of heavy metal distributions as tracers of anthropogenic contamination processes, lead (Pb) isotopes (204Pb, 206Pb, 207Pb, and 208Pb), whose relative abundances often diﬀer between natural and anthropogenic sources, make it possible to infer the source of Pb pollution in the environment (e.g., Boyle et al., 1986; Erel et al., 2002; Komárek et al., 2008). Indeed, the Pb isotopic composition of mussels has previously Despite numerous studies of the accumulation of heavy metals in mussels (e.g., Chan, 1989; Naimo, 1995; Yap et al., 2003; Fung et al., 2004; Zuykov et al., 2013; Liu and Wang, 2016), their June 2021 \| Volume 8 \| Article 669329 Frontiers in Marine Science \| www.frontiersin.org 2 Metals Depuration Dynamics in Mussels Benaltabet et al. FIGURE 1 \| (A) Location map. The location of the Interuniversity Institute (IUI) for Marine Sciences, where the mussels were relocated to, is marked by an empty triangle in panel (B). The location of the Peace Lagoon is presented in panel (C), including the exact site of sampling (white triangle). Map taken from Google Maps. FIGURE 1 \| (A) Location map. Frontiers in Marine Science \| www.frontiersin.org Experimental Setup and Sample Processing A batch of 18 adult (∼3 cm in length) Red Sea mussels B. pharaonis were collected from the Peace Lagoon in the north beach of Eilat (32◦54′84.4′′N 34◦96′82.3′′E) and translocated to a running seawater tank in the IUI (Figure 1) for the rest of the depuration experiment. The water tank volume was 120 liters with a ﬂowing water rate of 8.5 liters per minute. A pair of mussels was removed every week during the ﬁrst 6 weeks (Tables 1–3). The two last samples were removed after 12 and 13 weeks. A pair of mussels was sampled immediately after extraction from the Peace Lagoon (i.e., at “day 0”, without being exposed to the water tank). In this study, we explore the depuration process of heavy metals, REEs and Pb isotopes in mussels (B. pharaonis). To this end, a native population living in the Peace Lagoon (Figure 1) were relocated to pristine seawater tanks in the Interuniversity Institute (IUI) for Marine Sciences (Figure 1) where heavy metals concentrations were shown to be lower compared with the north shore, in the vicinity of the lagoon June 2021 \| Volume 8 \| Article 669329 3 Metals Depuration Dynamics in Mussels Benaltabet et al. Benaltabet et al. Metals Depuration Dynamics in Mussels TABLE 1 \| Heavy metal concentrations (µg/g dry weight) in mussel shells and soft tissue. Experimental Setup and Sample Processing Sample Day Al V Cr Mn Fe Co Ni Cu Zn Cd Pb Soft tissue EK-5 0 1148 3.1 2.1 28.7 1267 1.05 2.4 19.2 53.4 2.2 1.4 EK-6 0 735 1.6 1.3 33.6 725 0.46 1.1 21.3 41.7 0.6 0.7 EK-7 5 17.7 0.5 0.8 5.4 106 0.30 0.4 27.2 76.7 0.6 0.4 EK-8 5 83.8 0.7 1.4 42.8 183 0.31 0.5 28.1 66.3 0.6 0.6 EK-9 13 240 2.0 3.7 22.6 484 0.58 5.2 81.5 116 2.5 1.6 EK-10 13 135 2.3 3.9 13.5 487 0.59 2.0 28.2 187 3.8 1.1 EK-13 21 66.9 1.5 1.1 8.2 206 0.33 1.7 26.1 79.9 1.0 0.4 EK-14 21 92.1 1.4 1.1 7.1 159 0.26 2.1 26.6 63.2 1.5 0.4 EK-15 29 60.4 0.9 1.0 4.4 335 0.27 1.5 29.2 93.5 2.0 1.2 EK-16 29 136 1.0 1.4 5.8 222 0.30 1.3 54.4 59.4 1.2 0.5 EK-3 35 87.9 1.0 2.6 10.9 354 0.40 2.3 25.9 89.0 1.1 1.4 EK-4 35 55.5 0.8 0.5 9.8 119 0.16 0.8 5.5 16.2 0.5 0.3 EK-11 43 63.7 0.7 0.8 3.0 115 0.16 0.4 22.5 27.0 0.4 0.2 EK-12 43 50.7 0.8 0.9 5.5 133 0.28 0.6 93.5 98.1 1.1 0.4 EK-2 84 28.0 0.5 0.7 1.9 94.3 0.15 0.5 43.7 28.1 0.7 0.2 EK-17 91 34.1 0.6 0.5 2.6 105 0.15 0.5 19.4 52.9 0.9 0.5 EK-18 91 24.2 0.6 0.6 2.4 79.7 0.09 0.4 14.3 19.4 0.2 0.3 Shell EK-20 0 46.3 0.4 n.d 123 336 0.15 n.d. 0.6 n.d. 0.007 0.4 EK-24 0 38.4 0.3 n.d 68.1 247 0.12 n.d. 0.5 n.d. 0.003 0.3 EK-22 21 18.8 0.2 n.d 9.2 84.7 0.06 n.d. 0.5 n.d. 0.004 0.2 EK-19 35 4.9 0.1 n.d 11.2 14.4 0.06 n.d. 0.3 n.d. 0.007 0.3 EK-21 35 16.9 0.1 n.d 26.8 58.2 0.07 n.d. 0.4 n.d. 0.002 0.2 EK-23 42 4.6 0.1 n.d 17.3 51.8 0.05 n.d. 0.5 n.d. 0.002 0.2 EK-25 84 25.3 0.3 n.d 12.1 175 0.09 0.33 0.6 n.d. 0.004 0.3 TABLE 2 \| Rare earth element concentrations (ng/g dry weight) in mussel shells and soft tissue. Experimental Setup and Sample Processing Sample Day La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb Lu Soft tissue EK-5 0 1180 2486 255 985 178 38.7 172 23.3 126 24.9 64.1 9.4 54.6 7.5 EK-6 0 610 1222 141 525 103 19.8 88.5 12.5 61.4 13.3 33.7 4.5 25.9 4.4 EK-7 5 80.0 128 10.2 39.6 8.3 1.7 8.2 1.7 6.6 1.7 3.4 0.9 2.9 0.7 EK-8 5 133 262 23.4 89.9 18.6 2.9 17.5 2.3 11.7 2.4 6.4 0.8 4.5 0.4 EK-9 13 284 782 66.2 279 48.0 10.4 51.3 6.7 33.4 6.9 17.1 2.2 12.6 1.6 EK-10 13 321 958 71.8 302 50.7 9.6 52.2 5.6 29.1 5.0 13.8 1.5 9.9 1.2 EK-13 21 132 228 26.9 105 21.0 3.9 19.7 2.5 11.8 2.8 6.9 0.9 4.3 0.7 EK-14 21 108 213 21.5 88.1 14.1 2.5 14.5 1.7 11.0 2.2 5.2 0.7 3.5 0.4 EK-15 29 94.6 206 16.4 62.6 11.4 2.6 11.9 1.5 9.1 1.9 5.7 0.8 3.9 0.6 EK-16 29 163 321 28.3 106 21.7 4.3 19.2 2.4 12.9 2.9 8.2 1.1 7.0 0.9 EK-3 35 130 277 23.0 82.1 14.8 2.8 13.0 2.3 10.0 2.1 6.4 0.8 5.0 0.8 EK-4 35 78.0 120 12.1 49.3 11.2 2.1 8.0 1.4 8.4 1.4 3.8 0.5 2.7 0.5 EK-11 43 87.6 108 12.1 48.7 11.9 1.7 10.1 1.4 7.3 1.4 4.3 0.5 3.0 0.5 EK-12 43 100 132 13.2 51.3 9.9 2.0 12.7 1.2 7.2 1.4 3.7 0.3 2.6 0.3 EK-2 84 66.0 102 9.9 37.3 7.0 1.0 7.0 0.9 3.9 1.1 2.3 0.3 1.2 0.2 EK-17 91 62.0 96.0 9.0 32.8 6.1 1.1 6.0 0.7 4.1 0.8 2.3 0.3 1.6 n.d. EK-18 91 67.7 68.1 8.5 28.8 7.6 0.8 7.2 0.9 5.0 1.2 2.4 0.2 2.2 n.d. Shell EK-20 0 30.0 86.7 5.8 21.6 4.1 n.d. 3.8 0.5 2.1 0.6 0.8 0.1 0.7 0.2 EK-24 0 27.0 74.1 4.5 19.6 3.7 0.8 3.5 0.4 2.0 0.6 1.5 0.2 0.8 0.1 EK-22 21 27.2 64.5 4.6 16.9 2.6 0.7 4.1 0.5 1.8 0.6 1.4 0.2 0.8 0.1 EK-19 35 14.7 38.6 2.4 8.2 2.6 n.d. 1.3 0.2 0.7 0.1 0.5 0.1 n.d. n.d. EK-21 35 18.4 47.1 3.2 10.4 3.1 n.d. 1.9 0.2 0.8 0.3 1.0 n.d. 0.5 n.d. EK-23 42 13.4 35.9 2.0 9.7 3.7 n.d. 1.3 0.2 n.d. 0.2 0.6 0.1 n.d. n.d. Experimental Setup and Sample Processing EK-25 84 25.2 65.7 4.4 16.1 2.5 0.8 3.5 0.6 1.8 0.6 1.4 0.3 1.2 n.d. TABLE 1 \| Heavy metal concentrations (µg/g dry weight) in mussel shells and soft tissue. TABLE 2 \| Rare earth element concentrations (ng/g dry weight) in mussel shells and soft tissue. Sample Day La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb Lu Soft tissue EK-5 0 1180 2486 255 985 178 38.7 172 23.3 126 24.9 64.1 9.4 54.6 7.5 EK-6 0 610 1222 141 525 103 19.8 88.5 12.5 61.4 13.3 33.7 4.5 25.9 4.4 EK-7 5 80.0 128 10.2 39.6 8.3 1.7 8.2 1.7 6.6 1.7 3.4 0.9 2.9 0.7 EK-8 5 133 262 23.4 89.9 18.6 2.9 17.5 2.3 11.7 2.4 6.4 0.8 4.5 0.4 EK-9 13 284 782 66.2 279 48.0 10.4 51.3 6.7 33.4 6.9 17.1 2.2 12.6 1.6 EK-10 13 321 958 71.8 302 50.7 9.6 52.2 5.6 29.1 5.0 13.8 1.5 9.9 1.2 EK-13 21 132 228 26.9 105 21.0 3.9 19.7 2.5 11.8 2.8 6.9 0.9 4.3 0.7 EK-14 21 108 213 21.5 88.1 14.1 2.5 14.5 1.7 11.0 2.2 5.2 0.7 3.5 0.4 EK-15 29 94.6 206 16.4 62.6 11.4 2.6 11.9 1.5 9.1 1.9 5.7 0.8 3.9 0.6 EK-16 29 163 321 28.3 106 21.7 4.3 19.2 2.4 12.9 2.9 8.2 1.1 7.0 0.9 EK-3 35 130 277 23.0 82.1 14.8 2.8 13.0 2.3 10.0 2.1 6.4 0.8 5.0 0.8 EK-4 35 78.0 120 12.1 49.3 11.2 2.1 8.0 1.4 8.4 1.4 3.8 0.5 2.7 0.5 EK-11 43 87.6 108 12.1 48.7 11.9 1.7 10.1 1.4 7.3 1.4 4.3 0.5 3.0 0.5 EK-12 43 100 132 13.2 51.3 9.9 2.0 12.7 1.2 7.2 1.4 3.7 0.3 2.6 0.3 EK-2 84 66.0 102 9.9 37.3 7.0 1.0 7.0 0.9 3.9 1.1 2.3 0.3 1.2 0.2 EK-17 91 62.0 96.0 9.0 32.8 6.1 1.1 6.0 0.7 4.1 0.8 2.3 0.3 1.6 n.d. EK-18 91 67.7 68.1 8.5 28.8 7.6 0.8 7.2 0.9 5.0 1.2 2.4 0.2 2.2 n.d. Shell EK-20 0 30.0 86.7 5.8 21.6 4.1 n.d. 3.8 0.5 2.1 0.6 0.8 0.1 0.7 0.2 EK-24 0 27.0 74.1 4.5 19.6 3.7 0.8 3.5 0.4 2.0 0.6 1.5 0.2 0.8 0.1 EK-22 21 27.2 64.5 4.6 16.9 2.6 0.7 4.1 0.5 1.8 0.6 1.4 0.2 0.8 0.1 EK-19 35 14.7 38.6 2.4 8.2 2.6 n.d. Heavy Metals and REEs Elemental abundances were measured in a total of 17 soft tissue and seven shell samples (Tables 1, 2). A large suite of elements displays a general ongoing decrease in soft tissue concentrations with time from day 0 to 91. These include Al, V, Cr, Mn, Fe, Co, Ni (Figure 2), and the REEs (Table 2). Other elements show a more moderate decrease (Pb, Cd, and Zn) or a negligible change with time (Cu) (Figure 2). Regardless of the general trend, all elements (besides Mn and Cu) displayed a sharp increase in soft tissue concentrations at day 13, after which concentrations continued to gradually decrease. Compared to the soft tissue, the shell fraction concentration presented little change over time in all elements apart from Mn, which shows a coeval decrease in both fractions. Data Treatment The uncertainty for elemental concentrations is based on the standard deviation of duplicate samples (i.e., two biological replicates), except when duplicates were not available, in which case the relative uncertainty average of the rest of the samples was applied (Tables 1, 2 and Figure 2). The detection limits were deﬁned as three-fold the standard deviation of full procedural blanks processed and analyzed with the samples (n = 3). Samples below blank or detection limit values, the higher of the two, are marked by “nd” in Tables 1, 2. The uncertainty for Pb isotopic ratios was evaluated on the basis of a biological duplicate processing and analyses (sample from day 0 for the soft tissue and sample from day 35 for the shell). The standard deviation from the average of the duplicates was applied to the rest of the samples (Table 3 and Figure 3). The soft tissue was digested in acid cleaned Teﬂon (PFA) beakers (Savillex, United States) through several cycles of heated H2O2–HNO3 mixtures, while the shells were digested using 1 N HNO3. All reagents used in this study were ultrapure solutions (commercial or in-house double distilled) and their concentrations were adjusted with ultrapure MQ water. Generally, two biological replicates were processed and analyzed for soft tissue elemental abundances while one individual was used for soft tissue and shell Pb isotopic composition. The number of replicates used for elemental abundances and Pb isotopic composition at each sampling date is given in Tables 1–3. Analyses of Pb Isotopic Composition a yses o b so op c Co pos o The remaining solution after ICP-MS analysis was dried, re- dissolved in 1 N HBr, and then Pb puriﬁed using standard ion chromatography (e.g., Torfstein et al., 2018) and the details are summarized hereafter. The HBr solution was taken through a series of puriﬁcation steps to separate a puriﬁed Pb fraction. First, 100 µl of AG1X-8 R⃝100–200 mesh anion resin were loaded onto Teﬂon micro-columns and cleaned over repeated cycles of MQ water and 6 N HCl. The samples were then loaded to the columns and the matrix was removed using 1 N HBr and 2 N HCl. Pb was eluted into Teﬂon beakers using 6 N HCl. The samples were then dried and re-dissolved in 3% HNO3 doped to 50 ppb Tl to account for instrument mass fractionation. These aliquots were analyzed for their Pb isotopic composition on a Neptune Plus multi collector ICP-MS, together with repeated measurements of the NIST SRM-981 standard which was used for accuracy and instrumental drifts corrections. After removal from the water tank, the samples were carefully transferred to a clean lab where they were processed in a class 100 environment. Samples were ﬁrst rinsed in ultrapure MQ water (18.2 M cm) and sonicated over several cycles to remove external debris and particles. The shell and soft tissue were then separated and freeze-dried. After weighing the dry samples, the shell was gently leached with 0.05 N HNO3, to remove the outer rim that could have potentially been contaminated during the sampling, or contain external residue. We are aware that this might also be the part of the shell that could have formed during the experiment, but in terms of mass balance, this would be negligible and probably not observed when measuring the untreated bulk shell sample. Experimental Setup and Sample Processing 1.3 0.2 0.7 0.1 0.5 0.1 n.d. n.d. EK-21 35 18.4 47.1 3.2 10.4 3.1 n.d. 1.9 0.2 0.8 0.3 1.0 n.d. 0.5 n.d. EK-23 42 13.4 35.9 2.0 9.7 3.7 n.d. 1.3 0.2 n.d. 0.2 0.6 0.1 n.d. n.d. EK-25 84 25.2 65.7 4.4 16.1 2.5 0.8 3.5 0.6 1.8 0.6 1.4 0.3 1.2 n.d. June 2021 \| Volume 8 \| Article 669329 4 Frontiers in Marine Science \| www.frontiersin.org Metals Depuration Dynamics in Mussels Benaltabet et al. TABLE 3 \| Pb isotopes in mussel shells and soft tissue. Sample Day 206Pb/207Pb SD 208Pb/206Pb SD Soft tissue EK-5 0 1.201 0.003 2.052 0.001 EK-6 0 1.197 0.003 2.053 0.001 EK-8 5 1.186 0.003 2.067 0.001 EK-10 13 1.192 0.003 2.060 0.001 EK-13 21 1.187 0.003 2.066 0.001 EK-16 29 1.184 0.003 2.069 0.001 EK-4 35 1.174 0.003 2.078 0.001 EK-11 43 1.177 0.003 2.076 0.001 EK-2 84 1.184 0.003 2.068 0.001 EK-18 91 1.176 0.003 2.077 0.001 Shell EK-24 0 1.199 0.005 2.051 0.005 EK-22 21 1.197 0.005 2.054 0.005 EK-19 35 1.196 0.005 2.055 0.005 EK-21 35 1.203 0.005 2.048 0.005 EK-23 43 1.191 0.005 2.060 0.005 EK-25 84 1.197 0.005 2.053 0.005 TABLE 3 \| Pb isotopes in mussel shells and soft tissue. of the intra- and inter- session drift was achieved through the analyses of an in-house standard solution every 10–15 samples, yielding a long term precision of <2% (2σ). The results were corrected for procedural blank values. Analyses of Trace and Rare Earth Elements Concentrations The digested samples were dried on a hotplate, re-dissolved in 3% HNO3, and analyzed for their trace (Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd, Pb, and Th) and REE (La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu) abundances on an Agilent 7,500cx ICP-MS at the Institute of Earth Sciences, Hebrew University of Jerusalem. A multi-elemental standard solution (3% HNO3 matrix) was used for instrumental signal calibration. Each sample was spiked online with internal standards (Sc, Re, and Rh) to follow and correct for instrumental drifts. Additional monitoring June 2021 \| Volume 8 \| Article 669329 Frontiers in Marine Science \| www.frontiersin.org 5 Metals Depuration Dynamics in Mussels Benaltabet et al. URE 2 \| Elemental concentrations (µg/g dry weight) in the soft tissue (black squares) and shell (gray circles). The solid (black) and dashed (blue) curves represent wer law ﬁts for the elemental abundances from days 0–91 and 13–91, respectively, with the corresponding R2 values. Error bars mark the standard deviation of licate analyses (see section “Data treatment” for details). Ce is presented as ng/g (dry weight) and its temporal evolution is similar to that of the other REEs ble 2). I l d l h l l FIGURE 2 \| Elemental concentrations (µg/g dry weight) in the soft tissue (black squares) and shell (gray circles). The solid (black) and dashed (blue) curves represent power law ﬁts for the elemental abundances from days 0–91 and 13–91, respectively, with the corresponding R2 values. Error bars mark the standard deviation of duplicate analyses (see section “Data treatment” for details). Ce is presented as ng/g (dry weight) and its temporal evolution is similar to that of the other REEs (Table 2) to 2.077, respectively. At day 13, similar to the elemental abundance patterns, the 206Pb/207Pb and 208Pb/206Pb soft tissue compositions shift sharply toward higher and lower ratios, respectively. Thereafter, the isotopic ratios continue their initial trend and shift toward lower 206Pb/207Pb ratios and Pb Isotopes The isotopic composition of Pb was measured on a subset of 10 soft tissue and 6 shell samples (Table 3 and Figure 3). The soft tissue samples display a gradual shift in 206Pb/207Pb and 208Pb/206Pb compositions from 1.199 to 1.176 and 2.052 June 2021 \| Volume 8 \| Article 669329 Frontiers in Marine Science \| www.frontiersin.org 6 Metals Depuration Dynamics in Mussels Benaltabet et al. FIGURE 3 \| Pb isotopic compositions in the soft tissue (black squares) and shell (gray circles) for (A) 208Pb/206Pb and (B) 206Pb/207Pb. The solid (black) and dashed (blue) curves represent power law ﬁts for the elemental abundances from days 0–91 and 13–91, respectively, with the corresponding R2 values. Error bars mark the standard deviation of one duplicate analyses, which was applied to the rest of the samples (see section “Data treatment” for details). The pale blue bar represents the range of seawater Pb isotopic compositions in the GoA (Benaltabet et al., 2020). FIGURE 3 \| Pb i t i iti i th ft ti (bl k ) d (Tables 1, 2 and Figure 2), from the moment they were moved from the lagoon environment to the water tanks. Although not measured directly in this study, seawater elemental concentrations in the vicinity of the IUI and the lagoon can be estimated based on previous studies (Chase et al., 2011, 2006; Chien et al., 2019). Previously reported surface dissolved Al, Mn, Fe, Co, Cu, Zn, Cd, and Pb concentrations near the IUI coast are generally lower than surface concentrations at the north shore near the lagoon and are more similar to surface concentrations measured at the open sea (Figure 1, Station A), further away from shore (Supplementary Table 1). Moreover, Herut et al. (1999) have shown that native gastropods (Cellana rota) in the vicinity of the IUI presented lower levels of soft tissue Zn, Fe, Cu, and Mn when compared with specimens from the GoA north shore. These observations suggest that the decrease in soft tissue concentrations over time represents the elemental depuration as a result of the relocation from the lagoon to the pristine IUI environment. The decrease in elemental abundances is observed 5 days after the relocation to the water tanks, as soft tissue concentrations decreased by 51 to 86% between days 0 and 5. Pb Isotopes These rates are similar to the rapid depuration rates demonstrated by the mussel Perna virdis (Yap et al., 2003) and the bivalve Paphia undulata (El-Gamal, 2011). No signiﬁcant decrease trend is observed in the mussel’s shells because the fraction of the shell that formed during the experiment is negligible relative to its bulk weight. Hence, it appears that while the soft tissue can biomonitor short-term changes in the organism’s environment, the shell represents long-term chronic conditions. It is recognized that the elemental evolution trends are noisy, most likely reﬂecting the combined natural variability between diﬀerent specimens, their initial heterogeneity in the lagoon and their individual response to the relocation which could be inﬂuenced by their diﬀerent dry weights (ranging between 10–63 mg) and size, and possibly even their position in the water tank (Phillips, 1980). FIGURE 3 \| Pb isotopic compositions in the soft tissue (black squares) and shell (gray circles) for (A) 208Pb/206Pb and (B) 206Pb/207Pb. The solid (black) and dashed (blue) curves represent power law ﬁts for the elemental abundances from days 0–91 and 13–91, respectively, with the corresponding R2 values. Error bars mark the standard deviation of one duplicate analyses, which was applied to the rest of the samples (see section “Data treatment” for details). The pale blue bar represents the range of seawater Pb isotopic compositions in the GoA (Benaltabet et al., 2020). To better understand the dynamics of metal concentrations in the soft tissue, the Pearson correlation coeﬃcient (r) was examined between all studied metals in the samples collected after the relocation at days 5–91 (Table 4), as any deviation from a linear relationship between metals might suggest a diﬀerent depuration dynamic modulating soft tissue metal concentrations. Soft tissue Al, V, Cr, Fe, Co, Ni, Zn, Cd, and Pb concentrations between days 5 and 91 present positive correlations with one another (r = 0.76–0.98, p < 0.03). When the day 0 samples, which represent the lagoon compositions, are also considered in the calculation, the correlation yields signiﬁcantly lower values (Supplementary Table 2). Regardless of the calculation time span, it appears that the soft tissue passively records the ambient seawater composition of these metals, without an apparent preference. Inversely, poor correlations are presented for Mn and Cu when compared with the other metals suggesting that there might be other controls on their soft tissue abundances. higher 208Pb/206Pb ratios. DISCUSSION Soft tissue Cu concentrations do not present the same depuration trend similar to the rest of the metals, as they remain relatively constant over the course of the relocation experiment (Figure 2). This could be the result of chronically high dissolved Cu concentrations at the GoA western coast, which may be Pb Isotopes By contrast, and similar to elemental abundance patterns, the shell compositions display little variation regardless of the time the sample was exposed to pristine seawater. Moreover, this unchanging composition corresponds, within uncertainty, to the composition measured in the soft tissue at the start of the experiment, which represents the lagoon environment. Al-Normalized Ratios, REE, and Pb Isotopes as Proxies for Terrigenous Inputs Terrigenous inputs such as rivers, atmospheric aerosols and terrestrial and marine sediments are the main sources of metals to the oceans (Turekian, 1977; Bruland et al., 2013) with Al being a prominent proxy used to evaluate terrigenous ﬂuxes to the marine environment (Baker et al., 2016; Jickells et al., 2016). To better characterize the controls of terrigenous components over the elemental compositions discussed here, we present the mussel’s soft tissue metal concentrations normalized to Al (Supplementary Figure 1). The Al-normalized metal ratios allows a better estimation of relative elemental depletion/enrichment in various organic (Bekteshi et al., 2015) and inorganic phases (Shelley et al., 2015; Jickells et al., 2016). Following the relocation of the mussels from the lagoon to the water tanks, all metal/Al ratios between days 0 and 5 displayed a signiﬁcant increase and remained relatively constant thereafter. This shift clearly depicts the transition from the terrigenous dominated and Al rich shallow lagoon, where exchange with open seawater is limited, to the relatively Al- depleted seawater environment. Moreover, while most metal/Al ratios shifted by up to two order of magnitude, the increase in Fe/Al ratios was smaller (Supplementary Figure 1), reﬂecting the joint association of both Fe and Al with terrigenous material. For comparison, the Al-normalized Th abundances remains relatively stable throughout the entire duration of the experiment, reﬂecting the strong association of Th with terrigenous inputs. Out of the entire suite of metals studied, Mn is the only metal that presents higher or similar concentrations in the shell compared to the soft tissue (besides Al and Fe, which show similar concentrations at day 84). In addition, only Mn shell and soft tissue concentrations are signiﬁcantly correlated (r = 0.97, p < 0.01), implying that they are both modulated by the same mechanism. In their study of the mussel M. eduils, Freitas et al. (2016) have shown that shell Mn contents are not directly controlled by ambient dissolved and particulate Mn concentrations nor by kinetic eﬀects, but are mediated by a physiological mechanism associated with the extra-pallial ﬂuid (EPF). It is possible that the decrease in shell Mn after the The transition to a pristine environment is further illustrated in the mussel’s soft tissue Pb isotopic composition, which is considered in the context of previously reported GoA seawater Pb compositions (Figure 4) and its respective end members (Lee et al., 2015; Chien et al., 2019; Benaltabet et al., 2020). Long-Term Depuration Trends in Elemental Abundances Moreover, whi ratios shifted by up to two order of magnitude Fe/Al ratios was smaller (Supplementary Figu the joint association of both Fe and Al with terr For comparison the Al normalized Th abun coefﬁcient values (r) for soft tissue trace elements concentrations for samples collected between days 5 and 91 with correspondin TABLE 4 \| Pearson correlation coefﬁcient values (r) for soft tissue trace elements concentrations for samples collected between days 5 and 91 with corresponding p-values in brackets. relocation to the water tanks is coupled to the decrease in soft tissue Mn and related to the connections between the soft tissue and the shell through the EPF (Crenshaw, 1972; Freitas et al., 2016). This might also explain the lack of correlation between soft tissue Mn and other metals, given the high aﬃnity of the EPF to Mn2+ (Yin et al., 2005). Frontiers in Marine Science \| www.frontiersin.org Long-Term Depuration Trends in Elemental Abundances rota (Herut et al., 1999) near the IUI coast, while Cu concentrations are ∼15–40 times higher. Moreover, while Cr, Mn, Ni, Cd, and Pb soft tissue concentrations at day 91 are similar to the average natural values reported for in- situ B. pharaonis by Hamed et al. (2020) in the Mediterranean Sea, Fe, Cu, and Zn are higher by a factor of ∼12, 15, and 10, respectively. It is conceivable that when exposed to high ambient levels of bio-essential metals, B. pharaonis will actively retain optimal high soft tissue concentrations (White and Rainbow, 1982; Amiard et al., 2006). Out of the entire suite of metals studied, Mn is the only metal that presents higher or similar concentrations in the shell compared to the soft tissue (besides Al and Fe, which show similar concentrations at day 84) In addition only Mn relocation to the water tanks is coupled to the tissue Mn and related to the connections betwe and the shell through the EPF (Crenshaw, 19 2016). This might also explain the lack of correla tissue Mn and other metals, given the high aﬃn Mn2+ (Yin et al., 2005). Al-Normalized Ratios, REE, and Isotopes as Proxies for Terrigen Inputs Terrigenous inputs such as rivers, atmosphe terrestrial and marine sediments are the m metals to the oceans (Turekian, 1977; Brula with Al being a prominent proxy used to eval ﬂuxes to the marine environment (Baker et a et al., 2016). To better characterize the contro components over the elemental compositions we present the mussel’s soft tissue metal normalized to Al (Supplementary Figure 1). Th metal ratios allows a better estimation of re depletion/enrichment in various organic (Bekt and inorganic phases (Shelley et al., 2015; Jick Following the relocation of the mussels from the water tanks, all metal/Al ratios between displayed a signiﬁcant increase and remained re thereafter. This shift clearly depicts the tran terrigenous dominated and Al rich shallow exchange with open seawater is limited, to t depleted seawater environment. Long-Term Depuration Trends in Elemental Abundances To a ﬁrst order, all metals (except for Cu) and REEs studied here display a gradual decrease in the soft tissue concentrations June 2021 \| Volume 8 \| Article 669329 Frontiers in Marine Science \| www.frontiersin.org 7 Metals Depuration Dynamics in Mussels Benaltabet et al. TABLE 4 \| Pearson correlation coefﬁcient values (r) for soft tissue trace elements concentrations for samples collected between days 5 and 91 with p-values in brackets. Al V Cr Mn Fe Co Ni Cu Z V 0.93 (0.0007) Cr 0.95 (0.0004) 0.88 (0.004) Mn 0.44 (0.3) 0.37 (0.4) 0.55 (0.2) Fe 0.98 (0.00001) 0.88 (0.004) 0.95 (0.0003) 0.45 (0.3) Co 0.95 (0.0003) 0.89 (0.003) 0.95 (0.0003) 0.68 (0.06) 0.93 (0.0007) Ni 0.95 (0.0003) 0.97 (0.00007) 0.92 (0.001) 0.36 (0.4) 0.94 (0.0006) 0.90 (0.003) Cu 0.44 (0.3) 0.32 (0.4) 0.38 (0.3) −0.03 (0.9) 0.35 (0.4) 0.37 (0.4) 0.26 (0.5) Zn 0.96 (0.0001) 0.90 (0.002) 0.92 (0.001) 0.60 (0.1) 0.92 (0.001) 0.97 (0.00006) 0.87 (0.005) 0.48 (0.2) Cd 0.97 (0.00008) 0.92 (0.001) 0.91 (0.002) 0.32 (0.4) 0.94 (0.0004) 0.88 (0.003) 0.93 (0.0008) 0.50 (0.2) 0.92 Pb 0.90 (0.002) 0.76 (0.03) 0.90 (0.002) 0.52 (0.2) 0.96 (0.0001) 0.88 (0.004) 0.86 (0.007) 0.16 (0.7) 0.83 similar to those at the lagoon. However, as established before (Supplementary Table 1; Chase et al., 2011) high dissolved Cu concentrations are less plausible and a biological mechanism through which the soft tissue retains Cu might explain the constant Cu concentrations throughout the experiment. Similar observations were made by Lorenzo et al. (2003), who transferred mussels (Mytilus edulis) from a Cu enriched environment to clean seawater and reported low Cu depuration rates relative to the model expected rates, owing to biological regulation of Cu. It is worth noting that despite the diﬀerence in species, the ﬁnal soft tissue Cu concentrations reported by Lorenzo et al. (2003) were similar to those presented here (Figure 2). Furthermore, in a metal depuration experiment performed on the Mediterranean mussel Mytilus galloprovincialis, relatively low Cu depuration rates were observed (Anacleto et al., 2015). A possible explanation is that mollusks might actively retain high levels of Cu (and Zn) through metallothioneins, as these are biologically essential metals (Amiard et al., 2006). Similarly, soft tissue Zn, Cd, Fe, and Mn concentrations at day 91 are within the same order of magnitude as the values reported for the gastropod C. Al-Normalized Ratios, REE, and Pb Isotopes as Proxies for Terrigenous Inputs These include seaﬂoor sediments, open Red Sea waters and aerosols, June 2021 \| Volume 8 \| Article 669329 8 Metals Depuration Dynamics in Mussels Benaltabet et al. the latter being the most signiﬁcant source of anthropogenic Pb to the GoA (Chien et al., 2019; Benaltabet et al., 2020). The initial Pb isotopic composition of both the shell and the soft tissue at day 0 represents the lagoon end member, which presents a mixture between sediments and GoA open seawater. Five days after the translocation, the soft tissue presents open seawater compositions, portraying the organism’s rapid response to its ambient seawater Pb composition. However, this trend stops at day 13, when the soft tissue isotopic composition shifts toward the lagoon end member. The sample collected at the following week at day 21 and all subsequent samples, presents Pb compositions similar to open seawater, portraying the organism’s prolonged response to ambient pristine waters. The composition of the shell did not overlap with the GoA seawater ﬁeld throughout the entire experiment, depicting the limited response of the shells to the translocation. wastewaters inputs due to its use in magnetic resonance imaging, may result in a positive Gd anomaly in seawater (Kümmerer and Helmers, 2000; Nozaki et al., 2000; Hatje et al., 2014). The Ce and Gd anomalies are deﬁned as their deviation from an expected, PAAS-normalized ratio, as deﬁned by Eqs 1 (Mclennan, 1989) and 2 (de Baar et al., 1985): Ce anomaly = Ce/Ce∗ = CeN (LaN X PrN)0.5 (1) Gd anomaly = Gd/Gd∗ = 2GdN EuN + TbN (2) (2) where the PAAS-normalized concentrations are indicated by subscript N and ∗denotes the theoretical interpolated concentration based on neighboring elements. Accordingly, positive and negative Ce and Gd anomalies will feature Ce/Ce∗ and Gd/Gd∗ratios higher and lower than 1, respectively. The REE concentrations are normalized to a well-established reference composition, such as the Post Archean Australian Shale (PAAS, Taylor and McLennan, 1985), which helps reveal their relative enrichment and certain REE anomalies (e.g., Ce, Eu, and Gd), providing information regarding sources or sinks of certain elements (Elderﬁeld and Greaves, 1982; de Baar et al., 1983; Hatje et al., 2016). For example, dissolved Ce may be oxidized from Ce3+ to Ce4+, resulting in a decrease in solubility and a negative Ce anomaly in seawater (de Baar, 1983). Al-Normalized Ratios, REE, and Pb Isotopes as Proxies for Terrigenous Inputs By contrast, anoxic conditions may result in a positive Ce anomaly in sediment’s pore-waters (Elderﬁeld and Sholkovitz, 1987). External sources of anthropogenic Gd, associated with medical and industrial Figure 5 presents the distribution of soft tissue REEs relative to the composition of PAAS, displaying an enrichment of the light REEs (LREE, La-Eu) relative to the heavy REEs (HREE, Gd-Lu). Similar observations were made for clams (Bonnail et al., 2017), ﬁsh, crustaceans, and mollusks (Li et al., 2016; Wang et al., 2019) and were suggested to stem from biological fractionation that favors LREE over HREE (Wang et al., 2019). The samples collected at day 0 display high REE/PAAS ratios (an order of magnitude higher than the rest of the samples) with no anomalies (Figure 5A), reﬂecting the domination of terrigenous sources in the lagoon environment. FIGURE 4 \| The isotopic composition of 208Pb/206Pb versus 206Pb/207Pb in soft tissue (squares) and shells (circles). Colored markers represent the different samples through the length of the experiment. Shaded areas represent the regional end members: GoA seawater (blue ﬁeld; Benaltabet et al., 2020), atmospheric aerosols (Chien et al., 2019), GoA leached and residual sediment fractions (Benaltabet et al., 2020), which represent the carbonate (orange ﬁeld) and silicate (light blue ﬁeld) fractions of bottom sediments, respectively. Red Sea seawater compositions (purple ﬁeld) are assumed to correspond with Arabian Sea compositions (after Lee et al., 2015). For more information regarding the suggested end members the reader is referred to Chien et al. (2019) and Benaltabet et al. (2020). The day 0 shell and soft tissue composition represents the lagoon end member (gray ﬁeld). FIGURE 4 \| The isotopic composition of 208Pb/206Pb versus 206Pb/207Pb in soft tissue (squares) and shells (circles). Colored markers represent the different samples through the length of the experiment. Shaded areas represent the regional end members: GoA seawater (blue ﬁeld; Benaltabet et al., 2020), atmospheric aerosols (Chien et al., 2019), GoA leached and residual sediment fractions (Benaltabet et al., 2020), which represent the carbonate (orange ﬁeld) and silicate (light blue ﬁeld) fractions of bottom sediments, respectively. Red Sea seawater compositions (purple ﬁeld) are assumed to correspond with Arabian Sea compositions (after Lee et al., 2015). For more information regarding the suggested end members the reader is referred to Chien et al. (2019) and Benaltabet et al. (2020). The day 0 shell and soft tissue composition represents the lagoon end member (gray ﬁeld). Short-Term Perturbation During the depuration experiment, the soft tissue samples presented a 2–8-fold increase in elemental concentrations between days 5 and 13 (Figure 2), as well as a shift in Pb isotopic composition toward the lagoon and sedimentary end members (Figures 3, 4) and an increase in the PAAS- normalized REE concentrations (Figure 5). Moreover, when examining the subsequent samples, soft tissue Al, V, Cr, Mn, Fe, Co, Cd, Zn, Ni, and Pb concentrations gradually decrease from day 13 to 91, following a power law curve, with high R2 values of 0.56 for Pb to R2 = 0.95 for Co (Figure 2). When compared to the R2 values for the period between 0 and 91 days, all of the above mentioned R2 values for the period between 13 and 91 days are signiﬁcantly higher. The fact that the decrease in soft tissue concentrations after day 13 closely follows a well-deﬁned curve, suggests that the increase in concentrations in day 13 is associated with a compositional perturbation in the water tank’s seawater composition (rather than being associated with analytical noise) that eﬀectively reset the experiment at day 13. Moreover, 7 days after the day 13 perturbation, metal levels decreased by 33 to 70%, at rates similar to the initial decrease in concentrations following the relocation to the water tanks The cause of the perturbation between days 5 and 13 is unknown, and could be related to a natural change in the inﬂuxing seawater composition, possibly due to sediment resuspension along the coast, or to contamination of the water tank. The relatively high Ce anomaly at day 13 (Figure 5) may be indicative of a sedimentary source, as positive Ce anomalies are a common feature in some marine sediments (de Baar, 1983; Toyoda et al., 1990; Pattan et al., 2005). Moreover, several studies suggested that mollusks eﬀectively accumulate LREEs from sediments and suspended particles (Bonnail et al., 2017; Ma et al., 2019; Wang et al., 2019). Hence, a perturbation associated with a terrigenous source will be promptly recorded in the mussel’s soft tissue. This, coupled with the shift of the Pb isotopic composition toward sedimentary compositions (Figure 4) may suggest that the cause of the perturbation on day 13 is linked to a terrigenous source. Al-Normalized Ratios, REE, and Pb Isotopes as Proxies for Terrigenous Inputs June 2021 \| Volume 8 \| Article 669329 Frontiers in Marine Science \| www.frontiersin.org 9 Metals Depuration Dynamics in Mussels Benaltabet et al. (between days 0 to 5) and to previously reported depuration rates (Yap et al., 2003; El-Gamal, 2011). The rate of increase in soft tissue metal abundances in response to the perturbation is comparable with the increases in Cd and Zn reported by Yap et al. (2003), who exposed mussels (P. virdis) to high levels of seawater Cd and Zn in a controlled laboratory experiment and followed the change of accumulated metals over time. By contrast, the study carried out by Liu and Wang (2016), who translocated two oyster species from a natural to a contaminated environment and followed metal accumulation with time reported limited increases in the soft tissue metals concentrations after 5 days of exposure. However, the diﬀerence in metal accumulation rates compared to our results can probably be attributed to diﬀerent environmental conditions (Mubiana and Blust, 2007; Casas et al., 2008) and species types (Rainbow, 2002). Five days after the translocation, REE concentrations decrease signiﬁcantly, but shift back to higher values on day 13 while also displaying a positive Ce anomaly (Figure 5B). Afterward, the REE concentrations as well as the Ce anomaly at the samples collected at days 21–91, return to values that are similar to day 5. Moreover, following the relocation to the water tanks, the PAAS normalized pattern reveals an increasing positive Gd anomaly (Figures 5A,C), which suggests an anthropogenic source of Gd to the GoA waters. Short-Term Perturbation Interestingly, the Al normalized ratios of several elements (e.g., Ni, Co, Fe, Cr, V, Pb Cd, Zn, and FIGURE 5 \| (A) Soft tissue REE concentrations relative to PAAS (Taylor and McLennan, 1985). Colored symbols represent the different sampling days through the length of the experiment. Error bars mark the standard deviation of duplicate analyses (see section “Data treatment” for details). (B) Ce/Ce* ratios representing the Ce anomaly throughout the experiment. (C) Gd/Gd* ratios representing the Gd anomaly throughout the experiment. FIGURE 5 \| (A) Soft tissue REE concentrations relative to PAAS (Taylor and McLennan, 1985). Colored symbols represent the different sampling days through the length of the experiment. Error bars mark the standard deviation of duplicate analyses (see section “Data treatment” for details). (B) Ce/Ce* ratios representing the Ce anomaly throughout the experiment. (C) Gd/Gd* ratios representing the Gd anomaly throughout the experiment. June 2021 \| Volume 8 \| Article 669329 10 Frontiers in Marine Science \| www.frontiersin.org Metals Depuration Dynamics in Mussels Benaltabet et al. FIGURE 6 \| Soft tissue Zn (A) and Pb (B) concentrations, Ce/Ce* (C), and 206Pb/207Pb compositions (D) versus soft tissue Al concentrations. The dashed lines represent the mixing curves between the two terrigenous (Terr1 and Terr2; green and red triangles, respectively) and seawater (SW; pale blue circle) end members. Solid arrows represent the temporal trend throughout the experiment: the rapid depuration after the relocation from the lagoon environment (gray ﬁeld, purple squares) to the IUI (pink squares) between days 0 and 5, the brief perturbation toward Terr2 between days 5 and 13 (blue squares), and the long term depuration between days 21 and 91 (black squares). FIGURE 6 \| Soft tissue Zn (A) and Pb (B) concentrations, Ce/Ce* (C), and 206Pb/207Pb compositions (D) versus soft tissue Al concentrations. The dashed lines represent the mixing curves between the two terrigenous (Terr1 and Terr2; green and red triangles, respectively) and seawater (SW; pale blue circle) end members. Solid arrows represent the temporal trend throughout the experiment: the rapid depuration after the relocation from the lagoon environment (gray ﬁeld, purple squares) to the IUI (pink squares) between days 0 and 5, the brief perturbation toward Terr2 between days 5 and 13 (blue squares), and the long term depuration between days 21 and 91 (black squares). dominated by mixing of a terrigenous end member (“Terr1,” Figure 6) with the seawater end member (“SW,” Figure 6). Frontiers in Marine Science \| www.frontiersin.org Short-Term Perturbation While Terr1 features high metal concentrations and high 206Pb/207Pb ratios with no Ce anomaly (i.e., Ce/Ce∗≈1), the seawater end member is characterized by low metal concentrations (Chase et al., 2011; Chien et al., 2019; Benaltabet et al., 2020), a negative Ce anomaly, a common feature in oxygenated waters (Elderﬁeld and Greaves, 1982; Alibo and Nozaki, 1999) and low 206Pb/207Pb ratios (Benaltabet et al., 2020). After the relocation to the water tanks, the mussel’s soft tissue composition shifted rapidly to a separate mixing curve, with a diﬀerent terrigenous end member (“Terr2,” Figure 6), deﬁned by high metal abundances and lower Al contents relative to Terr1, a positive Ce anomaly and high 206Pb/207Pb ratios. Following the day 13 perturbation, the soft tissue compositions shifted toward Terr2, and thereafter subsided toward the composition of the seawater end member, Cu) display a relatively large shift between day 0 and day 5 but remain stable thereafter (Supplementary Figure 1), even after the day 13 perturbation. This implies that the terrigenous end member responsible for the perturbation has a diﬀerent composition, characterized by a higher metal/Al ratio, compared to the lagoon end member. In other words, one terrigenous end member dominates the lagoon area and a second dominates the IUI coastal waters. To better characterize the two terrigenous end members, the progress of the soft tissue depuration of various proxies versus Al is outlined in Figure 6. Zn and Pb were chosen to represent examples of anthropogenic metals in coastal waters (e.g., John et al., 2007; Boyle, 2019), while the shifts in the Ce anomaly value (Ce/Ce∗) and the isotopic composition of Pb (206Pb/207Pb) represent the competing inﬂuences of terrigenous and seawater end member values. The lagoon environment is June 2021 \| Volume 8 \| Article 669329 11 Metals Depuration Dynamics in Mussels Benaltabet et al. FIGURE 7 \| A conceptual representation of a perturbation/pollution event reﬂected by the ratio between soft tissue (Rtissue) and shell (Rshell) Pb isotopic compositions. (A) A perturbation represented by an exposure to end member B (dashed red line) will result in an increase in the Rtissue/Rshell ratio. Subsequently, if the contamination persists (chronic contamination), the Rtissue/Rshell ratio will remain high (until eventually the end member B signal starts dominating the bulk shell composition after a signiﬁcant period of time not considered here). Long-Term Biomonitoring Due to biological controls and the variability of elemental partitioning coeﬃcients between seawater, soft tissue and shell (White and Rainbow, 1982; Chong and Wang, 2001; Amiard et al., 2006), metal abundances in the soft tissue and shell are markedly diﬀerent (Figure 2). By contrast, the isotopic composition of the shell and soft tissue at day 0 overlap (Figures 3, 4) because Pb isotopic fractionation during assimilation is negligible (Russell Flegal and Stukas, 1987) and both phases acquired the long term local lagoon composition (Figure 4). The ratio between the Pb isotopic composition in the soft tissue and shell is expressed in Eq. 3: This approach can also be applied to other heavy isotopic systems where no isotopic fractionation is associated with the assimilation into the shell and soft tissue (e.g., Nd, U, and Th). However, the isotopic composition of Pb is especially useful for monitoring marine pollution given its sensitivity to anthropogenic inputs and ample reports of natural and anthropogenic end member compositions (e.g., Bollhöfer and Rosman, 2001, 2000; Labonne et al., 2001; Erel et al., 2006; Boyle et al., 2014; Dang et al., 2015). Rtissue Rshell = 206Pb 207Pb tissue 206Pb 207Pb shell (3) (3) Short-Term Perturbation If the perturbation is brief, the Rtissue/Rshell ratio will gradually return to a value of 1 (end member A, dashed blue line) following a power law curve. (B) Rtissue/Rshell ratio shortly after a contamination event, depicting how a partial set of observations (e.g., black squares) can be used to extract the timing of the suspected perturbation/contamination event through back extrapolation of the calculated power law depuration curve to the contaminating end member composition (empty square). FIGURE 7 \| A conceptual representation of a perturbation/pollution event reﬂected by the ratio between soft tissue (Rtissue) and shell (Rshell) Pb isotopic compositions. (A) A perturbation represented by an exposure to end member B (dashed red line) will result in an increase in the Rtissue/Rshell ratio. Subsequently, if the contamination persists (chronic contamination), the Rtissue/Rshell ratio will remain high (until eventually the end member B signal starts dominating the bulk shell composition after a signiﬁcant period of time not considered here). If the perturbation is brief, the Rtissue/Rshell ratio will gradually return to a value of 1 (end member A, dashed blue line) following a power law curve. (B) Rtissue/Rshell ratio shortly after a contamination event, depicting how a partial set of observations (e.g., black squares) can be used to extract the timing of the suspected perturbation/contamination event through back extrapolation of the calculated power law depuration curve to the contaminating end member composition (empty square). as the mussels gradually depurated the accumulated metals from the perturbation. be used for long-term biomonitoring in coastal environments, even if the initial isotopic composition is unknown, as any deviation from Rtissue/Rshell = 1 will represent a shift toward a new compositional end member (Figure 7). If the perturbation is of chronic nature, the Rtissue/Rshell ratio will remain constant, representing the current long-term composition of surrounding seawater, as is the case here, where the seawater composition changed after their relocation. Frontiers in Marine Science \| www.frontiersin.org Biomonitoring of Short-Term Pollution Events where Rtissue and Rshell are the 206Pb/207Pb ratios (or other Pb isotopic ratios) in the soft tissue and shell, respectively. In the lagoon samples (day 0), the Rtissue/Rshell ratio presents an approximate value of one and continuously decreases as the soft tissue registers lower 206Pb/207Pb seawater compositions (Figure 3B). Although serendipitously demonstrated here (i.e., by the perturbation between day 5 and 13), biomonitoring of short-term (daily) events is often challenged by the rapid compositional change in soft tissue compositions in the days following the event, hampering the use of mussels and other marine ﬁlter-feeding organisms as biomonitors of abrupt and short pollution events. Nevertheless, our results provide the possibility to overcome this shortcoming by extrapolating observations backward toward the onset of the event. Thus, mature mussels grown in stable conditions should display an identical Pb isotopic composition in both the shell and soft tissue, i.e., Rtissue/Rshell = 1. Accordingly, the coupled Pb isotopic composition of mussels soft tissue and shell can June 2021 \| Volume 8 \| Article 669329 Frontiers in Marine Science \| www.frontiersin.org 12 Metals Depuration Dynamics in Mussels Benaltabet et al. the shells remains rather constant throughout the experiment (206Pb/207Pb = 1.191 – 1.199, 208Pb/206Pb = 2.051 – 2.060), the soft tissue compositions (206Pb/207Pb = 1.174 – 1.199, 208Pb/206Pb = 2.052 – 2.078) shift gradually toward open seawater values from days 5 to 91, while brieﬂy shifting toward the sedimentary end member at day 13. This shift is also illustrated by the REEs pattern relative to PAAS, which features a positive Ce anomaly at day 13. Hence, we conclude that this compositional shift was driven by an environmental perturbation linked to a terrigenous source. By comparing soft tissue metal concentrations, Ce anomaly ratios, Pb isotopic compositions and Al concentrations, it is shown that following the relocation to the water tanks, the mussels were controlled by a separate mixing curve between GoA seawater and a local terrigenous end member (possibly marine sediments), which is diﬀerent than the terrigenous end member dominating the lagoon environment. Consider a pollution event that is identiﬁed along a coastline, but evidence (e.g., visual observations, smell, direct seawater analyses) only starts accumulating several days after the actual event. Thus, the exact timing and location of the contamination remain largely unknown. DATA AVAILABILITY STATEMENT In summary, mussels (B. pharaonis) growing in a contaminated lagoon were relocated to a pristine environment for depuration and their soft tissue and shells were analyzed for heavy metal and REE concentrations, as well as the isotopic composition of Pb. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. The shell retained its original composition and did not present signiﬁcant change in metal abundances (apart from Mn) during the experiment. On the other hand, most of the soft tissue metal concentrations (Al, V, Cr, Mn, Fe, Co, Ni, Zn, Cd, and Pb) and the REEs presented a gradual decrease in concentrations in the 91 days following the relocation from the lagoon to the water tanks, with the bulk majority of the decrease (51–86%) restricted to the ﬁrst 5 days. The decrease trend was disrupted by an abrupt increase in metal and REE concentrations at day 13, after which concentrations decreased gradually following a power law trend (R2 of up to 0.95). Biomonitoring of Short-Term Pollution Events Yet, following alerts of a potential pollution event, it is possible to initiate continuous sampling of in-situ living mussels at a daily resolution over a period of 1–2 weeks and analyze the isotopic compositions of their soft tissue and shell. The results will yield a partial segment of the longer-term depuration pattern during which the Rtissue/Rshell ratio gradually returns to a value of 1 following a power law curve (Figure 7B). A backward extrapolation of this curve, allows determining the timing of contamination, as long as the initial contamination value can be estimated, even if only roughly. The latter can be reasonably evaluated in coastal environments where recurring pollution events take place, or by assuming a regional anthropogenic Pb isotope compositional end member. Accordingly, the time of contamination (t) can be determined using Eq. 4 solved for t: The soft tissue of B. pharaonis may be used to biomonitor short-term environmental perturbations or pollution events. The ratio between the Pb isotopic composition of the soft tissue and shell sampled following an event may be used to determine the exact timing and possibly geographic location of the pollution, when both the latter are a-priori unknown. Rc Rshell = t−m (4) (4) Where Rc represents the composition of the contaminant, Rshell is the measured composition in the shell and m is the power constant given by the depuration curve. Alternatively, if the timing (t) of the pollution event is well known, Eq. 4 can be solved for Rc and cross-referenced with literature report to learn about the source of the contaminant end member (e.g., anthropogenic or natural/terrigenous). This approach assumes that the soft tissue is a sensitive recorder of ambient seawater compositions, as seen here and by others (e.g., Yap et al., 2003; El-Gamal, 2011; Anacleto et al., 2015). Moreover, if coupled with a spatial survey, this approach can also provide information regarding the geographic sources of the contaminants, and how these progress spatially and temporally. By implementing geochemical tools such as Pb isotopic compositions, Al-normalization and REE patterns, we demonstrated the potential of mussels as biomonitors of short-term ﬂuctuations or alternatively, of long-term seawater compositions. In both cases, the combined suite of geochemical proxies can be used to provide robust quantitative constraints on the sources and magnitude of pollution events, providing important tools for implementing and developing environmental management policies. AUTHOR CONTRIBUTIONS TB, EG-H, and AT wrote the manuscript. 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https://openalex.org/W2754605251	https://europepmc.org/articles/pmc5999171?pdf=render	English	null	Evaluation of current prediction models for Lynch syndrome: updating the PREMM5 model to identify PMS2 mutation carriers	Familial cancer	2,017	cc-by	6,658	Evaluation of current prediction models for Lynch syndrome: updating the PREMM5 model to identify PMS2 mutation carriers A. Goverde1,2 · M. C. W. Spaander2 · D. Nieboer3 · A. M. W. van den Ouweland1 · W. N. M. Dinjens4 · H. J. Dubbink4 · C. J. Tops5 · S. W. ten Broeke5 · M. J. Bruno2 · R. M. W. Hofstra1 · E. W. Steyerberg6 · A. Wagner1,7 Published online: 20 September 2017 © The Author(s) 2017. This article is an open access publication MMRpredict) and fair for MSH6 mutation carriers (0.69 for PREMM5 and 0.66 for MMRpredict). MMRpredict per- formed fair for PMS2 mutation carriers (AUC 0.72), while PREMM5 failed to discriminate PMS2 mutation carriers from non-mutation carriers (AUC 0.51). The only statisti- cally significant difference between PMS2 mutation carriers and non-mutation carriers was proximal location of colo- rectal cancer (77 vs. 28%, p < 0.001). Adding location of colorectal cancer to PREMM5 considerably improved the models performance for PMS2 mutation carriers (AUC 0.77) and overall (AUC 0.81 vs. 0.72). We validated these results in an external cohort of 376 colorectal cancer patients, including 158 LS patients. MMRpredict and PREMM5 cannot adequately identify PMS2 mutation carriers. Add- ing location of colorectal cancer to PREMM5 may improve the performance of this model, which should be validated in larger cohorts. Abstract Until recently, no prediction models for Lynch syndrome (LS) had been validated for PMS2 mutation car- riers. We aimed to evaluate MMRpredict and PREMM5 in a clinical cohort and for PMS2 mutation carriers specifi- cally. In a retrospective, clinic-based cohort we calculated predictions for LS according to MMRpredict and PREMM5. The area under the operator receiving characteristic curve (AUC) was compared between MMRpredict and PREMM5 for LS patients in general and for different LS genes specifi- cally. Of 734 index patients, 83 (11%) were diagnosed with LS; 23 MLH1, 17 MSH2, 31 MSH6 and 12 PMS2 mutation carriers. Both prediction models performed well for MLH1 and MSH2 (AUC 0.80 and 0.83 for PREMM5 and 0.79 for Electronic supplementary material The online version of this article (doi:10.1007/s10689-017-0039-1) contains supplementary material, which is available to authorized users. Keywords Lynch syndrome · Prediction models · Colorectal cancer · Hereditary cancer Keywords Lynch syndrome · Prediction models · Colorectal cancer · Hereditary cancer * A. Wagner a.wagner@erasmusmc.nl * A. Wagner a.wagner@erasmusmc.nl 1 Department of Clinical Genetics, Erasmus MC, University Medical Center, Rotterdam, The Netherlands 7 Department of Clinical Genetics, Erasmus MC, University Medical Center, Room Ee‑2018, P. O. Box 2040, 3000 CA Rotterdam, The Netherlands Familial Cancer (2018) 17:361–370 DOI 10.1007/s10689-017-0039-1 ORIGINAL ARTICLE Introduction 2 Department of Gastroenterology and Hepatology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands Lynch syndrome (LS) is a hereditary predisposition to colo- rectal cancer, endometrial cancer and other extra-colonic cancers at a young age [1, 2]. Morbidity and mortality of LS carriers can be significantly reduced by surveillance pro- grams [3–5]. Therefore identifying LS carriers is of great importance. 3 Department of Public Health, Erasmus MC, University Medical Center, Rotterdam, The Netherlands 4 Department of Pathology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands 5 Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands LS is caused by a germline mutation in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6 or PMS2, or in the 3′ end of the EPCAM gene and consequent hypermethylation of the MSH2 promoter region [6–10]. As a result, tumours in LS patients are characterized by 6 Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands 6 Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands 7 Department of Clinical Genetics, Erasmus MC, University Medical Center, Room Ee‑2018, P. O. Box 2040, 3000 CA Rotterdam, The Netherlands (0121 362 A. Goverde et al. failed or inconclusive analysis for MSI and IHC, a patho- genic mutation in APC or MUTYH, a variant of unknown clinical significance in one of the MMR genes or APC, and MSI or IHC suspect for LS while no MMR mutation was detected. To increase the number of LS families, 35 LS families outside our cohort, diagnosed after 2010, were also included in the analysis. microsatellite instability (MSI) and by loss of MMR pro- tein expression in immunohistochemistry (IHC) [11–13]. Analysis of MSI and IHC, combined with MLH1 promoter methylation analysis to exclude sporadic MMR deficient tumours, are used to identify patients with tumours likely caused by LS [13]. A definite diagnosis of LS is made when a pathogenic germline mutation is found. The revised Bethesda guidelines were based on a set of diagnostic criteria to select patients eligible for LS screen- ing in tumour tissue. However, due to limited sensitivity, many LS patients will likely be missed by these guidelines [14–17]. Several prediction models, such as MMRpro, MMRpredict and PREMM5 have also been developed to calculate an individual’s probability of carrying a germline MMR mutation [18–20]. Germline mutation analysis Patients with MMR deficient tumours suspect for LS under- went germline mutation analysis of the gene indicated by IHC. Germline mutation analysis of MLH1, MSH2 and MSH6 was performed by sequencing and multiplex ligation dependent probe amplification analyses. PMS2 mutation analysis was performed as described elsewhere [30]. In this study we aimed to evaluate MMRpredict and PREMM5 in a clinical cohort and for PMS2 mutation carri- ers specifically. Additionally, we aimed to identify clinical features useful for distinguishing PMS2 mutation carriers from non-mutation carriers. Methods In a retrospective, clinic-based cohort we assessed the performance of MMRpredict and PREMM5 in predicting LS mutations in general and for MLH1, MSH2, MSH6 and PMS2 mutations specifically. Additionally, we performed a univariate analysis to identify variables that can distin- guish PMS2 mutation carriers from patients with no MMR mutation. Family classification Tumour characteristics, age at diagnosis, results of molecular diagnostics and germline mutation analysis, and a detailed family history were collected from medical records. In every family the patient in whom MSI and/or IHC was analysed, was labelled the index patient. If more than one family mem- ber was screened for LS, the youngest CRC patient analysed was considered the index patient. Index patients with MMR proficient tumours or sporadic MMR deficient tumours, were labelled non-mutation carriers. Families identified with a pathogenic MMR mutation were labelled LS families. Analysis of MSI and IHC MSI analysis was carried out with five markers for MSI as described previously; up to 2007 the Bethesda panel [28] was used and from 2007 onwards our center performs Pro- mega pentaplex MSI analysis [29]. IHC for MLH1, MSH2, MSH6 and PMS2 protein was performed as described previ- ously [13]. Tumours without MSI or only a low degree of MSI and with all MMR proteins present, were considered MMR proficient. Tumours showing a high degree of MSI and/or absence of one or more MMR proteins, were consid- ered MMR deficient. MLH1 hypermethylation analysis was performed to distinguish between sporadic MMR deficient tumours and MMR deficient tumours suspect for LS. Introduction These models could aid in the selection of patients at high risk of having LS, for tumour analysis or direct germline mutation analysis. MMRpro is less useful in clinical practice since detailed information of all relatives is needed as input for the model [19]. However, MMRpredict and PREMM1,2,6 (a previous version of the newly developed PREMM5 model) both performed well in previous evaluations [21–27]. An advantage of PREMM5 is that it can also be used for individuals with extracolonic malignancies and healthy individuals, as opposed to MMR- predict, which can only be used for CRC patients. Until recently, all prediction models for LS were developed with cohorts of patients carrying a MLH1, MSH2, or MSH6 mutation. The recently published PREMM5 model is the only model that included PMS2 mutation carriers in its development. Validation of the extended PREMM5 model For external validation of the extended PREMM5 model, we used a cohort of 376 CRC patients. Of these patients, 218 were patients with MMR proficient CRC, that where analysed in the Erasmus Medical Center Rotterdam outside the dates of our initial cohort. LS patients (n = 158) in our validation cohort were CRC patients from Leiden University Medical Center in whom an MMR mutation was found and with known location of CRC. For all patients of the vali- dation cohort we calculated the probability of carrying an MMR mutation according to the original PREMM5 model and the extended model. The performance of both models were evaluated by comparing the AUC. Patient characteristics Patient characteristics for mutation-positive and mutation- negative patients are shown in Table 1. Significantly more mutation carriers developed multiple CRCs (21 vs. 10%, p = 0.005) and multiple LS-associated cancers in general (13 vs. 4%, p = 0.002) than non-mutation-carriers. CRC patients carrying an MMR mutation had a younger age of onset (49 vs. 53 years, p = 0.002) and more often had proximal CRCs (64 vs. 28%, p < 0.001) than non-mutation carriers. Among women, the frequency of EC was higher for mutation carri- ers than for non-mutation carriers (41 vs. 3%, p < 0.001). In the mutation positive group, first and second degree relatives developed CRC at a younger age than in the mutation nega- tive group (50 vs. 64 years, p < 0.001 and 47 vs. 62 years, p = 0.008). First degree relatives of mutation carriers had higher rates of EC than relatives of non-mutation carriers (19 vs. 5%, p < 0.001). Model updating Location of CRC is included in MMRpredict, but not in the PREMM5 model. To update the PREMM5 model, we used a previously proposed framework to update multi- nomial logistic regression models [31]. We extended the PREMM5 model using recalibration and extension. The PREMM5 model contains four linear predictors, each con- tributing weights to the probability of carrying a muta- tion in MLH1, MSH2 (or TACSTD1), MSH6 and PMS2. The coefficients of the linear predictors were constrained such that the linear predictor only contributed to the cal- culation of the corresponding mutation. Since the origi- nal PREMM5 model was developed on a population with no MSH6 mutation carriers with two or more CRCs, we developed two adaptations of the PREMM5 model. First we recalibrated the PREMM5 model and re-estimated the coefficient of the predictor ‘Two or more CRCs’ in the linear predictor for MSH6. In the second adaptation we also added side of CRC as an additional predictor to the original PREMM5 model. Discriminative ability of the prediction models was quantified using the AUC. Calcu- lations were done using R software (version 3.3.0), with estimation of the coefficients in the updated PREMM5 model using the VGAM package. Study population For each index patient the probability of carrying a LS muta- tion according to MMRpredict and PREMM5 was calculated as previously described [18, 20].f We collected data for all families that were referred for genetic counselling at Erasmus MC, Rotterdam, The Nether- lands, and in which colorectal cancer was analysed for MSI and/or IHC between 2000 and 2010. Exclusion criteria were: For PREMM5, the equation was slightly different from the published equation, based on personal communications 1 3 Evaluation of current prediction models for Lynch syndrome: updating the PREMM5 model to identify… 363 with F. Kastrinos. See Supplemental Material (Appen- dix 1) for the corrected PREMM5 equation. Statistical analysis Data were analyzed using SPSS statistical software version 21.0. Differences between mutation carriers and non-muta- tion carriers were compared using the Chi square test or Fishers’ exact test for frequencies, and by using the Mann Whitney U test for continuous data. These analysis were also performed to compare PMS2 mutation carriers with non-mutation carriers. P values < 0.01 were considered statistically significant. i Receiver operating characteristic curves were created for MMRpredict and PREMM5 by plotting the true posi- tive rate (sensitivity) against the false positive rate (1- specificity). Performance of MMRpredict and PREMM5 was evaluated by the area under the receiver operating characteristic curve (AUC). We compared the AUC of PREMM5 and MMRpredict for LS patients in general and for the different MMR genes specifically. Sensitiv- ity and specificity were calculated for cut-offs previously indicated by the developers of the models (5, 10, 20 and 40%). These values were compared with the sensitivity and specificity of the revised Bethesda guidelines. Results A total of 734 index patients were included in the study; 346 (47%) were male and mean age at time of diagnosis was 53 years (± 13 years). Overall, 569 (78%) patients fulfilled the revised Bethesda guidelines. Of the 734 index patients, 83 (11%) were diagnosed with a LS mutation; 23 MLH1, 17 MSH2, 31 MSH6 and 12 PMS2 mutation carriers. Discriminative ability of prediction models 0.73 [95% CI 0.66–0.79]). For MLH1 and MSH2 mutation carriers, both prediction models performed well, with AUC of 0.80 [95% CI 0.71–0.89] and 0.83 [95% CI 0.73–0.94] for PREMM5 and AUC of 0.79 [95% CI 0.69–0.89 and 0.67–0.91] for MMRpredict. Both models had a fair discriminative power for MSH6 mutation carriers (AUC of 0.69 [95% CI 0.58–0.80] for PREMM5 and AUC of 0.66 [95% CI 0.56–0.76] for MMRpredict). MMR- predict still had fair performance for PMS2 mutation carriers (AUC of 0.72 [95% CI 0.57–0.87]), while PREMM5 failed to discriminate PMS2 mutation carriers from non-mutation carriers at all with an AUC of 0.51 [95% CI 0.35–0.66]. 74% and the sensitivity for PMS2 mutation carriers was only 50%. For MMRpredict, at a 5% cut-off sensitivity for MLH1 and MSH2 mutation carriers were 74 and 77%, while sensi- tivity for PMS2 as well as MSH6 mutation carriers were 65 and 67%. For both models, using a cut-off of ≥ 20% failed to identify over 50% of the mutation carriers. and MMRpredict had similar overall performance (AUC 0.72 [95% CI 0.66–0.79] vs. 0.73 [95% CI 0.66–0.79]). For MLH1 and MSH2 mutation carriers, both prediction models performed well, with AUC of 0.80 [95% CI 0.71–0.89] and 0.83 [95% CI 0.73–0.94] for PREMM5 and AUC of 0.79 [95% CI 0.69–0.89 and 0.67–0.91] for MMRpredict. Both models had a fair discriminative power for MSH6 mutation carriers (AUC of 0.69 [95% CI 0.58–0.80] for PREMM5 and AUC of 0.66 [95% CI 0.56–0.76] for MMRpredict). MMR- predict still had fair performance for PMS2 mutation carriers (AUC of 0.72 [95% CI 0.57–0.87]), while PREMM5 failed to discriminate PMS2 mutation carriers from non-mutation carriers at all with an AUC of 0.51 [95% CI 0.35–0.66]. Sensitivity of the revised Bethesda guidelines decreased from 96% for MLH1 mutation carriers to 83% for PMS2 mutation carriers (Supplemental Table 2). Overall, the revised Bethesda guidelines had a sensitivity of 90% with a specificity of 24%. In order to reach the same sensitivity, PREMM5 and MMRpredict had a similar specificity (25%). Discriminative ability of prediction models Overall, PREMM5 predicted higher probabilities of car- rying a LS mutation than MMRpredict (median score 0.06 vs. 0.03, Supplemental Table 1). For mutation car- riers, risk scores varied from 0.02 to 0.99 for PREMM5 and from 0.002 to 0.99 for MMRpredict. Both prediction models could fairly discriminate between index patients with and without an MMR mutation.(Fig. 1) PREMM5 1 3 364 A. Goverde et al. Table 1 Index characteristics and family history by mutation status (n = 734) Mutation negative, % (n) Mutation positive, % (n) P value n 651 83 Revised Bethesda guidelines 76% (494) 90% (75) 0.003 Index characteristics Male gender 47% (305) 49% (41) 0.66 CRC Age CRC (median, IQR) 53 years [45–62] 49 years [39–59] 0.002 Proximal CRC 28% (185) 64% (53) < 0.001 ≥ 2 CRCs 10% (66) 21% (17) 0.005 Endometrial cancer 3% (11) 41% (17) < 0.001 Age EC (median, IQR) 55 years [50–75] 54 years [49–57] 0.18 Multiple LS cancers 4% (27) 13% (11) 0.002 First degree relatives CRC 55% (358) 51% (42) 0.45 ≥ 2 FDRs with CRC 16% (107) 17% (14) 0.92 Age CRC (median, IQR) 64 years [55–71] 50 years [43–57] < 0.001 Endometrial cancer 5% (35) 19% (16) < 0.001 ≥ 2 FDRs with EC 0.6% (4) 2% (2) 0.14 Age EC (median, IQR) 55 years [50–64] 50 years [45–57] 0.25 Other LS cancers 22% (142) 19% (16) 0.60 Second degree relatives CRC 33% (212) 35% (29) 0.66 ≥ 2 SDRs with CRC 12% (81) 12% (10) 0.92 Age CRC (median, IQR) 62 years [50–74] 47 years [38–64] 0.008 Endometrial cancer 3% (22) 7% (6) 0.12 ≥ 2 SDRs with EC 0.3% (2) 2% (2) 0.07 Age EC (median, IQR) 70 years [50–76] 49 years [44–51] 0.13 Other LS cancers 16% (104) 18% (15) 0.63 Mutation negative, % (n) Mutation positive, % (n) P value n Revised Bethesda guidelines Index characteristics Male gender CRC Age CRC (median, IQR) Proximal CRC ≥ 2 CRCs Endometrial cancer Age EC (median, IQR) Multiple LS cancers First degree relatives CRC ≥ 2 FDRs with CRC Age CRC (median, IQR) Endometrial cancer ≥ 2 FDRs with EC Age EC (median, IQR) Other LS cancers Second degree relatives CRC ≥ 2 SDRs with CRC Age CRC (median, IQR) Endometrial cancer ≥ 2 SDRs with EC Age EC (median, IQR) Other LS cancers and MMRpredict had similar overall performance (AUC 0.72 [95% CI 0.66–0.79] vs. Sensitivity and specificity Mutation carriers differed significantly from non-mutation carriers in many ways (Table 1). In contrast, there were almost no significant differences between PMS2 mutation carriers and non-mutation carriers. Only one significant dif- ference remained; PMS2 mutation carriers more often had proximal CRC than patients without an MMR mutation (83 vs. 28%, p < 0.001) (Table 2). Using a cut-off of 5% for both prediction models, PREMM5 had a higher sensitivity than MMRpredict (78 vs. 70%). This higher sensitivity came at the expense of a lower specificity (46 vs. 67%). For PREMM5, using a cut-off of 5%, resulted in a sensitivity for MLH1 and MSH2 mutations of 88 and 91%, while the sensitivity for MSH6 mutation carriers was 1 3 Evaluation of current prediction models for Lynch syndrome: updating the PREMM5 model to identify… luation of current prediction models for Lynch syndrome: updating the PREMM5 model to identify… 1 Performance of PREMM5 and MMRpredict in a clinical setting for all mutation carriers and for individual MMR mutatio 365 Fig. 1 Performance of PREMM5 and MMRpredict in a clinical setting for all mutation carriers and for individual MMR mutations Fig. 1 Performance of PREMM5 and MMRpredict in a clinical setting for all mutation carriers and for individual MMR mutations 1 3 366 A. Goverde et al. Sensitivity and specificity Mutation negative, % (n) PMS2 mutation posi- tive, % (n) P value n 651 12 Revised Bethesda guidelines 76% (494) 83% (10) 0.74 Index characteristics Male gender 47% (305) 50% (6) 0.83 CRC Age CRC (median, IQR) 53 years [45–62] 46 years [40–61] 0.21 Proximal CRC 28% (185) 83% (10) < 0.001 ≥ 2 CRCs 10% (66) 8% (1) 1.0 Endometrial cancer 3% (11) 0% (0) 1.0 Age EC (median, IQR) 55 years [50–75] Multiple LS cancers 4% (27) 0% (0) 1.0 First degree relatives CRC 55% (358) 42% (5) 0.36 ≥ 2 FDRs with CRC 16% (107) 8% (1) 0.70 Age CRC (median, IQR) 64 years [55–71] 62 years [45–90] 0.68 Endometrial cancer 5% (35) 17% (2) 0.14 ≥ 2 FDRs with EC 0.6% (4) 8% (1) 0.88 Age EC (median, IQR) 55 years [50–64] 37 years [–] 0.24 Other LS cancers 22% (142) 8% (1) 0.48 Second degree relatives CRC 33% (212) 17% (2) 0.35 ≥ 2 SDRs with CRC 12% (81) 8% (1) 1.0 Age CRC (median, IQR) 62 years [50–74] 39 years [39–] 0.12 Endometrial cancer 3% (22) 8% (1) 0.35 ≥ 2 SDRs with EC 0.3% (2) 8% (1) 0.05 Age EC (median, IQR) 70 years [50–76] 49 years [–] 0.67 Other LS cancers 16% (104) 17% (2) 1.0 Table 2 Index characteristics and family history for PMS2 mutation carriers compared with non-mutation carriers Improvement of the PREMM5 model PREMM5 model (AUC 0.81 [95% CI 0.76–0.86] vs. 0.72 [95% CI 0.66–0.79]) and MMRpredict (AUC 0.81 vs. 0.73 [95% CI 0.66–0.79]). The adjusted prediction model can be found as supplemental material. Since location of CRC was the only significant difference between PMS2 mutation carriers and non-mutation carri- ers, we incorporated this variable in the PREMM5 model, aiming to improve the prediction model. For PMS2 muta- tion carriers, the extended PREMM5 model had consider- ably better predictions than the original PREMM55 model (AUC 0.77 [95% CI 0.63–0.90] vs. 0.51 [95% CI 0.35–0.66]) (Fig. 2). At a 5% cut-off, the new PREMM5 model identified 5/6 PMS2 mutation carriers that would have been missed by PREMM5 and 3/4 PMS2 mutation carriers that would have been missed by MMRpredict at the same cut-off. At a 5% cut-off, sensitivity of the extended PREMM5 model was higher than the sensitivity of the original PREMM5 model (92 vs. 78%) with similar specificity (45 vs. 46%). Sensitivity and specificity of the extended PREMM5 model at a 5% cut off were both higher than those of the revised Bethesda guidelines (sensitivity 92 vs. 90% and specificity 45 vs. 24%). Validation of the extended PREMM5 model f Adding tumour location also improved the performance of PREMM5 for identifying MLH1 (AUC 0.92 [95% CI 0.88–0.97] vs. 0.80 [95% CI 0.71–0.89]) and MSH6 (AUC 0.75 [95% CI 0.65–0.84] vs. 0.69 [95% CI 0.58–0.80]) mutation carriers (Fig. 2). However, performance for MSH2 mutation carriers slightly decreased (AUC 0.80 [95% CI 0.69–0.91] vs. 0.83 [95% CI 0.73–0.94]). Overall, the adjusted PREMM5 model performed better than the original In our validation cohort, 60% of the patients were male and median age was 55 years (IQR 45–63 years). The cohort included 31 MLH1, 26 MSH2, 28 MSH6 and 73 PMS2 muta- tion carriers. Similar to the results in the initial cohort, the extended PREMM5 model had better predictions than the original PREMM5 model for PMS2 mutation carriers (AUC 0.90 [95% CI 0.86–0.94] vs. 0.82 [95% CI 0.76–0.87]) and 1 3 3 Evaluation of current prediction models for Lynch syndrome: updating the PREMM5 model to identify… 367 verall (AUC 0.92 [95% CI 0.89–0.95] vs. 0.87 [95% CI 84–0.91]). Performance for MLH1, MSH2 and MSH6 utation carriers was also slightly better for the extended PREMM5 model than for the original PR (AUC 0.97 [95% CI 0.94–1.00] vs. 0.95 [95% for MLH1,0.97 [95% CI 0.93–1.00] vs. g. 2 Performance of PREMM5 and the extended PREMM5 model in a clinical setting for all mutation carriers and for indiv ns Fig. 2 Performance of PREMM5 and the extended PREMM5 model in a clinical setting for all mutation carriers and for individual MMR muta- tions Fig. 2 Performance of PREMM5 and the extended PREMM5 model in a clinical setting for all mutation carriers and for individual MMR muta- tions Fig. 2 Performance of PREMM5 and the extended PREMM5 model in a clinical setting for all mutation carriers tions overall (AUC 0.92 [95% CI 0.89–0.95] vs. 0.87 [95% CI 0.84–0.91]). Performance for MLH1, MSH2 and MSH6 mutation carriers was also slightly better for the extended PREMM5 model than for the original PREMM5 model (AUC 0.97 [95% CI 0.94–1.00] vs. 0.95 [95% CI 0.91–0.99] for MLH1,0.97 [95% CI 0.93–1.00] vs. 0.96 [95% CI overall (AUC 0.92 [95% CI 0.89–0.95] vs. 0.87 [95% CI 0.84–0.91]). Performance for MLH1, MSH2 and MSH6 mutation carriers was also slightly better for the extended PREMM5 model than for the original PREMM5 model (AUC 0.97 [95% CI 0.94–1.00] vs. 0.95 [95% CI 0.91–0.99] for MLH1,0.97 [95% CI 0.93–1.00] vs. Discussion The results of our study indicate that while the models MMRpredict and PREMM5 can adequately predict whether an individual is likely to have Lynch syndrome, they fail to identify PMS2 mutation carriers. The performance of the PREMM5 model improved considerably by adding the location of CRC to the model. In our clinical cohort of 734 CRC patients as well as in a validation cohort of 376 CRC patients, this extended PREMM5 model not only identified PMS2 mutation carriers more accurately, its overall perfor- mance was also better than the original PREMM5 model and the MMRpredict model. Our results are in line with those of previous studies, where the PREMM1,2,6 model had a slightly better over- all performance than MMRpredict [22, 32, 33]. The first PREMM model, PREMM1,2 also performed better than MMRpredict in several studies [23, 24], but had similar [25, 26] or less accurate [21] predictions in other studies. A recent meta-analysis also found pooled AUCs to be higher for the PREMM model than for MMRpredict (AUC 0.84 vs. 0.81) [27]. The US Multi-Society Task Force on Colorectal Cancer recommends the use of either PREMM, MMRpredict or MMRpro to assess the probability of an individual carrying an MMR mutation [34]. Since we did not include the MMR- pro model in our analysis, we do not know how MMRpro would have performed in our cohort. However, MMRpro is less useful in clinical practice since extensive family data is needed as input for the model. Collection of this kind of data is very time consuming and therefore not suitable in clinical practice. PREMM5 and MMRpredict are web-based models that are easily accessible and therefore much easier to use. Also, multiple studies—including the recent meta- analysis—have shown MMRpro to have similar accuracy to PREMM1,2,6 [21–27, 32]. Although PREMM5 had better overall predictions, MMRpredict had a better performance for PMS2 mutation carriers specifically. An explanation for this could be that the location of CRC is incorporated in the MMRpredict model but not in the PREMM5 model. Proximal location of CRC is a known predictor for Lynch syndrome and in our cohort was the only significant difference between PMS2 mutation carriers and non-mutation carriers. After adding this new variable to the existing PREMM55 model, this new model performed better than MMRpredict for PMS2 mutation car- riers. Validation of the extended PREMM5 model 0.96 [95% CI PREMM5 model than for the original PREMM5 model (AUC 0.97 [95% CI 0.94–1.00] vs. 0.95 [95% CI 0.91–0.99] for MLH1,0.97 [95% CI 0.93–1.00] vs. 0.96 [95% CI 1 368 A. Goverde et al. 0.92–0.99] for MSH2 and 0.86 [95% CI 0.97–0.93] vs. 0.85 [95% CI 0.77–0.93] for MSH6 mutation carriers). Multi-Society Task Force on Colorectal Cancer recom- mends genetic evaluation if an individual’s risk of carrying an MMR gene mutation is ≥ 5% according to one of the prediction models MMRpro, MMRpredict or PREMM [34]. The American guideline recommends that all CRC patients undergo routine screening for LS by analysis of MSI and IHC [34], while current European guidelines recommend such routine screening in at least all CRC patients up to 70 years of age [35]. A recent study demonstrated that rou- tine screening for LS without an age cut-off is not cost-effec- tive [36]. A strategy using prediction models might lower the cost of screening for LS. In fact, two cost-effectiveness analyses found that strategies including prediction models were more cost-effective than those involving direct tumour testing of all CRC patients, if these prediction models were perfectly implemented [36, 37]. Additionally, prediction models could also be used in cases where no tumour tissue is available or where tumour tissue analysis failed, to assess whether an individual should be analysed for a germline MMR mutation. 1 3 References 1. Lynch HT, de la Chapelle A (2003) Hereditary colorectal cancer. N Engl J Med 348(10):919–932 1. Lynch HT, de la Chapelle A (2003) Hereditary colorectal cancer. N Engl J Med 348(10):919–932 2. Watson P, Lynch HT (1993) Extracolonic cancer in hereditary nonpolyposis colorectal cancer. Cancer 71(3):677–685 3. de Jong AE, Hendriks YM, Kleibeuker JH et al (2006) Decrease in mortality in Lynch syndrome families because of surveillance. Gastroenterology 130(3):665–671 4. Jarvinen HJ, Aarnio M, Mustonen H et al (2000) Controlled 15-year trial on screening for colorectal cancer in families with hereditary nonpolyposis colorectal cancer. Gastroenterology 118(5):829–834 5. Jarvinen HJ, Renkonen-Sinisalo L, Aktan-Collan K, Peltomaki P, Aaltonen LA, Mecklin JP (2009) Ten years after mutation testing for Lynch syndrome: cancer incidence and outcome in mutation- positive and mutation-negative family members. J Clin Oncol 27(28):4793–4797 , , A main strength of our study was the large cohort, which consisted of more than 700 index patient including 83 Lynch syndrome patients. Also, our cohort included patients with MSH6 and PMS2 mutations. Since 12 patients were identi- fied as a PMS2 mutation carrier, we were able to evaluate the prediction models for each MMR mutation specifically, admittedly with considerable uncertainty [39]. Furthermore, we validated the extended PREMM5 model in a separate cohort of 376 patients including 73 PMS2 mutation carriers. 6. Akiyama Y, Sato H, Yamada T et al (1997) Germ-line mutation of the hMSH6/GTBP gene in an atypical hereditary nonpolyposis colorectal cancer kindred. Cancer Res 57(18):3920–3923 7. Bronner CE, Baker SM, Morrison PT et al (1994) Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature 368(6468):258–261 8. Fishel R, Lescoe MK, Rao MR et al (1993) The human mutator gene homolog MSH2 and its association with hereditary nonpoly- posis colon cancer. Cell 75(5):1027–1038 9. Nicolaides NC, Papadopoulos N, Liu B et al (1994) Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature 371(6492):75–80 A limitation of our study was that germline mutation analysis was not done for all index patients. Patients who had microsatellite stable tumours with normal IHC were assumed to be non-mutation carriers. However, some of these patients might still have an MMR mutation. Also, the sample size per gene was still relatively small and it is unclear whether our results from a high-risk population apply to a population-based setting. 10. Compliance with ethical standards Compliance with ethical standards Conflict of interest None of the authors declare a conflict of interest. Conflict of interest None of the authors declare a conflict of interest. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea- tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. It is not known whether the current prediction models for Lynch syndrome are useful in non-Western populations. In a recent study among Korean patients, PREMM1,2,6 was more accurate than MMRpro and MMRpredict, but still only reached an AUC of 0.71 [32]. There was no associa- tion between tumour location and mutation status, so our extended PREMM5 model might not improve predictions in populations of non-Western ethnicity. However, germline analysis for PMS2 was not performed in the Korean study, so there might have been more mutation carriers in their cohort. Another non-Western population has been studied by Khan et al., who analysed the performance of prediction models in 15 African American patients [22]. In these patients, MMR- predict and PREMM1,2,6 both had a high AUC of 0.89. Discussion The extended PREMM55 model also performed better than the original model for MLH1, MSH2 and MSH6 muta- tion carriers and had a better overall performance. , , Both PREMM5 and MMRpredict were far more accurate for MLH1 and MSH2 mutation carriers than for LS patients carrying a mutation in MSH6 or PMS2. This finding is in line with a previous study that showed that carriers of muta- tions in MSH6 or PMS2 had lower risk scores than carriers of a mutation in MLH1 or MSH2 [21]. In our study, discrimi- nation between non-mutation carriers and PMS2 mutation carriers was the least accurate, in line with its more limited penetrance. In our validation cohort, all AUCs were much higher than in our original cohort, including those for PMS2 mutation carriers. Selection of patients for analysis of MSI and IHC may have been less stringent at the Erasmus Medical Center Rotterdam than at the Leiden University Medical Center. Therefore, mutation carriers in our validation cohort, who were all from Leiden University Medical Center, may have had a family history more suspect for Lynch syndrome than family history of the patients in our original cohort. This could explain the higher AUCs in the validation cohort. However, in both cohorts we showed that the extended PREMM5 had better performance. Around 15% of all Lynch syndrome cases are estimated to be caused by PMS2 mutations [38]. In our cohort, 14% (12/83) of the Lynch syndrome patients were PMS2 muta- tion carriers. To our knowledge, our study is the first to vali- date LS prediction models for PMS2 mutation carriers spe- cifically since the development of the PREMM5 model. At a 5% cut-off, our extended PREMM5 model was able to detect 5/6 PMS2 mutation carriers who would have been missed by the original PREMM5 model at the same cut-off. Identifica- tion of Lynch syndrome carriers is highly important, since Prediction models for Lynch syndrome are not yet reg- ularly used in current clinical practice. However, the US 1 3 Evaluation of current prediction models for Lynch syndrome: updating the PREMM5 model to identify… 369 identify patients with PMS2 mutations. Adding the location of CRC to the PREMM5 model improves the performance of the model for PMS2 mutation carriers as well as its overall performance. These findings should be validated in large cohorts from population-based settings. identify patients with PMS2 mutations. Discussion Adding the location of CRC to the PREMM5 model improves the performance of the model for PMS2 mutation carriers as well as its overall performance. These findings should be validated in large cohorts from population-based settings. this allows not only them, but also their family members carrying the same mutation, to undergo intensive surveil- lance in order to prevent the development of cancer. Our new model would also identify more Lynch syndrome patients overall than the original PREMM5 model. The performance of prediction models can differ between high-risk settings and population-based cohorts. Further validation studies should indicate whether our results can be generalized to settings with patients at low to median risk of having Lynch syndrome. Since patients in our study cohort were all referred for genetic counselling, family his- tories were obtained in detail and in many cases also verified by medical documents. In other settings where patients are at lower risk of having Lynch syndrome, family history is not verified and might be less reliable. Therefore, predic- tion models should also be validated in population-based cohorts. However, in a meta-analysis, prediction models performed better in population-based cohorts than in clinic- based cohorts [27]. Acknowledgements We thank M Nielsen and JT Wijnen for their contribution in the data collection of this study. Acknowledgements We thank M Nielsen and JT Wijnen for their contribution in the data collection of this study. References Niessen RC, Hofstra RM, Westers H et al (2009) Germline hyper- methylation of MLH1 and EPCAM deletions are a frequent cause of Lynch syndrome. Genes Chromosomes Cancer 48(8):737–744 11. Aaltonen LA, Salovaara R, Kristo P et al (1998) Incidence of hereditary nonpolyposis colorectal cancer and the feasi- bility of molecular screening for the disease. N Engl J Med 338(21):1481–1487 12. de la Chapelle A (2003) Microsatellite instability. N Engl J Med 349(3):209–210 In conclusion, we have shown that although MMRpredict and PREMM5 can accurately predict an individual’s risk of carrying a causative MMR mutation, neither model is able to 1 3 370 A. Goverde et al. 27. Win AK, Macinnis RJ, Dowty JG, Jenkins MA (2013) Criteria and prediction models for mismatch repair gene mutations: a review. J Med Genet 50(12):785–793 13. van Lier MG, Wagner A, van Leerdam ME et al (2010) A review on the molecular diagnostics of Lynch syndrome: a central role for the pathology laboratory. J Cell Mol Med 14(1–2):181–197f 28. Boland CR, Thibodeau SN, Hamilton SR et al (1998) A National Cancer Institute Workshop on Microsatellite Instability for can- cer detection and familial predisposition: development of interna- tional criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 58(22):5248–5257 14. Cross DS, Rahm AK, Kauffman TL et al (2013) Underutilization of Lynch syndrome screening in a multisite study of patients with colorectal cancer. Genet Med 15(12):933–940i 15. Julie C, Tresallet C, Brouquet A et al (2008) Identification in daily practice of patients with Lynch syndrome (hereditary nonpolypo- sis colorectal cancer): revised Bethesda guidelines-based approach versus molecular screening. Am J Gastroenterol 103(11):2825– 2835 (quiz 36) 29. Suraweera N, Duval A, Reperant M et al (2002) Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR. Gastroenterology 123(6):1804–1811i q 16. Perez-Carbonell L, Ruiz-Ponte C, Guarinos C et al (2012) Com- parison between universal molecular screening for Lynch syn- drome and revised Bethesda guidelines in a large population-based cohort of patients with colorectal cancer. Gut 61(6):865–872 30. van der Klift HM, Tops CM, Bik EC et al (2010) Quantification of sequence exchange events between PMS2 and PMS2CL pro- vides a basis for improved mutation scanning of Lynch syndrome patients. Hum Mutat 31(5):578–587f 17. Van Lier MG, De Wilt JH, Wagemakers JJ et al (2009) Underuti- lization of microsatellite instability analysis in colorectal cancer patients at high risk for Lynch syndrome. References Scand J Gastroenterol 44(5):600–604i 31. Van Hoorde K, Vergouwe Y, Timmerman D, Van Huffel S, Stey- erberg EW, Van Calster B (2014) Assessing calibration of multi- nomial risk prediction models. Stat Med 33(15):2585–2596 18. Barnetson RA, Tenesa A, Farrington SM et al (2006) Identifica- tion and survival of carriers of mutations in DNA mismatch-repair genes in colon cancer. N Engl J Med 354(26):2751–2763 32. Lee SY, Kim DW, Shin YK et al. (2015) Validation of prediction models for mismatch repair gene mutations in Koreans. Cancer Res Treat. doi:10.4143/crt.2014.288 g g 19. Chen S, Wang W, Lee S et al (2006) Prediction of germline mutations and cancer risk in the Lynch syndrome. JAMA 296(12):1479–1487 33. Kastrinos F, Ojha RP, Leenen C et al. (2016) Comparison of prediction models for Lynch syndrome among individuals with colorectal cancer. J Natl Cancer Inst. doi:10.1093/jnci/djv308 20. Kastrinos F, Uno H, Ukaegbu C et al (2017) Development and Validation of the PREMM5 Model for Comprehensive Risk Assessment of Lynch Syndrome. J Clin Oncol 35(19):2165–2172 34. Giardiello FM, Allen JI, Axilbund JE et al (2014) Guidelines on genetic evaluation and management of Lynch syndrome: a consen- sus statement by the US Multi-Society Task Force on Colorectal Cancer. Gastroenterology 147(2):502–526 21. Green RC, Parfrey PS, Woods MO, Younghusband HB (2009) Prediction of Lynch syndrome in consecutive patients with colo- rectal cancer. J Natl Cancer Inst 101(5):331–340 35. Vasen HF, Blanco I, Aktan-Collan K et al (2013) Revised guidelines for the clinical management of Lynch syndrome (HNPCC): recommendations by a group of European experts. Gut 62(6):812–823 22. Khan O, Blanco A, Conrad P et al (2011) Performance of Lynch syndrome predictive models in a multi-center US referral popula- tion. Am J Gastroenterol 106(10):1822–1827 (quiz 8) 36. Barzi A, Sadeghi S, Kattan MW, Meropol NJ (2015) Comparative effectiveness of screening strategies for Lynch syndrome. J Natl Cancer Inst. doi:10.1093/jnci/djv005 23. Monzon JG, Cremin C, Armstrong L et al (2010) Validation of predictive models for germline mutations in DNA mismatch repair genes in colorectal cancer. Int J Cancer 126(4):930–939 37. Ladabaum U, Wang G, Terdiman J et al (2011) Strategies to iden- tify the Lynch syndrome among patients with colorectal cancer: a cost-effectiveness analysis. Ann Intern Med 155(2):69–79 24. 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https://openalex.org/W2898448832	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0203016&type=printable	English	null	Correction: Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine	PloS one	2,018	cc-by	721	CORRECTION Correction: Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate- Phosphatidylcholine and Phosphatidylethanolamine Eriko Abe, Kazutaka Ikeda, Eri Nutahara, Masahiro Hayashi, Atsushi Yamashita, Ryo Taguchi, Kosaku Doi, Daiske Honda, Nozomu Okino, Makoto Ito The titles and legends for Figs 2 and 3 are swapped. Please see the correct Figs 2 and 3 here. CORRECTION OPEN ACCESS Citation: Abe E, Ikeda K, Nutahara E, Hayashi M, Yamashita A, Taguchi R, et al. (2018) Correction: Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate- Phosphatidylcholine and Phosphatidylethanolamine. PLoS ONE 13(8): e0203016. https://doi.org/10.1371/journal. pone.0203016 Correction: Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate- Phosphatidylcholine and Phosphatidylethanolamine Eriko Abe, Kazutaka Ikeda, Eri Nutahara, Masahiro Hayashi, Atsushi Yamashita, Ryo Taguchi, Kosaku Doi, Daiske Honda, Nozomu Okino, Makoto Ito The titles and legends for Figs 2 and 3 are swapped. Please see the correct Figs 2 and 3 here. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203016 August 23, 2018 Fig 3. Alignment of PLAT1, mLPCAT1, and mLPCAT2. PLAT1 (this work), mLPCAT1 (LPCAT1 from mouse), and mLPCAT2 (LPCAT2 from mouse) sequences were aligned using GENETYX ver.8.2.2. The conserved amino acids are shown by white characters on a black background. The four conserved AGPAT motifs are indicated by red boxes. Two transmembrane regions, predicted by TMHMM server v. 2.0 (www.cbs.dtu.dk/services/TMHMM/), are underlined in blue. Three EF hand Ca2+-binding motifs, predicted by PROSITE (www.expasy.ch/prosite/), are indicated by green dashed-lines. ER-retaining motifs are indicated by red characters. https://doi.org/10.1371/journal.pone.0203016.g002 Fig 3. Alignment of PLAT1, mLPCAT1, and mLPCAT2. PLAT1 (this work), mLPCAT1 (LPCAT1 from mouse), and mLPCAT2 (LPCAT2 from mouse) sequences were aligned using GENETYX ver.8.2.2. The conserved amino acids are shown by white characters on a black background. The four conserved AGPAT motifs are indicated by red boxes. Two transmembrane regions, predicted by TMHMM server v. 2.0 (www.cbs.dtu.dk/services/TMHMM/), are underlined in blue. Three EF hand Ca2+-binding motifs, predicted by PROSITE (www.expasy.ch/prosite/), are indicated by green dashed-lines. ER-retaining motifs are indicated by red characters. https://doi.org/10.1371/journal.pone.0203016.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203016 August 23, 2018 1. Abe E, Ikeda K, Nutahara E, Hayashi M, Yamashita A, Taguchi R, et al. (2014) Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate- Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine. PLoS ONE 9(8): e102377. https://doi.org/10.1371/journal.pone.0102377. PMID: 25090090 Published: August 23, 2018 Copyright: © 2018 Abe 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. 1 / 3 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203016 August 23, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203016 August 23, 2018 Fig 2. Phylogenetic tree o f LPLAT family members. The phylogenetic tree was drawn using CLUSTALW, DDBJ (http://clustalw.ddbj.nig.ac.jp/ top-j.html). LPLAT sequences are available on the NCBI database. The accession numbers are as follows: mGPAT1 (NP_032175), mGPAT2 (XP_130488), mGPAT3/LPAATh (NP_766303), mLPAATa (NP_061350), mLPAATb (NP_080488), mLPAATc (NP_443747), mLPAATd (NP_080920), mLPAATe (NP_081068), mGPAT4/LPAATf (NP_061213), mAT Like 1B (NP_081875), mLPGAT1 (NP_758470), mALCAT (Q3UN02), mLPCAT1 (BAE94687), mLysoPAFAT/LPCAT2 (BAF47695), mTafazzin (NP_852657), mMGAT1 (NP_080989), mMGAT2 (NP_803231), mDGAT2 (NP_080660), mDGAT2Like3 (NP_001074605), mDGAT2Like4 (NP_808414), mDGAT2Like6 (CAM19588), mLPCAT3/ MBOAT5 (NP_660112), mLPCAT4/MBOAT2 (NP_080313), mLPEAT1/MBOAT1 (NP_705774), mMBOAT4 (XP_134120), mDGAT1 (NP_034176), mACAT1 (NP_033256), mACAT2 (NP_666176), mPorcupine-a (NP_058609), mLRC4 (NP_084210), sLpt1 (BAF93897), and sAle1 (EWH15997); s, Saccharomyces cerevisiae, m, Mus musculus. https://doi org/10 1371/journal pone 0203016 g001 Fig 2. Phylogenetic tree o f LPLAT family members. The phylogenetic tree was drawn using CLUSTALW, DDBJ (http://clustalw.ddbj.nig.ac.jp/ top-j.html). LPLAT sequences are available on the NCBI database. The accession numbers are as follows: mGPAT1 (NP_032175), mGPAT2 (XP_130488), mGPAT3/LPAATh (NP_766303), mLPAATa (NP_061350), mLPAATb (NP_080488), mLPAATc (NP_443747), mLPAATd (NP_080920), mLPAATe (NP_081068), mGPAT4/LPAATf (NP_061213), mAT Like 1B (NP_081875), mLPGAT1 (NP_758470), mALCAT (Q3UN02), mLPCAT1 (BAE94687), mLysoPAFAT/LPCAT2 (BAF47695), mTafazzin (NP_852657), mMGAT1 (NP_080989), mMGAT2 (NP_803231), mDGAT2 (NP_080660), mDGAT2Like3 (NP_001074605), mDGAT2Like4 (NP_808414), mDGAT2Like6 (CAM19588), mLPCAT3/ MBOAT5 (NP_660112), mLPCAT4/MBOAT2 (NP_080313), mLPEAT1/MBOAT1 (NP_705774), mMBOAT4 (XP_134120), mDGAT1 (NP_034176), mACAT1 (NP_033256), mACAT2 (NP_666176), mPorcupine-a (NP_058609), mLRC4 (NP_084210), sLpt1 (BAF93897), and sAle1 (EWH15997); s, Saccharomyces cerevisiae, m, Mus musculus. https://doi.org/10.1371/journal.pone.0203016.g001 2 / 3 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203016 August 23, 2018 https://doi.org/10.1371/journal.pone.0203016.g002 Reference 1. Abe E, Ikeda K, Nutahara E, Hayashi M, Yamashita A, Taguchi R, et al. (2014) Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate- Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine. PLoS ONE 9(8): e102377. https://doi.org/10.1371/journal.pone.0102377. PMID: 25090090 3 / 3
https://openalex.org/W3197313757	https://europepmc.org/articles/pmc8446606?pdf=render	English	null	High-Throughput Phenotyping and Random Regression Models Reveal Temporal Genetic Control of Soybean Biomass Production	Frontiers in plant science	2,021	cc-by	14,328	ORIGINAL RESEARCH published: 03 September 2021 doi: 10.3389/fpls.2021.715983 ORIGINAL RESEARCH published: 03 September 2021 doi: 10.3389/fpls.2021.715983 High-Throughput Phenotyping and Random Regression Models Reveal Temporal Genetic Control of Soybean Biomass Production Fabiana Freitas Moreira1, Hinayah Rojas de Oliveira2, Miguel Angel Lopez1, Bilal Jamal Abughali3, Guilherme Gomes4, Keith Aric Cherkauer3, Luiz Fernando Brito2 and Katy Martin Rainey1* Fabiana Freitas Moreira1, Hinayah Rojas de Oliveira2, Miguel Angel Lopez1, Bilal Jamal Abughali3, Guilherme Gomes4, Keith Aric Cherkauer3, Luiz Fernando Brito2 and Katy Martin Rainey1* Keywords: digital agriculture, Glycine max, longitudinal traits, phenomics, plant breeding, time series, quantitative genetics Reviewed by: Karl Kunert, University of Pretoria, South Africa Johann Vollmann, University of Natural Resources and Life Sciences, Austria *Correspondence: Katy Martin Rainey krainey@purdue.edu Specialty section: This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science Received: 27 May 2021 Accepted: 26 July 2021 Published: 03 September 2021 Specialty section: This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science Received: 27 May 2021 Accepted: 26 July 2021 Published: 03 September 2021 1Department of Agronomy, Purdue University, West Lafayette, IN, United States, 2Department of Animal Sciences, Purdue University, West Lafayette, IN, United States, 3Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN, United States, 4Department of Statistics, Purdue University, West Lafayette, IN, United States Edited by: Sean Mayes, University of Nottingham, United Kingdom Understanding temporal accumulation of soybean above-ground biomass (AGB) has the potential to contribute to yield gains and the development of stress-resilient cultivars. Our main objectives were to develop a high-throughput phenotyping method to predict soybean AGB over time and to reveal its temporal quantitative genomic properties. A subset of the SoyNAM population (n = 383) was grown in multi-environment trials and destructive AGB measurements were collected along with multispectral and RGB imaging from 27 to 83 days after planting (DAP). We used machine-learning methods for phenotypic prediction of AGB, genomic prediction of breeding values, and genome-wide association studies (GWAS) based on random regression models (RRM). RRM enable the study of changes in genetic variability over time and further allow selection of individuals when aiming to alter the general response shapes over time. AGB phenotypic predictions were high (R2 = 0.92–0.94). Narrow-sense heritabilities estimated over time ranged from low to moderate (from 0.02 at 44 DAP to 0.28 at 33 DAP). AGB from adjacent DAP had highest genetic correlations compared to those DAP further apart. We observed high accuracies and low biases of prediction indicating that genomic breeding values for AGB can be predicted over specific time intervals. Genomic regions associated with AGB varied with time, and no genetic markers were significant in all time points evaluated. Thus, RRM seem a powerful tool for modeling the temporal genetic architecture of soybean AGB and can provide useful information for crop improvement. This study provides a basis for future studies to combine phenotyping and genomic analyses to understand the genetic architecture of complex longitudinal traits in plants. INTRODUCTION A simple repeatability (SR) model treats the individual measurements recorded over time as repeated records of the same trait (Meyer and Hill, 1997). This model assumes that the variances of different measurements are equal and the genetic correlations between all measurements are equal to one, which is an unrealistic assumption for most crop studies (Falconer and Mackay, 1996; Meyer and Hill, 1997; Littell et al., 1998). An alternative method that overcomes these restrictions is a multiple-trait model (MTM), which treats individual measurements over time as different traits. However, high- dimensional longitudinal data can lead to high correlations between consecutive measurements and over-parameterized models with high computational demands, restricting the application of MTM (Foster et al., 2006; Speidel, 2011). Random regression models (RRM) provide a robust framework for estimating breeding values and identifying alleles with time-specific effects for longitudinal traits (Oliveira et al., 2019a; Moreira et al., 2020) In summary, RRM use a given covariance function to describe the trajectory of the trait as a function of time (or environmental gradient), with no assumptions for constant variances and correlations (Kirkpatrick et al., 1990; Meyer and Hill, 1997; Schaeffer, 2016). RRM have some key advantages compared to other models, such as (1) greater computational efficiency, (2) prediction of breeding values for any time point within the range of data collection, and (3) more accurate breeding values (Oliveira et al., 2019a). RRM were originally proposed for use in livestock breeding programs and have been successfully used for genetic evaluation of longitudinal traits (Jamrozik and Schaeffer, 1997; Schaeffer, 2004; van Pelt et al., 2015; Englishby et al., 2016; Oliveira et al., 2019a), but have only recently been implemented in crops (Sun et al., 2017; Campbell et al., 2018, 2019). Thus, we hypothesized that RRM can be efficiently used to model temporal measurements of complex polygenic traits in crops. Measuring crop AGB across developmental stages is laborious, involving cutting, drying, and weighing plants from a target area, and is subject to errors and limitations resulting from (1) unrepresentative samples; (2) destructive sampling, which limits the number of samples that can be collected from a plot, and prevents longitudinal tracking of the same target area; and (3) extensive manual handling, which may lead to sample loss, and can be restrictive in large experiments (Jimenez- Berni et al., 2018). INTRODUCTION its life stages, and how it responds to the environment (Moreira et al., 2020). These measurements represent the crop in different “ages” or stages of development, with the mean and variance between measurements usually changing over time, characterizing the trait as longitudinal (Falconer and Mackay, 1996; Yang et al., 2006; Oliveira et al., 2019a). In animals, it has been shown that the phenotypic or additive polygenic effects of longitudinal traits are not constant during expression of longitudinal traits (Szyda et al., 2014; Brito et al., 2018; Oliveira et al., 2019a), so that breeders need an amenable statistical framework for genetic and genomic analysis that accounts for time-dependent genetic contributions to the phenotypes of longitudinal traits. Soybean [Glycine max (L.) Merr.] is one of the most economically important crops worldwide, being the primary source of plant-based protein, and the second largest source of vegetable oil (USDA, 2018). Advances in plant breeding and agronomic methods have substantially improved soybean yield over time (Anderson et al., 2019). Yield potential in any environment or cropping system can be expressed as a function of biomass produced, and the partitioning of biomass to the seeds, or harvest index (Monteith, 1972, 1977). Assessments of historical soybean germplasm have shown that increases in soybean grain yield over the last several decades are associated with increases in biomass production (Cregan and Yaklich, 1986; Frederick et al., 1991; Kumudini et al., 2001; De Bruin and Pedersen, 2009; Koester et al., 2014; Balboa et al., 2018). For instance, Koester et al. (2014) measured above-ground biomass (AGB) every 2 weeks in cultivars released between 1923 and 2007 and observed that biomass production per unit of absorbed light increased with the release year. Additionally, information on temporal biomass production provides insights into crop development and responses to multiple abiotic and biotic stressors (Bajgain et al., 2015; Jumrani and Bhatia, 2018). Increased temperatures and water stress have imposed vegetative and reproductive stage reduced AGB significantly and resulted in 28% and 74% reduction in soybean yield, respectively (Jumrani and Bhatia, 2018). Hence, understanding the genetic factors controlling the temporal dynamics of biomass accumulation may contribute to future soybean yield gains and the development of stress-resilient cultivars. p yp g Different approaches can be utilized for genomic evaluation of longitudinal traits (Moreira et al., 2020). Citation: Moreira FF, Oliveira HR, Lopez MA, Abughali BJ, Gomes G, Cherkauer KA, Brito LF and Rainey KM (2021) High-Throughput Phenotyping and Random Regression Models Reveal Temporal Genetic Control of Soybean Biomass Production. Front. Plant Sci. 12:715983. doi: 10.3389/fpls.2021.715983 September 2021 \| Volume 12 \| Article 715983 1 Frontiers in Plant Science \| www.frontiersin.org Random Regression Soybean Biomass Moreira et al. Plant Materials, Field Experiments, and Genotypic Data Radiometric calibration was done for every sampling date to remove atmospheric effects and potentially correct for any sensor sensitivity issues (Iqbal et al., 2018). During flight operations, we laid out four reflectance panels reflecting at a specific and consistent percentage of light (12, 22, 36, and 48% reflectance). A handheld spectrometer ASD FieldSpec® 4 (ASD, Boulder, CO, United States) was used to measure the true reflectance of the panels while the multispectral images were collected. We used the reflectance values from the panels, along with radiance values of the panels, extracted from the generated ortho-mosaics, to correct the radiance values for the plots using the empirical line method (Smith and Milton, 1999), which is crucial in producing reflectance data over the plots. The reflectance from the calibrated images was used to calculate vegetation indices (VI) using the VID 1.0 pipeline. Vegetation indices are typically used to estimate crop biomass, and for this study, we selected 14 VIs (Supplementary Table 1) previously reported in the literature to correlate with crop biomass (Babar et al., 2006; Bendig et al., 2015; Wang et al., 2016; Yue et al., 2017; Sankaran et al., 2018). In the biomass sampling panel, AGB was collected approximately every 10 days during the growing season between 27 to 83 DAP, from a linear section of 0.56 m in a row with borders. In 2017, we randomly picked plots to measure AGB in replication one for every sampling date, while in 2018, three full AGB sampling (~38, 58, and 84 DAP) were performed for both locations in the two full replications. The fresh AGB was dried at 80°C using a dry-air system until achieving constant weight. Finally, we obtained the dry AGB weight and rescaled it to g/m2. Figure 1 shows the data collection timeline for each environment and the respective phenological stage periods.h From the RGB imagery, we calculated canopy coverage (CC) using the software Progeny® (Progeny Drone Inc., West Lafayette, IN, United States) and the multilayer mosaic approach as described by Hearst (2019). The list of the imagery features used in this study is in Supplementary Table 1. All imagery features were calculated in intact and bordered plot rows not used for destructive biomass sampling. The SoyNAM founder parents were genotyped by Song et al. MATERIALS AND METHODS High-Throughput Phenotyping RGB and multispectral imagery were collected with fixed-wing SenseFly eBee unmanned aerial vehicle (UAV). RGB imagery was collected using a S.O.D.A. camera (SenseFly Parrot Group, Switzerland). Multispectral imagery was collected with a 1.2 MP Parrot Sequoia camera (MicaSense Inc., Seattle, United States), which captures four discrete spectral bands: green (wavelength = 550 nm, bandwidth = 40 nm), red (660 nm, 40 nm), red-edge (735 nm, 10 nm), and near-infrared (790 nm, 40 nm). Flights were performed close to solar noon at an altitude of approximately 120 m with both RGB and multispectral cameras. The forward and side overlap for flights were set to at least 85 and 70%, respectively. Ground control points were installed at the corners of the trials and their GPS coordinates were recorded using the TOPCON RTK (Topcon, Tokyo, Japan). High-Throughput Phenotyping INTRODUCTION To process the multispectral imagery from this experiment, two pipelines were built in MATLAB: Crop Image Extraction version 2 (CIE 2.0) and Vegetation Indices Derivation version 1 (VID 1.0; Lyu et al., 2019). The multispectral images were stitched using Pix4Dmapper (Pix4D SA, 2018) to produce a full ortho-mosaic of the experimental area. Individual plots were extracted from the ortho-mosaic using the CIE 2.0. Segmentation was performed to highlight the canopy of the vegetation using the Otsu’s method (Otsu, 1979). Radiometric calibration was done for every sampling date to remove atmospheric effects and potentially correct for any sensor sensitivity issues (Iqbal et al., 2018). During flight operations, we laid out four reflectance panels reflecting at a specific and consistent percentage of light (12, 22, 36, and 48% reflectance). A handheld spectrometer ASD FieldSpec® 4 (ASD, Boulder, CO, United States) was used to measure the true reflectance of the panels while the multispectral images were collected. We used the reflectance values from the panels, along with radiance values of the panels, extracted from the generated ortho-mosaics, to correct the radiance values for the plots using the empirical line method (Smith and Milton, 1999), which is crucial in producing reflectance data over the plots. The reflectance from the calibrated images was used to calculate vegetation indices (VI) using the VID 1.0 pipeline. Vegetation indices are typically used to estimate crop biomass, and for this study, we selected 14 VIs (Supplementary Table 1) previously reported in the literature to correlate with crop biomass (Babar et al., 2006; Bendig et al., 2015; Wang et al., 2016; Yue et al., 2017; Sankaran et al., 2018). From the RGB imagery, we calculated canopy coverage (CC) using the software Progeny® (Progeny Drone Inc., West Lafayette, IN United States) and the multilayer mosaic approach as Frontiers in Plant Science \| www.frontiersin.org Plant Materials, Field Experiments, and Genotypic Data We used a set of 383 recombinant inbred lines (RILs) representing 32 families from the Nested Association Mapping (SoyNAM) population (~12 RILs per family; Diers et al., 2018). The lines comprising the set were selected using breeding values for full maturity (R8; Fehr and Caviness, 1977) and grain yield, calculated from experiments performed in Indiana and Illinois from 2011 to 2014, in order to have a maturity-controlled panel (Xavier et al., 2016; Lopez et al., 2019). More details about the RIL panel selection and the full list of traits’ collection and distribution are described in Lopez et al. (2019). The RILs were grown under a randomized complete block design with two replications at the Purdue University Agronomy Center for Research and Education (ACRE), West Lafayette, IN, United States (40°28'20.5”N 86°59'32.3”W) and Romney, IN, United States (40°14'59.1'N 86°52'49.4'W). Planting occurred on May 31, 2017 and May 22, 2018 at ACRE, and May 17, 2018 at Romney. Soil fertility information and environmental conditions summarized by days after planting (DAP) for this experiment are described in Lopez et al. (2019). The combination of year and location where the experiment was grown was considered as an environment, resulting in three environments in this study (2017_ACRE, 2018_ACRE, and 2018_Romney). Experimental units consisted of a six-row plot (3.35 m with 0.76 m) with a targeted seeding rate of 35 seeds m−2. A total of 66 and 16 RILs were discarded in 2017 and 2018, respectively, because of poor emergence. In addition to the two full replications, we randomly selected 62 RILs in 2017 and 108 RILs in 2018 (the same 62 RILs in 2017 plus 46 others) to grow in a trail of eight-row plots (0.76 m × 3.35 m). This trail was defined as the biomass sampling panel and it was used as sampling plots for destructive AGB measurements throughout the growing season. To process the multispectral imagery from this experiment, two pipelines were built in MATLAB: Crop Image Extraction version 2 (CIE 2.0) and Vegetation Indices Derivation version 1 (VID 1.0; Lyu et al., 2019). The multispectral images were stitched using Pix4Dmapper (Pix4D SA, 2018) to produce a full ortho-mosaic of the experimental area. Individual plots were extracted from the ortho-mosaic using the CIE 2.0. Segmentation was performed to highlight the canopy of the vegetation using the Otsu’s method (Otsu, 1979). Plant Materials, Field Experiments, and Genotypic Data (2013) using the SoySNP50K BeadChip resulting in 42,509 segregating SNP markers that were imputed into the SoyNAM RILS using the Williams 82 reference genome (Wm82.a2.v1) bp positions by Diers et al. (2018). For genotypic quality control, we excluded SNPs with minor allele frequency lower than 0.05 and call rate lower than 0.90, resulting in 40,110 SNPs for the genome-wide analyses. INTRODUCTION High-throughput phenotyping platforms (HTPP) offer alternatives to ground-based AGB sampling, enabling collection of non-destructive data throughout the growing season in large experiments under actual field conditions (van Eeuwijk et al., 2018; Zhao et al., 2019). In some crops, such as wheat, barley, rice, and dry beans, AGB accumulation has been recognized as a potential target to increase yield gain, and the success of image-based AGB phenotyping has been demonstrated (Serrano et al., 2000; Babar et al., 2006; Tilly et al., 2014; Cheng et al., 2017; Neumann et al., 2017; Yue et al., 2017; Sankaran et al., 2018). In soybean, Maimaitijiang et al. (2019) used red, green and blue (RGB) imagery-derived metrics to predict AGB in production fields; however, there are no studies on the use of HTPP to estimate soybean AGB in experimental plots with different genotypes used for plant breeding. In this context, this study aimed to: (1) develop an HTTP methodology to estimate soybean AGB throughout the growing season; (2) reveal the genetic architecture and estimate time- dependent effects of single-nucleotide polymorphisms (SNPs) associated with this longitudinal trait using RRM; and (3) investigate the feasibility of implementing genomic selection for longitudinal traits in soybean using RRM. High-throughput phenotyping (HTP) allows time-series measurements that monitor the development of a crop through September 2021 \| Volume 12 \| Article 715983 Frontiers in Plant Science \| www.frontiersin.org 2 Random Regression Soybean Biomass Moreira et al. High-Throughput Phenotyping RGB and multispectral imagery were collected with fixed-wing SenseFly eBee unmanned aerial vehicle (UAV). RGB imagery was collected using a S.O.D.A. camera (SenseFly Parrot Group, Switzerland). Multispectral imagery was collected with a 1.2 MP Parrot Sequoia camera (MicaSense Inc., Seattle, United States), which captures four discrete spectral bands: green (wavelength = 550 nm, bandwidth = 40 nm), red (660 nm, 40 nm), red-edge (735 nm, 10 nm), and near-infrared (790 nm, 40 nm). Flights were performed close to solar noon at an altitude of approximately 120 m with both RGB and multispectral cameras. The forward and side overlap for flights were set to at least 85 and 70%, respectively. Ground control points were installed at the corners of the trials and their GPS coordinates were recorded using the TOPCON RTK (Topcon, Tokyo, Japan). Random Regression Models RRM were used to model AGB across 27 to 83 DAPs. Seven different models were tested: third-, fourth-, and fifth-order Legendre orthogonal polynomials (Kirkpatrick et al., 1990) and linear and quadratic B-splines (de Boor, 1980; Meyer, 2005) with one (at 55 DAP) or two knots (at 44 and 66 DAPs). In RRM, Legendre orthogonal polynomials and B-splines (segmented polynomials joined by knots) are used to describe the covariance structure of the data as a function of time (de Boor, 1980; Kirkpatrick et al., 1990; Meyer, 2005).h Regularization methods, such as LASSO, can reduce model complexity using a “penalty” parameter that minimizes the sum of squared error. As such, LASSO performs both regularization and variable selection, by shrinking variable coefficients to zero, and eliminating variables from the model when their coefficients reach zero. The PLSR is an extension of the multiple linear regression and principal component analysis that can also effectively handle the issue of multicollinearity among predictor variables (Wold et al., 2001). Essentially, PLSR performs simultaneous decomposition of the predictor and response variables into latent variables and then identifies key components that explain covariance between them (Abdi, 2010). For the PLSR, 10 principal components were selected so that the root mean squared error (RMSE) from cross-validation was minimized.h p y The general RRM can be described as: y Env b t a t e ijk k m m m m ij m m im m ij ijk = + ∅( )+ ∅( )+ = = ∑ ∑ 1 1 , where yijk is the predicted AGB of the ith RIL on DAP j within environment and replication combination k; Envk is the fixed effect of environment and replication combination; bm is the m fixed regression coefficient for modeling the average curve of the population; aim is the m random regression coefficient that describes the additive genetic effects for the ith RIL; tij is the time of data collection (DAP j) for the ith line; ∅( ) m ijt is a regression function according to DAP j (using Legendre or B-spline polynomials); and eijk is the random residual effect. The number of regression coefficients m varies according to the functions used for random regressions. For the Legendre orthogonal polynomials, ∅( ) m ijt is the mth Legendre orthogonal polynomial coefficient for DAP j (standardized for the −1 to 1 interval) from RIL i. Predicting Above-Ground Biomass g To predict the AGB for all DAP, including days when ground truth data were not available, we considered a linear model using the imagery features as the predictor variables within September 2021 \| Volume 12 \| Article 715983 3 Random Regression Soybean Biomass Moreira et al. FIGURE 1 \| Data collection timeline by environment 2017_ACRE, 2018_ACRE, and 2018_Romney. Planting date in parentheses below environment. UAV: unmanned aerial vehicle. Phenological stages (Fehr and Caviness, 1977): R1, beginning bloom; R5, beginning seed; R7, beginning maturity; and R8, full maturity. FIGURE 1 \| Data collection timeline by environment 2017_ACRE, 2018_ACRE, and 2018_Romney. Planting date in parentheses below environment. UAV: unmanned aerial vehicle. Phenological stages (Fehr and Caviness, 1977): R1, beginning bloom; R5, beginning seed; R7, beginning maturity; and R8, full maturity. prediction ability. Both models were implemented in the R software (R Core Team, 2019), using the package caret (Kuhn, 2008). each environment across all observed DAP. We observed that the distribution of the residuals was highly asymmetric, suggesting that a linear model was not suitable to fit the data (Thoni et al., 1990). To correct the asymmetry, we considered a Box-Cox transformation on the AGB, which led to the log-transformed values (data not shown, Box and Cox, 1964). The prediction of AGB was carried out using two different machine-learning methods: Least Absolute Shrinkage and Selection Operator (LASSO) Regression (Tibshirani, 1996) and Partial Least Squares Regression (PLSR; Wold et al., 2001). Both methods have been commonly used in building predictive models with HTP data (Montes et al., 2011; Bratsch et al., 2017; Wang et al., 2017; Vasseur et al., 2018; Fu et al., 2019). Frontiers in Plant Science \| www.frontiersin.org Random Regression Models ∅( )= ≤<    + ij m m t if T t T 1 1 , ∅( )= ≤<    + m ij m m t if T t T otherwise 1 0 , , ∅( )=  + m ijt f otherwise 0, otherwise 0, . Basis function for p> 0 can . Basis function for p> 0 can hj a a e j j 2 2 2 2 = + s s s    y ∅ ( )= − −      ∅ ( ) + − + − + + + m p ij m m p m m p ij m p m p t T T T t T t T , , t 1 1 + + + − −      ∅ ( ) 1 1 1 1 T t m m p ij , . h where saj 2 is the additive genetic variance for DAP j and where saj 2 is the additive genetic variance for DAP j and be represented by se 2 is the residual variance, which depends on the residual variance classes previously mentioned (when using the heterogeneity of residual variance). The genetic correlation between different DAP ( rj j, ¢) was obtained as: p yh individual segments were either linear or quadratic, with degree p = 1 or 2, respectively. The joined knots allow the function to become continuous. The models’ assumptions are as: rj j a a a j j j j , , , ′= ′ ′ √ +       s s s    2 2 var , a e G G I R      = ⊗ ⊗       0 0 0 where saj j, 2 is the genetic covariance between the DAP j and 2 2 where G0 is the (co)variance matrix of the genomic random regression coefficients, G is a genomic relationship matrix, I is an identity matrix, R represents a matrix containing residual variances, and Ä is the Kronecker product between matrices. The G matrix was calculated using the method presented by VanRaden (2008). The residual variances were allowed to be either homogeneous or heterogeneous. Genomic Prediction of Breeding Valuesh The performance of the genomic prediction of breeding values for AGB was investigated using a 5-fold cross-validation (CV) scheme. Briefly, all RILs were randomly separated into five equal-sized groups, where one group was retained as validation, and four groups were used as training. This procedure was repeated five times, with a unique group used exactly once as the validation set. Variance components and SNP marker effects were estimated based on the training set and used to predict GEBV in the validation set (reduced data). The prediction accuracy was measured using the Pearson’s correlation coefficient (r) estimated between the GEBV predicted using the full data (i.e., data including all training and validation RIL) and the reduced data, only for the validation RIL. To evaluate the genomic prediction bias, regression coefficients (b1) were estimated using linear regression of the GEBV estimated based on the full dataset on the GEBV estimated based on the reduced dataset from each CV fold (GEBV b b GEBV full reduced = + ∗ 0 1 ). Finally, prediction bias (b1) was calculated as the average of CV folds for each DAP. Random Regression Models We defined a different residual variance for each of the 18 DAP with AGB phenotypic data and grouped the remaining days based on their proximity to those DAP. The 18 heterogeneous residual variances classes are as follow: 27–33, 34–36, 37, 38–41, 42–43, 44–45, 46, 47–49, 50–53, 54–58, 59–61, 62, 63–65, 66–71, 72–74, 75–76, 77–80, 81–82, and 83. j¢ , and saj 2 and saj¢ 2 are the additive genetic variances for DAP j and j¢ , respectively. The vector of genomic estimated breeding values (GEBV i ) for all DAP of RIL i was obtained as (Oliveira et al., 2019a): GEBV T g i i = , where gi is the vector of predicted genomic values for the coefficients, for each RILi , and T is a matrix of covariates associated with the assumed function. The AIREMLF90 and BLUPF90 software from the BLUPF90 family (Misztal et al., 2002) were used to estimate the variance components and the solutions of the mixed model equations, respectively. The BLUPF90 family programs perform by default the single-step GBLUP (Misztal et al., 2009; Aguilar et al., 2010; Christensen and Lund, 2010); however, as all RILs were genotyped, the program was adapted to perform the traditional GBLUP (VanRaden, 2008), by using a dummy pedigree file. Akaike’s information criterion (AIC; Akaike, 1974) was used to compare the models’ performance, in which models with lower AIC values were preferred. Genetic Parametersh The genetic (co)variance matrix (Σ) for all DAP within the interval of AGB collection was obtained as (Oliveira et al., 2019a): Random Regression Models In the case of B-splines, ∅( ) m ijt is the mth interval given the previously mentioned knots associated with DAP from RIL i. According to Meyer (2005), the basis function of degree p=0 has values of unity for all points in a given interval (t) and zero otherwise. For the mth interval The performance of the predictive models was evaluated using a 10-fold cross-validation strategy, in which the dataset was randomly divided into a training set (90% of the plots) and validation set (10% of the plots). The predictive accuracy of the model was measured by the coefficient of determination (R2), which is equal to the fraction of AGB variance explained by the model, and by the RMSE, which measures the average error magnitude. Pearson’s correlation coefficient (r) was also considered to quantify the linear correlation between the observations and their estimates, being an indication of model jf The number of regression coefficients m varies according to the functions used for random regressions. For the Legendre orthogonal polynomials, ∅( ) m ijt is the mth Legendre orthogonal polynomial coefficient for DAP j (standardized for the −1 to 1 interval) from RIL i. In the case of B-splines, ∅( ) m ijt is the mth interval given the previously mentioned knots associated with DAP from RIL i. According to Meyer (2005), the basis function of degree p=0 has values of unity for all points in a given interval (t) and zero otherwise. For the mth interval September 2021 \| Volume 12 \| Article 715983 4 Random Regression Soybean Biomass Moreira et al. the proportion of phenotypic variance due to additive genetic variation, for each DAP ( hj 2 ) was obtained as: the proportion of phenotypic variance due to additive genetic variation, for each DAP ( hj 2 ) was obtained as: the proportion of phenotypic variance due to additive genetic given by knots Tm and Tm+1 , with T t T m m ≤< +1 , ariation, for each DAP ( hj 2 ) was obtained as: variation, for each DAP ( hj 2 ) was obtained as: ∅( )= ≤<     + m ij m m t if T t T otherwise 1 0 1 , , . u = DZ ZDZ GEBV m m ′( ) -1 u = DZ ZDZ GEBV m m ′( ) -1 Predicting Above-Ground Biomass Predicting Above-Ground Biomass We used two methods to quantify the ability of image-based features to statistically predict the AGB in soybean: LASSO regression and PLSR. Both methods were evaluated using a 10-fold CV strategy and we obtained high prediction performance for AGB estimation with both methods. Figure 2 shows the statistical distributions of R2 and RMSE values for each CV fold, in each environment. In general, similar performance was observed for both methods in all environments. It was found that LASSO and PLSR had the same R2 averages for 2017_ACRE (0.94), 2018_ACRE (0.92), and 2018_Romney (0.94). However, the PLSR presented a smaller RMSE average for 2017_ACRE (0.23 vs. 0.24 for PLSR and LASSO, respectively), and LASSO presented a smaller RMSE average for 2018_ACRE (0.28 and 0.29 for LASSO and PLSR, respectively). Both models presented the same RMSE average for 2018_ACRE (0.24). SNP =Tu , s s Genome-Wide Association Studyf Genome-Wide Association Study For the GWAS, SNP effects were derived from GEBVs for each additive random regression coefficient using the POSTGSF90 software (Aguilar et al., 2014). The prediction of SNP effects ( um  ) for the mth random regression coefficient was calculated as (Wang et al., 2012): For the GWAS, SNP effects were derived from GEBVs for each additive random regression coefficient using the POSTGSF90 software (Aguilar et al., 2014). The prediction of SNP effects ( um  ) for the mth random regression coefficient was calculated as (Wang et al., 2012): S= ′ TGT , S= ′ TGT , where T is a matrix of covariates associated with the function assumed for RIL i and G is the genetic (co)variance matrix for the coefficients. The narrow-sense heritability, defined as September 2021 \| Volume 12 \| Article 715983 Frontiers in Plant Science \| www.frontiersin.org 5 Random Regression Soybean Biomass Moreira et al. selected as relevant SNPs. The exploration of candidate genes was carried out in the range of ± 25 kb from the location of the selected SNP. Potential candidate genes and their associated functional annotation were determined using the genomic position and gene models based on Glyma. Wm82.a2.v1 genome in the soybean database SoyBase (Soybase, 2020). RESULTS where D is a diagonal matrix of weights accounting for variances of SNPs markers (assumed as an identity matrix in this study), Z is a matrix relating genotypes of each locus, and GEBV m  is the vector of GEBV for the mth random regression coefficient. Finally, the SNP effects for all DAP were obtained as (Oliveira et al., 2019c): where D is a diagonal matrix of weights accounting for variances of SNPs markers (assumed as an identity matrix in this study), Z is a matrix relating genotypes of each locus, and GEBV m  is the vector of GEBV for the mth random regression coefficient. Finally, the SNP effects for all DAP were obtained as (Oliveira et al., 2019c): SNP =Tu , s s where SNPs  is the vector that contains the SNP effects estimated for each DAP of the sth SNP, us  is the vector of SNP solutions for all random regression coefficients related to the sth SNP, and T is a matrix of covariates associated with the assumed function. where SNPs  is the vector that contains the SNP effects estimated for each DAP of the sth SNP, us  is the vector of SNP solutions for all random regression coefficients related to the sth SNP, and T is a matrix of covariates associated with the assumed function. The SNPs were selected to be further investigated based on the magnitude of their effects, as suggested by Oliveira et al. (2019c). In this context, the top 10 SNPs that showed the highest magnitude of SNP effect in each DAP were FIGURE 2 \| Performance of above-ground biomass prediction for each environment. Predictions were performed using the least absolute shrinkage and selection operator (LASSO) regression, and the partial least squares regression methods. The performance of predictions was evaluated using the root mean squared error (RMSE) and coefficient of determination (R2), using a 10-fold cross-validation set. The y-axis represents the values for RMSE and R2 and x-axis indicates each cross-validation fold. FIGURE 2 \| Performance of above-ground biomass prediction for each environment. Predictions were performed using the least absolute shrinkage and selection operator (LASSO) regression, and the partial least squares regression methods. The performance of predictions was evaluated using the root mean squared error (RMSE) and coefficient of determination (R2), using a 10-fold cross-validation set. The y-axis represents the values for RMSE and R2 and x-axis indicates each cross-validation fold. FIGURE 2 \| Performance of above-ground biomass prediction for each environment. Predictions were performed using the least absolute shrinkage and selection operator (LASSO) regression, and the partial least squares regression methods. The performance of predictions was evaluated using the root mean squared error (RMSE) and coefficient of determination (R2), using a 10-fold cross-validation set. The y-axis represents the values for RMSE and R2 and x-axis indicates each cross-validation fold September 2021 \| Volume 12 \| Article 715983 Frontiers in Plant Science \| www.frontiersin.org 6 Moreira et al. Random Regression Soybean Biomass FIGURE 3 \| Narrow-sense heritability estimated for each day after planting. FIGURE 3 \| Narrow-sense heritability estimated for each day after planting. SNP =Tu , s s FIGURE 4 \| Estimated genetic correlation of above-ground biomass between days after planting. within each environment, by DAP, is presented in Supplementary Figure 4 and Supplementary Figure 5, respectively. Genetic Parameters Supplementary Table 2 shows the AIC values calculated for all seven RRM using both homogeneous and heterogeneous residual variance. The best model was using linear B-spline with 2 knots and heterogeneous residual variance and it was selected for subsequent genome-wide analyses. The genetic architecture of predicted AGB was assessed by estimating the narrow-sense heritabilities (h2) across the 57 days (from 27 to 83 DAP; Figure 3) from the RRM. Narrow-sense heritability estimates for AGB were low to moderate and varied over time (ranging from 0.02 at 44 DAP to 0.28 at 33 DAP). The genetic correlation between AGB on different DAP was also estimated, and it is showed in Figure 4. Adjacent DAP showed the highest genetic correlations, while those further apart exhibited lower correlations. For instance, the lowest genetic correlation between 27 and 83 DAP was 0.16 and the highest genetic correlation between 48 to 50 DAP was 1.00. FIGURE 4 \| Estimated genetic correlation of above-ground biomass between days after planting. Genomic Prediction of Breeding Values Genomic Prediction of Breeding Values The genomic prediction accuracy for AGB over time is presented in Figure 5. Overall, the prediction accuracies were high considering the heritabilities estimated across all DAP, ranging from 0.21 at 83 DAP to 0.55 at 27 DAP. We observed a decreasing trend in prediction accuracy over time, indicating that it is more difficult to predict AGB for latter DAPs compared to early DAPs. From 27 DAP to 44 DAP, the prediction accuracy steadily decreased, reaching a slight plateau between 44 to 66 DAP, and decreased again until the end of the surveyed time. These findings suggest that longitudinal phenotypes can be accurately predicted using RRM. Regression coefficients’ patterns were used to access the bias of GEBV over DAP (Supplementary Figure 6). Overall, regression coefficients closer to 1.0 were found in earlier DAP. The most biased estimates with regression coefficients deviating from 1.0 were observed toward the end of the surveyed time. The correlation between AGB predicted from UAV-based imagery and observed from ground samples was high (r ≥ 0.91) in all environments for both methods, implying that the methods captured the relationship among image-based features and AGB (Supplementary Figure 1). Based on these findings, and because it makes a simpler and more direct connection between the response and predictor variables, the LASSO method was chosen to predict AGB for all plots of the two full replications on all flight dates in this study. Supplementary Figure 2 shows the relative importance of each predictor variable for the LASSO method, which indicates that the model utilized information from different predictor variables for each environment. In addition, we performed a CV leaving one environment out to assess the models’ ability to predict AGB for a new environment. In this scenario, the performance of both methods declined greatly (Supplementary Figure 3). The phenotypic distribution of the predicted AGB across environments and September 2021 \| Volume 12 \| Article 715983 Frontiers in Plant Science \| www.frontiersin.org 7 Random Regression Soybean Biomass Moreira et al. Moreira et al. Random Regression Soybean Biomass Genome-Wide Association Study considered as important SNPs for less than 10 consecutive FIGURE 5 \| Genomic prediction accuracy based on Pearson’s correlation coefficient (r) for each day after planting. FIGURE 6 \| Effects for the selected single-nucleotide polymorphisms (SNPs) across days after planting, in each duration category. Genomic Prediction of Breeding Values Duration categories were defined as long-duration (SNPs present for more than 30 consecutive days), mid-duration (SNPs present for more than 10 consecutive days but less than 30), short-duration (SNPs present for less than 10 consecutive days), and intermittent (SNPs at different non-consecutive intervals). Each y-axis point corresponds to one SNP represented by the chromosome number and position in the soybean Williams 82 reference genome (Wm82.a2.v1; Diers et al., 2018). The blue scale represents negative effects and the red scale represents positive effects. The gray color indicates a zero effect. FIGURE 5 \| Genomic prediction accuracy based on Pearson’s correlation coefficient (r) for each day after planting FIGURE 5 \| Genomic prediction accuracy based on Pearson’s correlation coefficient (r) for each day after planting. FIGURE 6 \| Effects for the selected single-nucleotide polymorphisms (SNPs) across days after planting, in each duration category. Duration categories were defined as long-duration (SNPs present for more than 30 consecutive days), mid-duration (SNPs present for more than 10 consecutive days but less than 30), short-duration (SNPs present for less than 10 consecutive days), and intermittent (SNPs at different non-consecutive intervals). Each y-axis point corresponds to one SNP represented by the chromosome number and position in the soybean Williams 82 reference genome (Wm82.a2.v1; Diers et al., 2018). The blue scale represents negative effects and the red scale represents positive effects. The gray color indicates a zero effect. FIGURE 6 \| Effects for the selected single-nucleotide polymorphisms (SNPs) across days after planting, in each duration category. Duration categories were defined as long-duration (SNPs present for more than 30 consecutive days), mid-duration (SNPs present for more than 10 consecutive days but less than 30), short-duration (SNPs present for less than 10 consecutive days), and intermittent (SNPs at different non-consecutive intervals). Each y-axis point corresponds to one SNP represented by the chromosome number and position in the soybean Williams 82 reference genome (Wm82.a2.v1; Diers et al., 2018). The blue scale represents negative effects and the red scale represents positive effects. The gray color indicates a zero effect. FIGURE 6 \| Effects for the selected single-nucleotide polymorphisms (SNPs) across days after planting, in each duration category. Duration categories were defined as long-duration (SNPs present for more than 30 consecutive days), mid-duration (SNPs present for more than 10 consecutive days but less than 30), short-duration (SNPs present for less than 10 consecutive days), and intermittent (SNPs at different non-consecutive intervals). Genomic Prediction of Breeding Values Each y-axis point corresponds to one SNP represented by the chromosome number and position in the soybean Williams 82 reference genome (Wm82.a2.v1; Diers et al., 2018). The blue scale represents negative effects and the red scale represents positive effects. The gray color indicates a zero effect. High-Throughput Phenotyping of Soybean Above-Ground Biomass Besides being an important yield component, plant biomass is a foundation for unraveling several complex processes of plant growth, development, and environmental response (De Bruin and Pedersen, 2009; Koester et al., 2014; Balboa et al., 2018; Jumrani and Bhatia, 2018). The capacity to non-destructively estimate soybean AGB enables capturing these data in a temporal fashion leading to insights about AGB dynamics. Previously, satellite-derived vegetation indices were used separately to predict soybean AGB with high predictive abilities (Kross et al., 2015; Richetti et al., 2019). However, both studies are from production fields with no significant genetic variation. Recently, Maimaitijiang et al. (2019) used UAV-based RGB imagery- derived spectral, structural, and volumetric information to predict AGB in production fields with three cultivars, but the study did not represent the genetic diversity or small plot formats typical of breeding programs. To our best knowledge, this is the first study estimating soybean AGB of experimental plots and diverse genotypes, demonstrating the feasibility to measure and use this trait in plant breeding programs.f Genome-Wide Association Studyh Genome-Wide Association Studyh considered as important SNPs for less than 10 consecutive days), and intermittent (they were considered as important SNPs on different non-consecutive intervals; Figure 6). These SNP classes were nearly evenly distributed as long- (9 SNPs), mid-(8 SNPs), and short-duration (9 SNPs). The intermittent category had the lowest number of relevant SNPs (4 SNPs). The majority of mid-duration SNPs was detected toward the beginning of the DAP. Interestingly, the SNPs classified in the short- and mid-duration categories were found either toward the beginning or end of the studied time period. y Thirty unique SNPs were selected as the most relevant SNPs for AGB. Figure 6 shows the chromosome number, position, period of occurrence, and the SNP effects for selected SNPs. None of the SNPs selected were significant across all time points. In general, the magnitude of effects over time increased for most of the selected SNPs. According to the duration of the SNP effect across all 57 predicted days, the selected SNPs were classified as long-duration (they were considered as important SNPs for more than 30 consecutive days), mid-duration (they were considered as important SNPs for more than 10 consecutive days but less than 30), short-duration (they were A comprehensive list of positional candidate genes related to the selected SNPs can be found in Supplementary Table 3. September 2021 \| Volume 12 \| Article 715983 Frontiers in Plant Science \| www.frontiersin.org 8 Random Regression Soybean Biomass Moreira et al. TABLE 1 \| Selected single-nucleotide polymorphisms (SNPs) associated with above-ground biomass mapped inside potential candidate genes in the soybean genome. Duration Category SNP Chr. Pos. (bp) Selected candidate genes Annotation Description Long 2:5777782 2 5,777,782 Glyma.02 g064500 Rhomboid protein-related Glyma.02 g064600 Agenet domain-containing protein Short 3:5150181 3 5,150,181 Glyma.03 g040800 Regulator of chromosome condensation (RCC1) family with FYVE zinc finger domain Long 7:6108702 7 6,108,702 Glyma.07 g067900 Disease resistance protein (TIR-NBS- LRR class), putative Mid 7:6523718 7 6,523,718 Glyma.07 g071800 cytidine/deoxycytidylate deaminase family protein Short 7:15340513 7 15,340,513 Glyma.07 g128300 – Long 13:24980935 13 24,980,935 Glyma.13 g137200 ROP interactive partner 3 Short 15:36306421 15 36,306,421 Glyma.15 g217500 CTP synthase family protein Long 16:4353954 16 4,353,954 Glyma.16 g046000 DEAD/DEAH box helicase, putative Chr, Chromosome; Pos (bp), position in base pair; – annotation not available. Genome-Wide Association Studyh Chromosome; Pos (bp), position in base pair; – annotation not available As expected, due to the high number of SNPs selected, the number of candidate genes identified was also high. No positional candidate genes within ± 25 kb were found for five selected SNPs: 3:14985662, 4:10352467, 4:14549891, 7:27576963, and 15:36870472. Among the selected SNPs, eight fell within potential candidate genes in the soybean genome (Table 1). Wang et al., 2017; Maimaitijiang et al., 2019; Li et al., 2020). For instance, spectral indices and plant height were used to predict barley, wheat, and potato AGB (Bendig et al., 2015; Yue et al., 2017; Li et al., 2020); and spectral and structural data fusion was applied for AGB estimation in maize (Wang et al., 2017). In this study, we compared two methods, LASSO regression and PLSR, combining 19 features (Supplementary Table 1) extracted from RGB and multispectral imagery captured with UAV to predict soybean AGB. Our results showed that both methods presented similar performances in all environments (Figure 2). When assessing the importance of the individual variables from the LASSO regression (Supplementary Figure 2), we observed that this method used information from different predictor variables for each environment. For example, the relative importance of CC was higher for 2018_ACRE and 2018_Romney than 2017_ACRE. On the other hand, NDVI was only included in the model to predict AGB at 2017-ACRE. This is also supported by the results of the CV leaving one environment out which indicates that new environments could not be predicted accurately (Supplementary Figure 3). These results provided a solid basis for constructing different models for each environment to enhance the strengths of each imagery feature by the environment. Frontiers in Plant Science \| www.frontiersin.org Genetic Architecture of Soybean Temporal Above-Ground Biomasshi For longitudinal traits, such as AGB, genetic effects are expected to vary over time and studies have shown that the additive polygenic effects of longitudinal traits are not constant over time (Brito et al., 2017; Oliveira et al., 2019a). The RRM approach improves statistical power to detect loci associated with longitudinal traits over other methods because the entire collection of phenotypic observations is considered, capturing the genetic changes throughout the time period considered (Ning et al., 2017; Oliveira et al., 2019a). Therefore, RRM longitudinal GWAS can detect time-dependent significant SNPs that might not be detected when using independent analyses of individual time points.f q y We observed that the heritability for AGB fluctuates over DAP (Figure 3), indicating that the proportion of genetic variance responsible for the phenotypic variation changes across DAP, which is expected due to differential growth patterns and fluctuation of some environmental variables across development and across three locations. Using RRM on phenotypes collected in a controlled-environment, Campbell et al. (2018) found heritabilities ranging from 0.60 to 0.77 for shoot biomass in rice. Studies using independent analyses of individual time points of phenotypes from controlled- environments found high broad-sense heritabilities for AGB in barley (Neumann et al., 2017), maize (Muraya et al., 2017), and canola (Knoch et al., 2020). Lack of environmental variation throughout growth likely contributes to the high heritabilities observed in these studies. Under the field conditions of multi- environment trials, as in our study, the genetic contribution to the observed phenotypes is both variable and reduced due to environmental fluctuations. Regarding genetic correlation of phenotypes across days (Figure 4), Campbell et al. (2018) and Baba et al. (2020) observed the same trend that we did, We observed SNP effects were generally small and time- specific (Figure 6), and no SNPs had a significant association with soybean AGB throughout the observed time period, suggesting the trait is regulated by small effect loci and their interactions. This highlights the importance of the temporal assessment of longitudinal traits, as many associations could not have been discovered if AGB had been evaluated at the end of the experiment or at individual time points. Previous studies have explored the dynamic genetic architecture of AGB in other crops (Campbell et al., 2017, 2019; Muraya et al., 2017; Knoch et al., 2020), but none at field scale or with high temporal resolution. Campbell et al. Genetic Architecture of Soybean Temporal Above-Ground Biomasshi Among the RRM tested here, the model using quadratic B-spline with one knot and homogeneous residual variance failed to converge, which indicates that this model did not fit the data well (Supplementary Table 2). The models using fifth-order Legendre polynomial and quadratic B-spline with two knots also did not achieve convergence when heterogeneous residual variance was used, probably because of the higher complexity of the models (i.e., they are more parameterized) and the dataset size. Usually, more parametrized models require a higher number of observations to accurately estimate their parameters (Thoni et al., 1990). As the number of parameters increases, problems with convergence and estimation, as well as an increase in computational demand, can be expected. The model that seemed to be the most suitable to fit the data was the model fitting linear B-spline with two knots and heterogeneous residual variance. Hence, this model was selected to describe the genetic architecture of AGB over time in subsequent analyses. We observed that the heritability for AGB fluctuates over DAP (Figure 3), indicating that the proportion of genetic variance responsible for the phenotypic variation changes across has not kept pace with the ability to generate high-throughput phenotypic data (Momen et al., 2019). Most of the studies using longitudinal traits mainly performed statistical genetic or genomic analysis for each time point independently (Würschum et al., 2014; Pauli et al., 2016; Xavier et al., 2017; Zhang et al., 2017; Wang et al., 2019; Knoch et al., 2020), ignoring the existing temporal genetic correlation and dependency during trait development. RRM are deemed the most effective alternative to genetically evaluate longitudinal traits in numerous livestock breeding programs (Oliveira et al., 2019a). This approach uses the covariance between each time point with no assumptions of constant variances or correlations, resulting in more accurate breeding values compared to other methods (Sun et al., 2017; Oliveira et al., 2019a). We combined HTP data, high-density genomic information, and RRM to carry out longitudinal analysis and understand the genetics of the development of AGB in soybean. In this context, this study provides the first application of RRM for genomic analyses of longitudinal traits in soybean, as well as the first genetic study on soybean AGB. where the highest correlations were observed between adjacent time points. Genetic Architecture of Soybean Temporal Above-Ground Biomasshi Using RRM allowed us to specify the residual variance structure over time, and what we chose to apply likely contributes to the heritability fluctuations we observed. We grouped interpolated AGB phenotypes with observed phenotypes for the DAP nearest in time, which may not reflect the true residual variance of the longitudinal data. Nonetheless, all models with the heterogeneity of residual variance structure outperformed the models with homogeneous residual variance (Supplementary Table 2), agreeing with other studies (Brito et al., 2017; Campbell et al., 2018). The residual variance is affected by many factors that change with DAP, for instance, as the plants grow the scale of AGB phenotypes increases dramatically from approximately 10 to 940 g/m2. Thus, when considering the genetic architecture of longitudinal traits it is crucial to assess the need of a heterogeneous residual variances structure over time points, since there can be improvements in the partition of the total variation, yielding better estimates of genetic parameters (Brito et al., 2017). In this context, it is important to emphasize that this approach is often performed in studies using RRM (Brito et al., 2017; Campbell et al., 2018). yi g y y Among the RRM tested here, the model using quadratic B-spline with one knot and homogeneous residual variance failed to converge, which indicates that this model did not fit the data well (Supplementary Table 2). The models using fifth-order Legendre polynomial and quadratic B-spline with two knots also did not achieve convergence when heterogeneous residual variance was used, probably because of the higher complexity of the models (i.e., they are more parameterized) and the dataset size. Usually, more parametrized models require a higher number of observations to accurately estimate their parameters (Thoni et al., 1990). As the number of parameters increases, problems with convergence and estimation, as well as an increase in computational demand, can be expected. The model that seemed to be the most suitable to fit the data was the model fitting linear B-spline with two knots and heterogeneous residual variance. Hence, this model was selected to describe the genetic architecture of AGB over time in subsequent analyses. In this study, time was introduced as an additional dimension to association studies enabling the observation of the effects of individual markers over 57 days of soybean AGB development from late vegetative up to mid reproductive stages between 27 and 83 DAP. Frontiers in Plant Science \| www.frontiersin.org Genetic Architecture of Soybean Temporal Above-Ground Biomasshi The identification of the genetic causes underlying phenotypic variation is a major step toward crop improvement. By implementing an HTPP that is capable of collecting non-destructive data in large populations throughout the season under actual field conditions, researchers and plant breeders are able to quantify and understand more thoroughly the dynamics of temporal variation of traits and thereby better optimize genotypes through selection in breeding programs (Pauli et al., 2016). It is important to note that the effort and investment in HTTP demand equal effort to properly analyze the data. Nevertheless, the improvement of statistical methodologies to analyze image-based longitudinal phenotypes Many different techniques and HTPP have been used to estimate AGB in different crops (Bendig et al., 2015; Wang et al., 2016; Zhang et al., 2017; Jimenez-Berni et al., 2018; Maimaitijiang et al., 2019). Using information from multiple sensors is a common practice to predict AGB because it improves trait estimation by combining the advantages of the spectral, spatial, and structural metrics derived from different sensors (Bendig et al., 2015; Chen et al., 2016; September 2021 \| Volume 12 \| Article 715983 Frontiers in Plant Science \| www.frontiersin.org 9 Random Regression Soybean Biomass Moreira et al. has not kept pace with the ability to generate high-throughput phenotypic data (Momen et al., 2019). Most of the studies using longitudinal traits mainly performed statistical genetic or genomic analysis for each time point independently (Würschum et al., 2014; Pauli et al., 2016; Xavier et al., 2017; Zhang et al., 2017; Wang et al., 2019; Knoch et al., 2020), ignoring the existing temporal genetic correlation and dependency during trait development. RRM are deemed the most effective alternative to genetically evaluate longitudinal traits in numerous livestock breeding programs (Oliveira et al., 2019a). This approach uses the covariance between each time point with no assumptions of constant variances or correlations, resulting in more accurate breeding values compared to other methods (Sun et al., 2017; Oliveira et al., 2019a). We combined HTP data, high-density genomic information, and RRM to carry out longitudinal analysis and understand the genetics of the development of AGB in soybean. In this context, this study provides the first application of RRM for genomic analyses of longitudinal traits in soybean, as well as the first genetic study on soybean AGB. Genetic Architecture of Soybean Temporal Above-Ground Biomasshi (2017) used power function parameters as the pseudo- phenotypes in a multiple-trait GWAS to study AGB in rice during early and active tillering stages. Using RRM, several loci with both transient and persistent effects were found controlling rice AGB during early vegetative development in a green-house (Campbell et al., 2019). Knoch et al. (2020) used time point data and relative growth rates for a GWAS of canola AGB under controlled-environment conditions and observed that September 2021 \| Volume 12 \| Article 715983 Frontiers in Plant Science \| www.frontiersin.org 10 Random Regression Soybean Biomass Moreira et al. several medium and many small effect loci controlled the trait, most of which act during short periods. We evaluated the effectiveness of RRM-based genomic selection for longitudinal soybean AGB. Using CV, we found that it was possible to model longitudinal AGB with RRM (Figure 5). Prediction accuracy varied across DAP, with a decreasing trend over time. Accuracy of GS is dependent on many factors, such as the level of linkage disequilibrium (LD) in the population, effective population size, the number of markers, trait heritability, and the number of QTL influencing the trait (Lin et al., 2014; Wang et al., 2018). Since the LD, population size and number of markers were held constant in our study, the difference in prediction accuracy across DAP can be largely attributed to the differences in heritability. Considering the heritability values, in general, we obtained better prediction accuracy than Campbell et al (2018) observed when predicting AGB in rice using RRM. Prediction bias for the GEBVs also varied over DAP, suggesting that selection based on different days produces different results (Supplementary Figure 6). This is in agreement with our GWAS results because it implies that different genes can be expressed by DAP and that selection based on different days can have distinct genetic implications on AGB (Oliveira et al., 2019b). One possible reason for the decrease in prediction accuracy and bias over time could be decreasing quality of the phenotypes as the season progresses and the plot canopy closes, because it is difficult to quantify accurate phenotypic differences between plots. Phenotyping accuracy can be improved by enhancing imagery resolution and adding volume and height metrics. Another reason may be our limited population size (n = 383). Increasing population and training set size generally increase the accuracy of predictions, especially for low heritability traits (Goddard, 2009; Wang et al., 2018). Genetic Architecture of Soybean Temporal Above-Ground Biomasshi Xavier et al. (2016) found that training population size was the most relevant factor in improving prediction accuracy in the SoyNAM population, with optimal populations size between 1,000 and 2000 individuals. g p Among the selected SNPs positioned within candidate genes in soybean (Table 1), some may have a direct impact on AGB. The Glyma.02 g064600 candidate gene potentially codes a protein belonging to the Agenet domain family, which is known as chromatin remodeling proteins (Brasil et al., 2015). In Arabidopsis thaliana, Agenet/Tudor domain family proteins were associate with regulating gene expression by DNA methylation (Brasil et al., 2015; Zhang et al., 2018). Interestingly, an Agenet domain-containing protein in A. thaliana was highly expressed in reproductive tissues and its downregulation delayed flower development timing (Brasil et al., 2015). In our study, the effect of the SNP associates with Glyma.02 g064600 started to be present at 43 DAP, which overlaps with the average beginning of the blooming (R1) period, and the magnitude of its effects increases with time. Also, on chromosome two, Glyma.02 g064500 possibly corresponds to rhomboid protein- related that in A. thaliana is a putative cellular component in the Golgi apparatus with unknown function. Ban et al. (2019) reported that Glyma.07 g067900, which codes a disease resistance protein, was upregulated when studying the regulation of genes in mutant dwarf soybeans related to plant growth. It is known that the over-expression of disease resistance and other immune- responsive genes tend to divert resources to generate protection metabolites, thus reducing overall growth (Ban et al., 2019). Glyma.07 g071800 is predicted to have biological functions involved in the riboflavin biosynthetic process. In plants, Riboflavin is known to be involved in disease defense (Nie and Xu, 2016), therefore Glyma.07 g071800 may be associated with the trade-off between the defense response and plant growth as mentioned before. Glyma.16 g046000 is a putative DEAD/DEAH box helicase. Some proteins of this family are known to play a role in plant growth and development, and in response to stresses in plants (Wang et al., 2000; Zhu et al., 2015). These results improve our understanding of the genetic control of soybean AGB and bridge gaps in understanding the relationship between genotype and phenotype. Further studies are necessary to validate the potential candidate genes and understand their contribution to soybean AGB. Genetic Architecture of Soybean Temporal Above-Ground Biomasshi In summary, based on the prediction accuracy and bias, our results indicate that AGB is a potential candidate for genomic selection in soybeans. The ability to predict temporal-based GEBV allows targeting specific intervals in the growing season or selecting plants with specific growth patterns. For instance, increased temperatures and water stress can reduce AGB significantly, resulting in reduction in soybean yield (Jumrani and Bhatia, 2018); using genomic selection to increase AGB during vegetative stages and making the plant more robust may improve stress resilience. Moreover, even if a longitudinal trait itself is not the target of selection, but its genetically correlated to economic traits, such as yield, it has the potential of being used for early indirect selection or to improve genomic prediction accuracy in a MTM (Sun et al., 2017; Moreira et al., 2020). The genetic correlation between longitudinal soybean AGB and grain yield is currently being investigated. Given HTPP’s power to simultaneously collect multiple temporal traits, multiple-trait RRM may be powerful tools for joint genomic prediction of multiple longitudinal traits (Oliveira et al., 2016; Baba et al., 2020; Moreira et al., 2020). Therefore, RRM and HTPP have a great potential to accelerate the rate of genetic gain in soybean breeding programs. Potential of Genomic Selection to Improve Soybean Temporal Above-Ground Biomass Genomic selection has been proved to be a powerful tool in plant and livestock breeding (Meuwissen et al., 2016; Crossa et al., 2017). HTPP allow crop scientists to generate high- quality phenotypic data and effectively characterize large training populations throughout the growing season. Thus, the combination of GS and HTPP has the potential to increase accuracy and throughput, while reducing costs and minimizing labor (Araus et al., 2018). Several studies in animals have demonstrated that RRM improve genomic prediction accuracy of longitudinal traits compared to single-time point and MTM (Oliveira et al., 2019a; Moreira et al., 2020) and more recently, this has been demonstrated in plants (Campbell et al., 2018; Momen et al., 2019). REFERENCES Ban, Y. W., Roy, N. S., Yang, H., Choi, H. K., Kim, J. H., Babu, P., et al. (2019). Comparative transcriptome analysis reveals higher expression of stress and defense responsive genes in dwarf soybeans obtained from the crossing of G. max and G. soja. Genes Genomics 41, 1315–1327. doi: 10.1007/ s13258-019-00846-2 Abdi, H. (2010). Partial least squares regression and projection on latent structure regression (PLS regression). Wiley Interdiscip. Rev. Comput. Stat. 2, 97–106. doi: 10.1002/wics.51 Bendig, J., Yu, K., Aasen, H., Bolten, A., Bennertz, S., Broscheit, J., et al. (2015). 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FM and KR conceived and designed the study. ML assisted with field data collection. KC and BA conducted the multispectral images analyses. GG assisted with AGB phenotypic prediction. HO and LB assisted with the random regression model analyses. HO, LB, and KR critically revised and improved the manuscript. All authors read and approved the manuscript. We express our gratitude to the soybean breeding laboratory at Purdue for their assistance in the field work, and Stuart Smith for his contributions to managing the UAS imagery. We thank the North Central Soybean Research Program (NCSRP) and the United Soybean Board (USB) for funding the development of the Soybean Nested Association Panel. FUNDING The Supplementary material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fpls.2021.715983/ full#supplementary-material We thank the Indiana Corn and Soybean Innovation Center (ICSIC) endowment funds and the Indiana Soybean Alliance REFERENCES “Soybean [Glycine max (L.) Merr.] breeding: history, improvement, production and future opportunities,” in Advances in Plant Breeding Strategies: Legumes. eds. J. Al-Khayri, S. Jain, and D. Johnson. (Cham: Springer International Publishing), 431–516. Brito, L. F., Gomes da Silva, F., Rojas de Oliveira, H., Souza, N., Caetano, G., Costa, E. V., et al. (2017). Modelling lactation curves of dairy goats by fitting random regression models using Legendre polynomials or B-splines. Can. J. Anim. Sci. 98, 73–83. doi: 10.1139/CJAS-2017-0019 Araus, J. L., Kefauver, S. C., Zaman-Allah, M., Olsen, M. S., and Cairns, J. E. (2018). Translating high-throughput phenotyping into genetic gain. 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(2017). Estimation of winter wheat above-ground biomass using unmanned aerial vehicle-based snapshot hyperspectral sensor and crop height improved models. Remote Sens. 9:708. doi: 10.3390/rs9070708 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. Zhang, C., Du, X., Tang, K., Yang, Z., Pan, L., Zhu, P., et al. (2018). Arabidopsis AGDP1 links H3K9me2 to DNA methylation in heterochromatin. Nat. Commun. 9:4547. doi: 10.1038/s41467-018-06965-w 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. Zhang, X., Huang, C., Wu, D., Qiao, F., Li, W., Duan, L., et al. (2017). High- throughput phenotyping and qtl mapping reveals the genetic architecture of maize plant growth. Plant Physiol. September 2021 \| Volume 12 \| Article 715983 Frontiers in Plant Science \| www.frontiersin.org REFERENCES 173, 1554–1564. doi: 10.1104/pp.16.01516 Zhao, C., Zhang, Y., Du, J., Guo, X., Wen, W., Gu, S., et al. (2019). Crop Phenomics: current status and perspectives. Front. Plant Sci. 10:714. doi: 10.3389/fpls.2019.00714 Copyright © 2021 Moreira, Oliveira, Lopez, Abughali, Gomes, Cherkauer, Brito and Rainey. 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. Zhu, M., Chen, G., Dong, T., Wang, L., Zhang, J., Zhao, Z., et al. (2015). SlDEAD31, a putative DEAD-Box RNA helicase gene, regulates salt and drought tolerance and stress-related genes in tomato. PLoS One 10:e0133849. doi: 10.1371/journal.pone.0133849 September 2021 \| Volume 12 \| Article 715983 Frontiers in Plant Science \| www.frontiersin.org 15
https://openalex.org/W2263991409	https://jfootankleres.biomedcentral.com/track/pdf/10.1186/s13047-016-0136-7	English	null	Journal of Foot and Ankle Research reviewer acknowledgement 2015	Journal of foot and ankle research	2,016	cc-by	694	Alan Borthwick1* and Hylton Menz2 Alan Borthwick1* and Hylton Menz2 Borthwick and Menz Journal of Foot and Ankle Research (2016) 9:5 DOI 10.1186/s13047-016-0136-7 Borthwick and Menz Journal of Foot and Ankle Research (2016) 9:5 DOI 10.1186/s13047-016-0136-7 Open Access © 2016 Borthwick and Menz. 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. Journal of Foot and Ankle Research reviewer acknowledgement 2015 Alan Borthwick1* and Hylton Menz2 Contributing reviewers The editors of Journal of Foot and Ankle Research would like to thank all our reviewers who have contributed to the journal in Volume 8 (2015). Andrew Buldt Australia Felix Burden United Kingdom Joshua Burns Australia Paolo Caravaggi Italy Matt Carroll New Zealand Paul Chadwick United Kingdom Lindsey Cherry United Kingdom Nachiappan Chockalingam United Kingdom Richard Collier United Kingdom Mark Cornwall United States of America Matthew Cotchett Australia Stephen Cousins Australia Emma Cowley United Kingdom Muaaze Ahmad United Kingdom © 2016 Borthwick and Menz. 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 3 Borthwick and Menz Journal of Foot and Ankle Research (2016) 9:5 George S Murley Australia Vanessa Nube Australia Simon Otter United Kingdom Kade Paterson Australia Smadar Peleg Israel Byron Perrin Australia Jill Phethean United Kingdom Stephen Preece United Kingdom Carina Price United Kingdom Trevor D Prior United Kingdom Anita Raspovic Australia Michael Skovdal Rathleff Denmark Anthony Redmond United Kingdom William Ribbans United Kingdom Jody Riskowski United Kingdom Edward Roddy United Kingdom Keith Rome New Zealand Dominique Rouleau Canada Dale Schuit United States of America Debbie Sharman United Kingdom Heidi Siddle United Kingdom Martin Spink Australia Tim Kilmartin Ireland Dorit Kunkel United Kingdom Toshiyuki Kurihara Japan Peter Lazzarini Australia Alberto Leardini Italy Pazit Levinger Australia Kirsten Leyland United Kingdom Anmin Liu United Kingdom Vincent Luboz France John Ludlow United States of America Ryan Mahaffey United Kingdom Matthew Malone Australia Michelle Marshall United Kingdom Joan Marti Spain Ian Mathieson United Kingdom Marlene Mauch Switzerland joanne McCardle United Kingdom Keith McCormick United Kingdom Julie McDonald Australia Karen Mickle Australia Anna Moran Australia Stewart Morrison United Kingdom Dominic Gehring Germany Moshi Geso Australia Kellie Gibson United Kingdom Nikolaos Gougoulias United Kingdom Andrea Graham United Kingdom Kelly Gray Australia Rodney Green Australia Martin Gronbech Jorgensen Denmark Jill Halstead United Kingdom Joseph Hamill United States of America Farina Hashmi United Kingdom Gordon Hendry United Kingdom C. Collin Herb United States of America jj Hermans Netherlands Claire Hiller Australia Brian Horsak Austria Sheree Hurn Australia Kasper Janssen Netherlands Sara Jones Australia David Kachlik Czech Republic Anne-Maree Keenan United Kingdom Robert Kidd Australia Page 3 of 3 Page 3 of 3 Borthwick and Menz Journal of Foot and Ankle Research (2016) 9:5 Marinus Winters Netherlands James Woodburn United Kingdom Gavin Wylie United Kingdom Michael Yelland Australia Maria Young United Kingdom Bernhard Zipfel South Africa Author details 1University of Southampton, Southampton, UK. 2La Trobe University, Bundoora, Australia. Kate Springett United Kingdom Michelle Spruce United Kingdom David Stephensen United Kingdom Martijn Steultjens United Kingdom Simon Taylor Australia Scott Telfer United Kingdom Masafumi Terada United States of America Jo Tweed United Kingdom Stephen Urry Australia Jaap van Netten Netherlands Edgar Vieira United States of America Scott Wearing Australia Caleb Wegener Australia Michael Wilding Singapore Richard Wilkins United Kingdom Cylie Williams Australia Stephen Urry Australia Jaap van Netten Netherlands Edgar Vieira United States of America Scott Wearing Australia Caleb Wegener Australia Michael Wilding Singapore Richard Wilkins United Kingdom Cylie Williams Australia
https://openalex.org/W4210743897	https://jmir.org/api/download?alt_name=resprot_v9i1e16320_app2.pdf&filename=f2ae7a090f712619820914456f8c71e8.pdf	English	null	Testing the Effectiveness of Enhanced Alcohol Warning Labels and Modifications Resulting From Alcohol Industry Interference in Yukon, Canada: Protocol for a Quasi-Experimental Study (Preprint)	null	2,019	cc-by	416	December 14,2017 Vice-President Research University of Victoria Room A110, Administrative Services Building, 3800 Finnerty Rd. (Ring Road) Victoria BC V8P 5C2 December 14,2017 Vice-President Research University of Victoria Room A110, Administrative Services Building, 3800 Finnerty Rd. (Ring Road) Victoria BC V8P 5C2 Subject: Dr. Tim Stockwell – Yukon / NWT Northern Territories Alcohol Study – Ethics Board Approval Inquiry I have learned through media reports that a project is being undertaken in the Yukon that involves surveying beverage alcohol consumers for awareness of alcohol warning labels being tested as part of pilot that feeds into a larger study. I have a significant interest in this project and in particular the nature of the pre-pilot survey structure and post-pilot follow-up. I am very concerned that the project has been designed to ensure an outcome that aligns with the researchers’ agenda. I am also concerned that the labels being piloted are false, misleading, conflicted and potentially dangerous. Here are my concerns with two of the three labels I have seen in the media: • “Alcohol can cause cancer” is a false statement intended to mislead and alar • “Alcohol can cause cancer” is a false statement intended to mislead and alarm consumers. • “2 drinks a day for women and three for men” might be intended to convey the Low Risk Drinking Guidelines but could be interpreted by consumers to mean the amount that is safe to drink and still drive. This is dangerous. • “2 drinks a day for women and three for men” might be intended to convey the Low Risk Drinking Guidelines but could be interpreted by consumers to mean the amount that is safe to drink and still drive. This is dangerous. As the Executive Director of the British Columbia Craft Brewers Guild who represents over 100 independently owned and operated businesses I want to ensure this research will be fair and unbiased. As the Attorney General of BC Mr. Eby has spoken about this pilot, it makes it relevant to our craft beer community. I would like to know if the project, where the University of Victoria’s name is used and Dr. Tim Stockwell is one of the principal investigators and spokespeople, was cleared through University of Victoria’s Ethics board. If it was, could I get a copy of the Board’s review. If not, can you help me understand why it would not have gone through the Ethics Board. Thank You Thank You Ken Beattie Executive Director – BC Craft Brewers Guild Ken@bccraftbeer.com
W2929211900.txt	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0214506&type=printable	en		Non-perfusing cardiac rhythms in asphyxiated newborn piglets	PloS one	2,019	cc-by	5,028	RESEARCH ARTICLE Non-perfusing cardiac rhythms in asphyxiated newborn piglets Anne Lee Solevåg ID1, Deandra Luong2,3, Tze-Fun Lee2,4, Megan O’Reilly2,4, PoYin Cheung2,4, Georg M. Schmölzer2,4 1 Department of Paediatric and Adolescent Medicine, Akershus University Hospital, Lørenskog, Norway, 2 Centre for the Studies of Asphyxia and Resuscitation, Neonatal Research Unit, Royal Alexandra Hospital, Edmonton, Alberta, Canada, 3 Faculty of Science, University of Alberta, Edmonton, Alberta, Canada, 4 Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a.l.solevag@medisin.uio.no Abstract Aim OPEN ACCESS Citation: Solevåg AL, Luong D, Lee T-F, O’Reilly M, Cheung P-Y, Schmölzer GM (2019) Non-perfusing cardiac rhythms in asphyxiated newborn piglets. PLoS ONE 14(4): e0214506. https://doi.org/ 10.1371/journal.pone.0214506 Editor: Ahmad N. Al-Dissi, Western College of Veterinary Medicine, University of Saskatchewan, CANADA Received: August 23, 2018 Accepted: March 14, 2019 Published: April 4, 2019 Copyright: © 2019 Solevåg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: GMS is a recipient of the Heart and Stroke Foundation/University of Alberta Professorship of Neonatal Resuscitation, a National New Investigator of the Heart and Stroke Foundation Canada and an Alberta New Investigator of the Heart and Stroke Foundation Alberta. This research has also been facilitated by the Women and Children’s Health Research We recently demonstrated that asphyxiated piglets commonly had bradycardia displayed on electrocardiography (ECG) while no carotid blood flow (CBF) or audible heart sounds could be detected. Such pulseless electrical activity (PEA) in newborn infants has not previously been thoroughly described. The aim of this study was to further investigate the occurrence of non-perfusing cardiac rhythms in asphyxiated piglets and the potential implications for the success of cardiopulmonary resuscitation (CPR) and short-term survival. Methods Neonatal piglets (1–4 days, 1.7–2.4kg) had their right common carotid artery exposed and enclosed with a real-time ultrasonic flow probe. Heart rate (HR) was continuously measured and recorded using ECG. This allowed simultaneous monitoring of HR via ECG and CBF. The piglets were asphyxiated until cardiac arrest, defined as no CBF and no audible beat upon precordial auscultation. CPR was performed until return of spontaneous circulation (ROSC, defined as a HR �100 bpm). ECG traces were retrospectively assessed. Results Nine out of 21 piglets (43%) had QRS-complexes on their ECG while no CBF and no audible heart sounds could be detected. Five (56%) of the piglets with PEA and 12/12 (100%) piglets with asystole at cardiac arrest obtained ROSC (p = 0.02). Thirty-three per cent of the piglets with PEA versus 58% with asystole survived to 4 hours post-ROSC (p = 0.39). Conclusion Cardiac arrest in the presence of a non-perfusing cardiac rhythm on ECG is common in asphyxiated piglets. Clinical arrest in the presence of a non-perfusing cardiac rhythm on ECG may reduce the success of CPR. PLOS ONE \| https://doi.org/10.1371/journal.pone.0214506 April 4, 2019 1 / 10 Neonatal non-perfusing rhythms Institute through the generous support of the Stollery Children’s Hospital Foundation. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: OHCA, Out-of-hospital cardiac arrests; VF, Ventricular fibrillation; CPR, Cardiopulmonary resuscitation; PEA, Pulseless electrical activity; pVT, Pulseless ventricular tachycardia (pVT); ECG, Electrocardiography; CA, Carotid artery; HR, Heart rate; CBF, Carotid blood flow; PPV, Positive pressure ventilation; CC, Chest compression; ROSC, Return of spontaneous circulation. Introduction Adult out-of-hospital cardiac arrests (OHCA) are commonly of primary cardiac origin with ventricular fibrillation (VF) as the cause of arrest. Thus, rhythm diagnosis and defibrillation are important features of adult cardiopulmonary resuscitation (CPR) [1]. In contrast, paediatric cardiac arrest is usually of respiratory aetiology, and the initial rhythm is often non-shockable including asystole and pulseless electrical activity (PEA) [2]. Therefore, the initial focus for paediatric OHCA has been on rescue breathing and chest compressions whereas rhythm diagnosis and defibrillation have received less emphasis. However, in observational studies, VF was diagnosed as the initial rhythm in 4–19% of paediatric cardiac arrests [3, 4]. When direct evidence is lacking, guidelines for paediatric CPR are developed with consideration of the evidence from adults [5]. In the neonatal subpopulation, even less direct evidence exists, and guidelines for neonatal resuscitation are rather simplified compared to adult guidelines; e.g., adrenaline (epinephrine) is the only drug in the neonatal resuscitation algorithm [6, 7]. Antiarrhythmic medications, such as amiodarone and lidocaine, or defibrillation are not considered during neonatal CPR; mainly because shockable arrhythmias such as VF and pulseless ventricular tachycardia (pVT) have not been recognized in newborn infants with cardiac arrest. Pulseless electrical activity (PEA) is organized cardiac electrical activity without associated mechanical activity [8]. Treatment includes reversing the cause of cardiac arrest [9], in addition to providing assisted ventilation and chest compression. The PEA rhythm may be sinus, atrial, junctional, or ventricular in origin, but is broadly categorized as narrow QRS-complex (70% of cases) and wide complex PEA [9]. Narrow complex PEA on electrocardiography (ECG) may be caused by a mechanical problem due to right ventricle inflow or outflow obstruction (e.g., cardiac tamponade, tension pneumothorax, mechanical lung hyperinflation, and pulmonary embolism), whereas wide complex PEA is more likely to be due to a metabolic condition (e.g., hyperkalaemia and sodium channel blocker overdose), left ventricular failure (due to ischemia), or an agonal rhythm (clinically regarded as asystole with equivalent treatment approach) [10]. PEA may also be caused by hypovolaemia, tachydysrhythmias, and cardiomyopathy [8]. It is stated that only a very small percentage of PEA arrests are caused by asphyxia [11]. However, we recently demonstrated that in severely asphyxiated piglets, 23/54 (43%) of the animals had distinct QRS-complexes on the ECG without a detectable carotid blood flow or an audible heartbeat on precordial auscultation [12, 13]. In addition, recent case reports [14, 15] reported five cases of PEA in newborn infants during neonatal resuscitation in the delivery room. Most concerning, 4/5 infants died during resuscitation. The aim of the present study was to further examine the occurrence of PEA and potentially other arrhythmias in asphyxiated piglets. Based on the poor outcome after PEA in adults [16] and older children [5], we hypothesized that PEA negatively influences the success of CPR and short-term survival of asphyxiated piglets. Materials and methods Secondary analysis of a previously published randomized animal trial in asphyxiated piglets using different methods of CPR [17]. Subjects Newborn mixed breed piglets (1–4 days, 1.7–2.4 kg, n = 41) were obtained on the day of experimentation from the Swine Research Technology Center, University of Alberta. All experiments were conducted by certified University of Alberta Animal User Training Program researchers, and conducted in accordance with the guidelines. The research was approved by PLOS ONE \| https://doi.org/10.1371/journal.pone.0214506 April 4, 2019 2 / 10 Neonatal non-perfusing rhythms the Animal Care and Use Committee (Health Sciences), University of Alberta and presented according to the ARRIVE guidelines [18]. The protocol is presented in [17]. Animal preparation The piglets were anesthetized with Isoflurane 1–5%, tracheotomised and mechanically ventilated (Sechrist infant ventilator model IV-100; Sechrist Industries, Anaheim, CA) at a 25/min rate, peak inspiratory pressure of 25cmH2O and positive end-expiratory pressure of 5cmH2O. After central vascular access was obtained, hydration was maintained with 5% Dextrose and 0.9% NaCl, and anaesthesia was changed to intravenous morphine 50-200mcg/kg/h and propofol 0.1–0.2mg/kg/h. A bolus of morphine (0.15mg/kg) was given before tracheotomy. Piglets recovered from surgical instrumentation for 1h during which the ventilator rate and airway pressure were adjusted to keep paCO2 35–45mmHg. Surgical procedures A 5-French Argyle single-lumen catheter (Covidien, Dublin, Ireland) was inserted into the left common carotid artery (CCA) for continuous blood pressure monitoring and blood sampling. A 5-French Argyle double-lumen catheter (Covidien) was inserted in the external jugular vein on the same side for fluid and medication infusion. The piglet was tracheotomised and a 3.5 uncuffed endotracheal tube was inserted and fixed to the trachea. A real-time ultrasonic flow probe (2SB; Transonic Systems Inc., Ithaca, NY) was placed around the right CCA. Systemic arterial pressure and heart rate (HR) were measured continuously with a Hewlett Packard 78833B monitor (Hewlett Packard Co., Palo Alto, CA). Experimental protocol Asphyxia was induced as described in [17] by reducing FiO2 to 0.08 and reducing the ventilator rate by 10/min every 10min until a rate of 0/min was reached. Ten minutes later, the ventilator was disconnected and the endotracheal tube clamped until cardiac arrest/asystole, defined as carotid blood flow <5 mL/min and no audible HR upon auscultation of the precordium [17]. Thirty seconds after cardiac arrest was diagnosed, we provided positive pressure ventilation (PPV) with air for 30sec with a Neopuff T-Piece (Fisher & Paykel, Auckland, NZ) with peak inspiratory pressure 25cmH2O and positive end-expiratory pressure 5cmH2O before chest compression (CC) was started. Manual CC was performed and PPV provided at a 30/min rate. If there was no return of spontaneous circulation (ROSC) after 30sec of CC, adrenaline (0.02mg/kg) was given intravenously and repeated every 3min as needed (maximum 4 doses). CPR was discontinued if ROSC was not achieved after 15min. As previously described [19], ROSC was defined as an unassisted HR � 100 bpm demonstrated by arterial blood pressure waveforms. After ROSC, piglets were observed for 4h and euthanized (within five minutes) with IV phenobarbital (100 mg/kg), unless death occurred earlier. Humane endpoints included a decrease in HR <100 bpm or hypotension, and decrease in haemoglobin <5.5 g/ dL. No animal died before meeting criteria for euthanasia. Data collection and analysis We recorded age, weight and sex of the piglets. Transonic flow probe, HR and pressure transducer outputs were digitized and recorded (PowerLab LabChart software (ADInstruments, Dunedin, NZ)). Cardiac output was measured with echocardiography (Vivid 7/5S probe (GE Healthcare, Buckinghamshire, UK)) at baseline, during asphyxiation, and 30 min and 4 h after PLOS ONE \| https://doi.org/10.1371/journal.pone.0214506 April 4, 2019 3 / 10 Neonatal non-perfusing rhythms ROSC as described in [17]. Markers for cardiac arrest were placed within the LabChart program to indicate the time of cardiac arrest before initiation of the resuscitation protocol. This marker was then used to compare timing of onset of arrest as determined by auscultation, ECG and CBF. The study is based on secondary analyses of a ROSC study in asphyxiated piglets [17]. Continuous variables are presented as median (interquartile range (IQR)). Data was compared between groups using the Mann-Whitney U test for continuous variables, and χ2 for categorical variables. P-values were 2-sided and p<0.05 was considered statistically significant. Statistical analyses were performed with IBM SPSS 25 for Mac (IBM Corporation, Armonk, NY). Results Thirty-two piglets were analysed with respect to ECG rhythm at the time of cardiac arrest. At cardiac arrest, median (IQR) arterial pH was 6.6 (6.6–6.7), paCO2 was 91 (54–101) mmHg, base excess -28 (-30-(-25)) mmol/L, and lactate was 18 (17–20) mmol/L. In 11 (34%) piglets, the ECG tracings were of insufficient quality for an interpretation to be made. In the piglets where ECG failed to give a reliable signal at cardiac arrest, the duration of hypoxia/asphyxia had been longer than in the piglets with ECG tracings of good quality (42 (32–43) vs 33 (26– 34) min, p = 0.007). However, pH at cardiac arrest (p = 1.00), and time to ROSC (p = 0.89) were not different between piglets with insufficient vs. good quality ECG tracings. Piglets with good quality ECG tracings survived the whole experiment in 10/21 (48%) of cases, vs. 2/11 (18%) piglets with insufficient quality ECG (p = 0.14). Of the 21 piglets with good quality ECG, nine (43%) had identifiable QRS-complexes on their ECG while no CBF and no audible heart rate could be detected (Fig 1A). The QRS-rate ranged from 38 to 190 beats per minute (median: 66 beats per minute). In all cases, the QRS-complexes were interpreted as narrowcomplex PEA. None of the piglets had VF or pVT. Twelve (57%) piglets were asystolic with no QRS-complexes visible on the ECG at the time of arrest (Fig 1B). Characteristics of the piglets with good quality ECG-recordings are presented in Table 1. pH (6.5 (6.5–6.8) vs. 6.6 (6.6–6.7), p = 0.42) and lactate (18 (14–20) mmol/L vs. 18 (17–20) mmol/L, p = 0.88) at the time of cardiac arrest were similar in piglets with PEA and asystole, respectively. There was no difference in the distribution of CPR interventions (original study of different oxygen fractions and CC methods) between piglets with PEA and asystole. Time to ROSC was not different between piglets with PEA and asystole, but the fraction of piglets obtaining ROSC was lower in piglets with PEA compared to asystole (Table 1). Three out of nine (33%) piglets with PEA survived to 4 hours post-ROSC, whereas seven out of 12 (58%) piglets with asystole survived (Table 1). There was no difference in HR (Table 1) and MAP (Table 2) at 4 hours post-ROSC between piglets with PEA and asystole (Table 1). Fig 2 is a Kaplan-Meyer survival graph showing that piglets with PEA died earlier during the course of the experiment than the piglets with asystole (p = 0.04). Discussion In this study of asphyxia-induced cardiac arrest, we observed that piglets frequently had detectable QRS-complexes on ECG while there were no CBF and audible heart contractions (auscultation). Our findings are similar to previous reports in asphyxiated piglets with 40–50% having PEA after asphyxia-induced cardiac arrest [12, 13]. Initial non-shockable rhythms (PEA or asystole) account for about two-thirds of adult OHCA with an increasing incidence [11] compared to initial shockable rhythms (VF and pVT) [20, 21]. Overall survival after adult OHCA is about 8% [22], with a worse prognosis with PEA compared to initial shockable rhythms [16, 23–28]. Even if the rhythm converts PLOS ONE \| https://doi.org/10.1371/journal.pone.0214506 April 4, 2019 4 / 10 Neonatal non-perfusing rhythms Fig 1. Waveforms of carotid artery (CA) blood flow (CBF) and electrocardiogram (ECG). Panel a: ECG showing bradycardia in the absence of CBF and no audible sound. Panel b: Asystole correctly assessed with absence of CBF, ECG and no audible heart sound. https://doi.org/10.1371/journal.pone.0214506.g001 from non-shockable to shockable during CPR, outcomes (e.g., survival to hospital discharge) do not improve [21, 29]. Similarly, during paediatric cardiac arrest, a shockable rhythm (VT/ pVT) is a predictor for improved outcome [5]. We previously reported that 1/54 asphyxiated piglets had VT/VF with no CBF or audible heart sounds [12]. In the present study, no piglet had a shockable rhythm. In all the piglets with PEA, we only observed narrow QRS-complexes on the ECG. We speculate that asphyxia, and potentially hypovolaemia, are associated with narrow-complex PEA. Similar to human adults and older children, PEA resulted in less asphyxiated piglets achieving ROSC and survival compared to asystole. A chart review of 262 adults with cardiac arrest and an initial rhythm of PEA reported that neither electrical rate nor QRS width was associated with survival or neurologic outcome [9]. However, there was a trend toward improved survival in bradycardic PEA compared to other PEA rhythms (i.e., normocardic or tachycardic PEA). Unorganized PEA may represent a final common preterminal electrical rhythm. PEA in our piglets had an electric QRS heart rate ranging between 38 to 190 per minute. However, the sample was too small to be stratified to bradycardic versus normocardic versus tachycardic PEA. Our study is hypothesis generating how the initial ECG-rhythm might affect the prognosis of asphyxiated newborn infants that require delivery room CPR. Questions that remain PLOS ONE \| https://doi.org/10.1371/journal.pone.0214506 April 4, 2019 5 / 10 Neonatal non-perfusing rhythms Table 1. Characteristics of piglets with pulseless electrical activity (PEA) versus asystole on electrocardiogram at cardiac arrest. PEA (n = 9) Asystole (n = 12) p-value Sex (female/male) 3/6 7/5 0.39 Age (days) 2 (2–3) 2 (1–3) 1.00 Weight (kg) 1,9 (1,8–2,3) 2,0 (1,7–2,3) 1.00 Baseline HR (bpm) 230 (202–268) 198 (179–238) 0.31 Hypoxia/asphyxia time (min) 33 (31–37) 31 (26–33) 0.68 ROSC (Y/N) 5/4 12/0 0.02 Time to ROSC (sec) 170 (92–182) 117 (95–25) 0.92 Adrenaline doses (n) 2 (0.5–4) 1 (0–1) 0.22 Survival to 4 hours (n (%)) 3 (33) 7 (58) 0.39 HR at 4 hours (bpm) 249 (196-)� 229 (219–234) 0.55 Continuous variables are reported as median (interquartile range) HR–heart rate ROSC–return of spontaneous circulation � not able to calculate interquartile range (n = 3) https://doi.org/10.1371/journal.pone.0214506.t001 unanswered include i) whether there is a difference in disease severity between infants with asystole versus PEA, or ii) whether PEA in itself affects the myocardial response to resuscitative measures. The piglets with PEA had the same hypoxia time and similar biochemical signs of asphyxia compared to piglets with asystole. However, piglets with PEA had a poorer response to CPR with only about half the piglets obtaining ROSC. Newborn piglets have similar anatomy and pathophysiology to newborn infants at nearterm gestation. In addition to anaesthetic and surgical confounding factors, all piglets had already undergone foetal to neonatal transition, and their responses to severe asphyxia may not be entirely comparable to infants during foetal-to-neonatal transition. A perivascular flow probe was placed around the right CCA while the left CCA was cannulated for MAP measurements and blood sampling. Although this approach has been used in previous animal models of perinatal asphyxia [30], occluding the left CCA could potentially change the flow through Table 2. Hemodynamic variables in piglets with pulseless electrical activity (PEA) versus asystole reported as median (interquartile range). 30 min after ROSC 4 h after ROSC PEA Baseline Asystole PEA 20 min asphyxia Asystole PEA Asystole PEA Asystole CA flow (mL/min) 86 (72–104) 76 (61–94) 78 (56–93) 68 (36–81) 31 (22-)� 39 (23–50) 12 (0-)� 16 (3–23) MAP (mmHg) 80 (76–90) 76 (69–85) 70 (45–76) 55 (48–64) 70 (67-)� 55 (53–63) 32 (31-)� 43 (24–54) CVR (mmHg�mL�min-1) 0,97 (0,93-)� 1,07 (0,90–1,32) 0,82 (0,79-)� 0,79 (0,65–1,19) 2,17 (1,16-)� 1,64 (1,06–2,35) 2,69 (2,58-)� 2,78 (1,65–9,75) CO (mL/kg/min) 309 (257–450) 341 (237–345) 196 (156–581) 216 (95–299) 240 (227-)� 216 (188-)� 66 (7-)� 134 (111-)� The differences between asystole and PEA were not significant for all variables at all time points. CA–carotid artery MAP–mean arterial blood pressure CVR–carotid artery vascular resistance CO–cardiac output ROSC–return of spontaneous circulation � not able to calculate interquartile range (n = 3) https://doi.org/10.1371/journal.pone.0214506.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0214506 April 4, 2019 6 / 10 Neonatal non-perfusing rhythms Fig 2. Kaplan–Meier survival graph for piglets with pulseless electrical activity (PEA) and asystole p = 0.04. 1 = start of experiment, 2 = CPR, 3 = 1h after return of spontaneous circulation (ROSC), 4 = 2h after ROSC, 5 = 3h after ROSC, 6 = 4h after ROSC. https://doi.org/10.1371/journal.pone.0214506.g002 the right CCA, resulting in abnormal flow values relative to a non-occluded state. We still argue that the lack of difference in CCA flow between asystolic and PEA piglets may be valid. The difference in survival to 4 hours after ROSC between the PEA and asystole groups did not reach statistical significance, which was potentially due to a small sample size. As our results are based on secondary analyses of a study with a different endpoint, a power calculation was not performed for 4-hour survival. Clinical applicability ECG was only recently introduced to the delivery room [6]. Recent guidelines have suggested the potential benefit of ECG monitoring as standard of care due to the faster acquisition of a HR signal in preterm infants [31], and better accuracy compared to pulse oximetry [32]. However, the clinical data was collected mainly in non-asphyxiated infants. Our findings in piglets indicate that in one-third of the cases, ECG fails to provide a signal when the asphyxia becomes severe. In piglets with good quality ECG recordings, ECG demonstrated a non-perfusing rhythm, so-called PEA, in more than a third of cases. During perinatal asphyxia, any ECG HR without simultaneously assessing clinical signs of perfusion using auscultation or palpation should be considered suspicious of PEA. Based on the high incidence of a non-perfusing rhythm observed in our asphyxiated piglets, ECG rates alone might not be optimal to guide CPR interventions in asphyxiated infants. For adult use, efforts are made to develop devices and methods that may facilitate rhythm interpretation and decrease hands off time during CC [33]. In newborn infants, novel methods for HR assessment include digital stethoscopes or Doppler ultrasound [34–37]. Both technologies can obtain a HR faster than pulse oximetry [35–37] and have a good correlation with ECG HR [35, 37]. Bowel gas or movement of the infant might interfere with the signal acquisition using Doppler [36], while crying can decrease the accuracy of digital stethoscope [37]. However, neither movements nor crying are present in unresponsive newborn infants who require resuscitation. While both technologies have been assessed in healthy term and preterm infants, neither was assessed in asphyxiated infants. Further studies are needed before they could be introduced into clinical care. PLOS ONE \| https://doi.org/10.1371/journal.pone.0214506 April 4, 2019 7 / 10 Neonatal non-perfusing rhythms HR assessment remains central to neonatal CPR. However, failure to recognize the difference between PEA and bradycardia on an ECG when assessing HR without using other signs of systemic perfusion might be detrimental. The focus of neonatal CPR should remain on ventilation and chest compressions, as shockable rhythms are very rare in this population. Conclusion Cardiac arrest in the presence of a non-perfusing cardiac rhythm (PEA) on ECG was common in asphyxiated piglets. Piglets with PEA had lower rates of ROSC and lower 4 h survival compared to asystole, but this did not reach statistical significance. We recommend against the use of ECG as the sole method for assessing HR in the delivery room. Our study indicates that a combination of techniques or methods should be used to assess perfusion during neonatal resuscitation. Our results should guide future efforts to investigate heart rhythm disturbances and arrhythmias in newborn infants in the delivery room. Supporting information S1 Dataset. The dataset generated for this study. (SAV) Acknowledgments We would like to thank the public for donating money to our funding agencies. Author Contributions Conceptualization: Anne Lee Solevåg, Deandra Luong, Tze-Fun Lee, Megan O’Reilly, Po-Yin Cheung, Georg M. Schmölzer. Data curation: Anne Lee Solevåg, Deandra Luong, Tze-Fun Lee, Megan O’Reilly, Po-Yin Cheung, Georg M. Schmölzer. Formal analysis: Anne Lee Solevåg, Deandra Luong, Tze-Fun Lee, Megan O’Reilly, Po-Yin Cheung, Georg M. Schmölzer. Investigation: Anne Lee Solevåg, Deandra Luong, Tze-Fun Lee, Megan O’Reilly, Po-Yin Cheung, Georg M. Schmölzer. Methodology: Anne Lee Solevåg, Deandra Luong, Tze-Fun Lee, Megan O’Reilly, Po-Yin Cheung, Georg M. Schmölzer. Project administration: Megan O’Reilly, Po-Yin Cheung, Georg M. Schmölzer. Supervision: Po-Yin Cheung, Georg M. Schmölzer. Writing – original draft: Anne Lee Solevåg, Deandra Luong, Tze-Fun Lee, Megan O’Reilly, Po-Yin Cheung, Georg M. Schmölzer. Writing – review & editing: Anne Lee Solevåg, Deandra Luong, Tze-Fun Lee, Megan O’Reilly, Po-Yin Cheung, Georg M. Schmölzer. References 1. Larsen MP, Eisenberg MS, Cummins RO, Hallstrom AP. Predicting survival from out-of-hospital cardiac arrest: a graphic model. Ann Emerg Med. 1993; 22: 1652–8. PMID: 8214853 2. Eisenberg M, Bergner L, Hallstrom A. 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https://openalex.org/W4381335234	https://jurnal.yudharta.ac.id/v2/index.php/studi-arab/article/download/3497/2453	English	null	Can Mentimeter Become an Innovative Media in Sharf Learning?	Studi Arab	2,022	cc-by-sa	6,196	Studi Arab with CC BY-SA 4.0 license. Copyright © 2022, the author(s) Sanggupkah Mentimeter Menjadi Media Inovatif dalam Pembelajaran Shorof? Faizmailiatus Sofa1, Zainal Arifin Ahmad2, Nasiruddin3, Syiva Nurul Afifah4 Sunan Kalijaga State Islamic University1, 2, 3, Al-Azhar University Cairo, Egypt4 faizsofa1@gmail.com1, zainal.a@uin-suka.ac.id2, nasircahaya03@gmail.com3, afifahsyifa@gmail.com4 corresponding author Article History: Received: 21 November 2022 Revised: 2 Desember 2022 Accepted: 14 Desember 2022 Abstract The main objective of this research is to describe whether Mentimeter can be an innovative solution environment applied to Sharaf learning which is caused by the unenthusiasm and inactivity of students in Sharf learning in the Arabic Language Education study program at Sunan Kalijaga Islamic State University which results in ineffectiveness in learning, which should be learning using the presentations and questions and answers can increase student enthusiasm which makes learning effective. The study was conducted in a qualitative descriptive manner and its subject was Sharf's learning. This technique gathering data for this research in the form of documents, interviews, and observations. And the analysis technique uses presentation, condensation, and inference of data. Mentimeter is capable and capable of being an innovative medium for learning Sharf. With mentimeter, it will raise student attention so that students will focus during learning. This can increase student enthusiasm for learning, making them carry out teaching and learning activities to the fullest. However, due to limited time, the use of multimeters in Sharf learning has not been able to run optimally and not all students have answered the questions posed in mentimeter. Keywords: Mentimeter; Innovative Media; Sharf Kata Kunci: Mentimeter; Media Inovatif; Sharf Kata Kunci: Mentimeter; Media Inovatif; Sharf STUDI ARAB P-ISSN: 2086-9932 E-ISSN: 2502-616X STUDI ARAB P-ISSN: 2086-9932 E-ISSN: 2502-616X Vol. 13, No. 2, Desember 2022 pp. 51-61 doi: https://doi.org/10.35891/sa.v13.i2.3497 Vol. 13, No. 2, Desember 2022 pp. 51-61 doi: https://doi.org/10.35891/sa.v13.i2.3497 Abstrak Abstrak Tujuan dilakukannya kajian ini adalah untuk mendiskripsikan apakah mentimeter dapat menjadi media inovatif solutif atas terjadinya ketidakantusiasan dan ketidakaktifan mahasiswa pada pembelajaran Sharf dalam prodi Pendidikan Bahasa Arab yang ada di UIN Sunan Kalijaga yang menjadikan ketidakefektifan dalam pembelajaran, yang seharusnya pembelajaran dengan metode presentasi dan tanya jawab dapat meningkatkan keantusiasan siswa yang menjadikan efektifnya pembelajaran. Kajian dilakukan secara kualitatif deskriptif dengan subjek berupa pembelajaran Sharf. Teknik pengumpulan informasi penelitian berupa observasi, wawancara, dan dokumentasi. Dan teknik analisisnya menggunakan penyajian, kondensasi, serta penyimpulan data. Mentimeter sanggup dan mampu menjadi media inovatif dalam pembelajaran Sharf. Dengan mentimeter maka akan memunculkan atensi mahasiswa sehingga mahasiswa akan fokus selama pembelajaran. Hal itu dapat meningkatkan antusiasme mahasiswa dalam belajar yang menjadikannya melakukan kegiatan belajar mengajar secara maksimal. Akan tetapi dikarenakan waktu yang terbatas maka penggunaan mentimeter dalam pembelajaran Sharf belum dapat berjalan secara maksimal dan belum seluruh mahasiswa menjawab pertanyaan yang diajukan dalam mentimeter. 2 \| Vol. 13, No. 2, Desember 2022 2 Annisa Nidaur Rohmah, “Belajar Dan Pembelajaran (Pendidikan Dasar),” CENDEKIA Media Komunikasi Penelitian Dan Pengembangan Pendidikan Islam 09, no. 02 (2017). Introduction There are components in learning that must be present and mutually sustainable.1 These elements are students, teachers, learning objectives, materials, methods, media, as well as evaluation.2 All of these components are an integration that affects each other.3 If one of these components does not have a role or is not adjusted, it will affect the quality of the learning carried out.4 With the development of learning, it will train teachers to be creative and innovate to develop interesting learning for students,5 one of which is with learning media. In the language learning system, strategies, methods, and media are needed in their implementation.6 Learning media is a strategic tool in determining the success of the process.7 Media use as a learning component is necessary for teaching and learning activities.8,9 Although media is not the main element in learning,10 The existence of media in teaching and learning activities can help teachers to interact with students in delivering learning materials to achieve learning objectives.11 The use of appropriate learning environments can also help students become more active in the classroom allowing effective active learning.12 The dynamics of technology hold tremendous control over science.13 The technology that had been invented decades ago began to be replaced with new, more advanced technologies.14 4 Sutera dkk, “Analisis Sikap Siswa Dalam Proses Pembelajaran Dengan Pendekatan Saintifik Pada Kurikulum 2013 Tema Sejarah Peradaban Indonesia Kelas v Di Sekolah Dasar Negeri 28 Dangin Puri,” E-Journal PGSD Universitas Pendidikan Ganesha Jurusan PGSD Volume: 3 No: 1 Tahun 2015, 2015. ( ) p g 6 Nasir Salasa and Hasan Syaiful Rizal, “Penerapan Permainan Al-Asrar Al-Mutasalsilah Dalam Pembelajaran Maharah Istima’ Di SMP Darussalam 2 Watukosek Gempol,” Studi Arab 13, no. 1 (2022), https://doi.org/10.35891/sa.v13i1.3131. m Wijaya and Siti Maisuroh, “Pengembangan Bahan Ajar Kosakata Dengan Media Kartu Di MIN 1 ggo,” Studi Arab 13, no. 1 (2022), https://doi.org/10.35891/sa.v13i1.3137. 8 Junaidi Junaidi, “Peran Media Pembelajaran Dalam Proses Belajar Mengajar,” Diklat Review : Jurnal Manajemen Pendidikan Dan Pelatihan 3, no. 1 (2019), https://doi.org/10.35446/diklatreview.v3i1.349. ( ) p g 9 Samad Umarella M. Sahrawi Saimima Saddam Husein, “Urgensi Media Dalam Proses Pembelajaran,” Al-Iltizam: Jurnal Pendidikan Agama Islam 3, no. 2 (2018), https://doi.org/10.33477/alt.v3i2.605. g ( ) p g 10 Herka Maya Jatmika, “Pemanfaatan Media Visual Dalam Menunjang Pembelajaran Pendidikan Jasmani Di Sekolah Dasar,” Jurnal Pendidikan Jasmani Indonesia 3, no. 1 (2005). 4 Sutera dkk, “Analisis Sikap Siswa Dalam Proses Pembelajaran Dengan Pendekatan Saintifik Pada Kurikulum 2013 Tema Sejarah Peradaban Indonesia Kelas v Di Sekolah Dasar Negeri 28 Dangin Puri,” E-Journal PGSD Universitas Pendidikan Ganesha Jurusan PGSD Volume: 3 No: 1 Tahun 2015, 2015. H. M. Jufri Dolong, “Teknik Analisis Dalam Komponen Pembelajaran,” Jurnal UIN Alauddin 5, no. 2 (2016). Linda Khuroidah, The Transformation of Arabic Learning Language Majors in High School, Studi Arab 13, no. 2 (2022), https://doi.org/10.35891/sa.v13i2.3393. 6 Nasir Salasa and Hasan Syaiful Rizal, “Penerapan Permainan Al-Asrar Al-Mutasalsilah Dalam Pembelajaran Maharah Istima’ Di SMP Darussalam 2 Watukosek Gempol,” Studi Arab 13, no. 1 (2022), https://doi.org/10.35891/sa.v13i1.3131. 13 Wiwin Hartanto, “Penggunaan E-Learning Sebagai Media Pembelajaran,” Jurnal Pendidikan Ekonomi 10, no. 1 (2016). 14 Duma Megaria Elisabeth, “Kajian Terhadap Peranan Teknologi Informasi Dalam Perkembangan Audit Komputerisasi (Studi Kajian Teoritis),” METHOMIKA: Jurnal Manajemen Informatika & Komputerisasi Akuntansi 3, no. 1 (2019). 8 Junaidi Junaidi, “Peran Media Pembelajaran Dalam Proses Belajar Mengajar,” Diklat Review : Jurnal Manajemen Pendidikan Dan Pelatihan 3, no. 1 (2019), https://doi.org/10.35446/diklatreview.v3i1.349. 1 H. M. Jufri Dolong, “Teknik Analisis Dalam Komponen Pembelajaran,” Jurnal UIN Alauddin 5, no. 2 (2016). , ( ), p // g/ / 9 Samad Umarella M. Sahrawi Saimima Saddam Husein, “Urgensi Media Dalam Proses Pembelajaran,” Al-Iltizam: Jurnal Pendidikan Agama Islam 3, no. 2 (2018), https://doi.org/10.33477/alt.v3i2.605. g ( ) ti Fujiawati, “Pemahaman Konsep Kurikulum Dan Pembelajaran Dengan Peta Konsep Bagi Mahasiswa an Seni,” Jurnal Pendidikan Dan Kajian Seni 1, no. 1 (2016). p g 7 Mu’alim Wijaya and Siti Maisuroh, “Pengembangan Bahan Ajar Kosakata Dengan Media Kartu D Probolinggo,” Studi Arab 13, no. 1 (2022), https://doi.org/10.35891/sa.v13i1.3137. 12 Nina Sundari, “Penggunaan Media Gambar Dalam Meningkatkan Keaktifan Siswa Dalam Pembelajaran Pengetahuan Sosial Di Sekolah Dasar,” EduHumaniora \| Jurnal Pendidikan Dasar Kampus Cibiru 5, no. 1 (2016), https://doi.org/10.17509/eh.v5i1.2836. 1 H. M. Jufri Dolong, “Teknik Analisis Dalam Komponen Pembelajaran,” Jurnal UIN Alauddin 5, no. 2 (2016). 2 Annisa Nidaur Rohmah, “Belajar Dan Pembelajaran (Pendidikan Dasar),” CENDEKIA Media Komunikasi Penelitian Dan Pengembangan Pendidikan Islam 09, no. 02 (2017). 3 Fuja Siti Fujiawati, “Pemahaman Konsep Kurikulum Dan Pembelajaran Dengan Peta Konsep Bagi Mahasiswa Pendidikan Seni,” Jurnal Pendidikan Dan Kajian Seni 1, no. 1 (2016). 4 Sutera dkk, “Analisis Sikap Siswa Dalam Proses Pembelajaran Dengan Pendekatan Saintifik Pada Kurikulum 2013 Tema Sejarah Peradaban Indonesia Kelas v Di Sekolah Dasar Negeri 28 Dangin Puri,” E-Journal PGSD Universitas Pendidikan Ganesha Jurusan PGSD Volume: 3 No: 1 Tahun 2015, 2015. 5 Linda Khuroidah, “The Transformation of Arabic Learning Language Majors in High School,” Studi Arab 13, no. 2 (2022), https://doi.org/10.35891/sa.v13i2.3393. 6 Nasir Salasa and Hasan Syaiful Rizal, “Penerapan Permainan Al-Asrar Al-Mutasalsilah Dalam Pembelajaran Maharah Istima’ Di SMP Darussalam 2 Watukosek Gempol,” Studi Arab 13, no. 1 (2022), https://doi.org/10.35891/sa.v13i1.3131. 7 Mu’alim Wijaya and Siti Maisuroh, “Pengembangan Bahan Ajar Kosakata Dengan Media Kartu Di MIN 1 Probolinggo,” Studi Arab 13, no. 1 (2022), https://doi.org/10.35891/sa.v13i1.3137. 8 Junaidi Junaidi, “Peran Media Pembelajaran Dalam Proses Belajar Mengajar,” Diklat Review : Jurnal Manajemen Pendidikan Dan Pelatihan 3, no. 1 (2019), https://doi.org/10.35446/diklatreview.v3i1.349. 9 Samad Umarella M. Sahrawi Saimima Saddam Husein, “Urgensi Media Dalam Proses Pembelajaran,” Al-Iltizam: Jurnal Pendidikan Agama Islam 3, no. 2 (2018), https://doi.org/10.33477/alt.v3i2.605. 10 Herka Maya Jatmika, “Pemanfaatan Media Visual Dalam Menunjang Pembelajaran Pendidikan Jasmani Di Sekolah Dasar,” Jurnal Pendidikan Jasmani Indonesia 3, no. 1 (2005). 11 Teni Nurrita, “Pengembangan Media Pembelajaran Untuk Meningkatkan Hasil Belajar Siswa,” MISYKAT: Jurnal Ilmu-Ilmu Al-Quran, Hadist, Syari’ah Dan Tarbiyah 3, no. 1 (2018), https://doi.org/10.33511/misykat.v3n1.171. 12 Nina Sundari, “Penggunaan Media Gambar Dalam Meningkatkan Keaktifan Siswa Dalam Pembelajaran Pengetahuan Sosial Di Sekolah Dasar,” EduHumaniora \| Jurnal Pendidikan Dasar Kampus Cibiru 5, no. 1 (2016), https://doi.org/10.17509/eh.v5i1.2836. 13 Wiwin Hartanto, “Penggunaan E-Learning Sebagai Media Pembelajaran,” Jurnal Pendidikan Ekonomi 10, no. 1 (2016). 14 Duma Megaria Elisabeth, “Kajian Terhadap Peranan Teknologi Informasi Dalam Perkembangan Audit Komputerisasi (Studi Kajian Teoritis),” METHOMIKA: Jurnal Manajemen Informatika & Komputerisasi Akuntansi 3, no. 1 (2019). 11 Teni Nurrita, “Pengembangan Media Pembelajaran Untuk Meningkatkan Hasil Belajar Siswa,” MISYKAT: Jurnal Ilmu-Ilmu Al-Quran, Hadist, Syari’ah Dan Tarbiyah 3, no. 1 (2018), https://doi.org/10.33511/misykat.v3n1.171. j g g , J Pendidikan Ganesha Jurusan PGSD Volume: 3 No: 1 Tahun 2015, 2015. 5 Linda Khuroidah, “The Transformation of Arabic Learning Language Majors in High School,” Studi Arab 13, no. 2 (2022), https://doi.org/10.35891/sa.v13i2.3393. 6 Nasir Salasa and Hasan Syaiful Rizal, “Penerapan Permainan Al-Asrar Al-Mutasalsilah Dalam Pembelajaran Maharah Istima’ Di SMP Darussalam 2 Watukosek Gempol,” Studi Arab 13, no. 1 (2022), https://doi.org/10.35891/sa.v13i1.3131. 7 Mu’alim Wijaya and Siti Maisuroh, “Pengembangan Bahan Ajar Kosakata Dengan Media Kartu Di MIN 1 Probolinggo,” Studi Arab 13, no. 1 (2022), https://doi.org/10.35891/sa.v13i1.3137. 8 Junaidi Junaidi, “Peran Media Pembelajaran Dalam Proses Belajar Mengajar,” Diklat Review : Jurnal Manajemen Pendidikan Dan Pelatihan 3, no. 1 (2019), https://doi.org/10.35446/diklatreview.v3i1.349. 9 Samad Umarella M. Sahrawi Saimima Saddam Husein, “Urgensi Media Dalam Proses Pembelajaran,” Al-Iltizam: Jurnal Pendidikan Agama Islam 3, no. 2 (2018), https://doi.org/10.33477/alt.v3i2.605. 10 Herka Maya Jatmika, “Pemanfaatan Media Visual Dalam Menunjang Pembelajaran Pendidikan Jasmani Di Sekolah Dasar,” Jurnal Pendidikan Jasmani Indonesia 3, no. 1 (2005). 11 Teni Nurrita, “Pengembangan Media Pembelajaran Untuk Meningkatkan Hasil Belajar Siswa,” MISYKAT: Jurnal Ilmu-Ilmu Al-Quran, Hadist, Syari’ah Dan Tarbiyah 3, no. 1 (2018), https://doi.org/10.33511/misykat.v3n1.171. 12 Nina Sundari, “Penggunaan Media Gambar Dalam Meningkatkan Keaktifan Siswa Dalam Pembelajaran Pengetahuan Sosial Di Sekolah Dasar,” EduHumaniora \| Jurnal Pendidikan Dasar Kampus Cibiru 5, no. 1 (2016), g ( ) p g 10 Herka Maya Jatmika, “Pemanfaatan Media Visual Dalam Menunjang Pembelajaran Pendidikan Jasmani Di Sekolah Dasar,” Jurnal Pendidikan Jasmani Indonesia 3, no. 1 (2005). Introduction 11 Teni Nurrita, “Pengembangan Media Pembelajaran Untuk Meningkatkan Hasil Belajar Siswa,” MISYKAT: Jurnal Ilmu-Ilmu Al-Quran, Hadist, Syari’ah Dan Tarbiyah 3, no. 1 (2018), https://doi.org/10.33511/misykat.v3n1.171. 12 Nina Sundari, “Penggunaan Media Gambar Dalam Meningkatkan Keaktifan Siswa Dalam Pembelajaran Pengetahuan Sosial Di Sekolah Dasar,” EduHumaniora \| Jurnal Pendidikan Dasar Kampus Cibiru 5, no. 1 (2016), https://doi.org/10.17509/eh.v5i1.2836. 13 Wiwin Hartanto, “Penggunaan E-Learning Sebagai Media Pembelajaran,” Jurnal Pendidikan Ekonomi 10, no. 1 (2016). 14 Duma Megaria Elisabeth, “Kajian Terhadap Peranan Teknologi Informasi Dalam Perkembangan Audit Komputerisasi (Studi Kajian Teoritis),” METHOMIKA: Jurnal Manajemen Informatika & Komputerisasi Akuntansi 3, no. 1 (2019) Vol. 13, No. 2, Desember 2022 \| 53 Vol. 13, No. 2, Desember 2022 \| 53 \| 53 Vol. 13, No. 2, Desember 2022 \| Learning media is also increasingly varied along with the development of technology.15 There is always new media created and developed all the time.16 Ever-evolving media is in the form of technology,17 whether it is physical or nonphysical.18 The development of physical media in educational technology is the discovery of sophisticated tools that assist the learning and teaching activity.19 At the same time, the development of non-physical media can be found in applications that are constantly updated and developed every year.20 Learning media is also increasingly varied along with the development of technology.15 There is always new media created and developed all the time.16 Ever-evolving media is in the form of technology,17 whether it is physical or nonphysical.18 The development of physical media in educational technology is the discovery of sophisticated tools that assist the learning and teaching activity.19 At the same time, the development of non-physical media can be found in applications that are constantly updated and developed every year.20 In the span of decades, applications have grown very rapidly.21 The developed application has made a lot of progress.22 It is inseparable from the role of the internet.23 Applications that used to be limited to only being able to be used are growing with the emergence of internet-based applications.24 This makes the use of the application unlimited so that the application can be used in the field of life, it includes learning media.25 An application used as an online medium to support learning and teaching activities is Mentimeter. Introduction Mentimeter is an online polling application (polling tool) that acts as an aggregator of student responses.26 This web-based application can help collect students' opinions and questions.27 Using Mentimeter as a tool that can increase student engagement in learning activities can be done by measuring and assessing opinions, engaging in discussions, raising attention, and submitting questions.28 J y ( ) 16 Rudi Susilana and Cepi Riyana, Media Pembelajaran: Hakikat, Pengembangan, Pemanfaatan, Dan Penilaian (Bandung: CV Wacana Prima, 2008). ) 17 M A M Ardiansyah, “Inovasi Pemanfaatan Teknologi Informasi Dan Komunikasi Sebagai Media Pembelajaran Efektif,” Semnas Ristek (Seminar Nasional … 9924 (2021). 18 Alan Suud Maadi, “Digitalisasi Manajemen Pendidikan Islam Dan Ekonomi Syariah Di Perguruan Tinggi,” FIKROTUNA 7, no. 1 (2018), https://doi.org/10.32806/jf.v7i1.3185. p g j n Teknologi Dalam Pembelajaran,” Jakarta: Kencana, 201 ( ) p g j 19 R Benny A Pribadi, “Media Dan Teknologi Dalam Pembelajaran,” Jakarta: Kencana, 2017. 20 Titi Anjarini, “Strategi, Model, Media Dan Teknologi Pembelajaran Di Sekolah Dasar,” ELSE : Jurnal Pendidikan Dan Pembelajaran Sekolah Dasar Universitas Muhammadiyah Purworejo 1, no. Strategi, Model, Media dan Teknologi Pembelajaran di Sekolah Dasar (2017). 21 Muhamad Ngafifi, “Kemajuan Teknologi Dan Pola Hidup Manusia Dalam Perspektif Sosial Budaya,” Jurnal Pembangunan Pendidikan: Fondasi Dan Aplikasi 2, no. 1 (2014), https://doi.org/10.21831/jppfa.v2i1.2616. g p ( ) p g jpp 22 Selamet Samsugi et al., “Internet of Things Untuk Peningkatan Pengetahuan Teknologi Bagi Siswa,” Journal of Technology and Social for Community Service (JTSCS) 2, no. 2 (2020). 23 Unik Hanifah Salsabila et al., “Peran Teknologi Dalam Pembelajaran Di Masa Pandemi Covid-19,” Al-Mutharahah: Jurnal Penelitian Dan Kajian Sosial Keagamaan 17, no. 2 (2020), https://doi.org/10.46781/al-mutharahah.v17i2.138. wi Abdillah, L. A. et al., Aplikasi Teknologi Informasi: Konsep Dan Penerapan (Medan: Yayasan Kita Menulis, 2020) 25 Erfin Nurfalah, “Optimalisasi E-Learning Berbasis Virtual Class Dengan Google Classroom Sebagai Media Pembelajaran Fisika,” Physics Education Research Journal 1, no. 1 (2019), https://doi.org/10.21580/perj.2019.1.1.3977. 26 Kat Sarah Anne Vallely and Poppy Gibson, “Engaging Students on Their Devices with Mentimeter,” Compass: Journal 25 Erfin Nurfalah, “Optimalisasi E-Learning Berbasis Virtual Class Dengan Google Classroom Sebagai Media Pembelajaran Fisika,” Physics Education Research Journal 1, no. 1 (2019), https://doi.org/10.21580/perj.2019.1.1.3977. Erfin Nurfalah, Optimalisasi E-Learning Berbasis Virtual Class Dengan Google Classroom Sebagai Media Pembelajaran Fisika,” Physics Education Research Journal 1, no. 1 (2019), https://doi.org/10.21580/perj.2019.1.1.3977. 26 Kat Sarah Anne Vallely and Poppy Gibson, “Engaging Students on Their Devices with Mentimeter,” Compass: Journal of Learning and Teaching 11, no. 2 (2018), https://doi.org/10.21100/compass.v11i2.843. J y ( ) 16 Rudi Susilana and Cepi Riyana, Media Pembelajaran: Hakikat, Pengembangan, Pemanfaatan, Dan Penilaian (Bandung: CV Wacana Prima, 2008). , ) 17 M A M Ardiansyah, “Inovasi Pemanfaatan Teknologi Informasi Dan Komunikasi Sebagai Media Pembelajaran Efektif,” Semnas Ristek (Seminar Nasional … 9924 (2021). Jurnal Ilmu Pendidikan PKn Dan Sosial Budaya 2, no. 1 (2019). 16 Rudi Susilana and Cepi Riyana, Media Pembelajaran: Hakikat, Pengembangan, Pemanfaatan, Dan Penilaian (Bandung: CV Wacana Prima, 2008). 15 Sodiq Anshori, “Pemanfaatan Teknologi Informasi Dan Komunikasi Sebagai Media Pembelajaran,” Civic-Culture: Jurnal Ilmu Pendidikan PKn Dan Sosial Budaya 2, no. 1 (2019). g ( ) p g j 28 Alison Skoyles and Erin Bloxsidge, “Have You Voted? Teaching OSCOLA with Mentimeter,” Legal I Management 17, no. 4 (2017), https://doi.org/10.1017/s1472669617000457. 27 J. I. Pichardo et al., “A Brief Review of Mentimeter – a Student Response System,” Journal of Applied Learning & Teaching 1, no. 1 (2017), https://doi.org/10.37074/jalt.2018.1.1.5. 54 \| Vol. 13, No. 2, Desember 2022 54 \| Vol. 13, No. 2, Desember 2022 Effective learning should be supported by student activity.29 That applies to every learning,30 including Sharf learning. However, in the observations that have been made, learning in the Sharf course in the Arabic language education program at Sunan Kalijaga State Islamic University shows students' inactivity in learning as evidenced by the absence of students asking questions and some students not paying attention to presentations made by other students. Therefore, solutions are needed that can provoke students to be active in learning. There are several scientific studies that discuss the success of Mentimeter as an innovative medium that can increase student activity in the classroom. Like Emma Mayhew's research31 that applied it in political science, research conducted by Pei Miin Wong32 that applied it to writing skills, as well as a scientific study conducted by Angela Bayu Pertama Sari33 Based on several studies that have succeeded in increasing student activity through Mentimeter, this study intends to analyze whether Mentimeter can be an innovative medium that can provoke student enthusiasm in class in Sharf learning as a development in the world of educational technology so that it can be a solution to the gap between ideal conditions in learning and reality that exists in Sharf's teaching and learning activities. With this research, it will lead to the development and expansion of the use of Mentimeter in teaching Arabic, especially Sharf. 29 Ramdanil Mubarok, “Dinamika Lembaga Pendidikan Dasar Dalam Pengelolaan Pembelajaran Daring,” Pedagogi: Jurnal Ilmu Pendidikan 21, no. 1 (2021), https://doi.org/10.24036/pedagogi.v21i1.1033. g, g , ( ), p // g/ / j 33 Angela Bayu Pertama Sari, “The Impacts of Mentimeter-Based Activities on EFL Students’ Engagement In Indonesia,” LLT Journal: A Journal on Language and Language Teaching 24, no. 1 (2021), https://doi.org/10.24071/llt.v24i1.3025. J ( ) p g p g g 30 Kartini Hutagaol, “Pembelajaran Kontekstual Untuk Meningkatkan Kemampuan Representasi Matematis Siswa Sekolah Menengah Pertama,” Infinity Journal 2, no. 1 (2013), https://doi.org/10.22460/infinity.v2i1.27. Introduction j j 26 Kat Sarah Anne Vallely and Poppy Gibson, “Engaging Students on Their Devices with Mentimeter,” Compass: Journal of Learning and Teaching 11, no. 2 (2018), https://doi.org/10.21100/compass.v11i2.843. y ppy , g g g Learning and Teaching 11, no. 2 (2018), https://doi.org/10.21100/compass.v11i2.843. 27 J. I. Pichardo et al., “A Brief Review of Mentimeter – a Student Response System,” Journal of Applied Learning & Teaching 1, no. 1 (2017), https://doi.org/10.37074/jalt.2018.1.1.5. g ( ) p g j 28 Alison Skoyles and Erin Bloxsidge, “Have You Voted? Teaching OSCOLA with Mentimeter,” Legal Information Management 17, no. 4 (2017), https://doi.org/10.1017/s1472669617000457. 31 Emma Mayhew, “No Longer a Silent Partner: How Mentimeter Can Enhance Teaching and Learning Within Political Science,” Journal of Political Science Education 15, no. 4 (2019), https://doi.org/10.1080/15512169.2018.1538882. 32 Pei Miin Wong and Melor Md Yunus, “Enhancing Writing Vocabulary Using Mentimeter,” International Journal of Learning, Teaching and Educational Research 19, no. 3 (2020), https://doi.org/10.26803/ijlter.19.3.7. 29 Ramdanil Mubarok, “Dinamika Lembaga Pendidikan Dasar Dalam Pengelolaan Pembelajaran Daring,” Pedagogi: Jurnal Ilmu Pendidikan 21, no. 1 (2021), https://doi.org/10.24036/pedagogi.v21i1.1033. 30 Kartini Hutagaol, “Pembelajaran Kontekstual Untuk Meningkatkan Kemampuan Representasi Matematis Siswa Sekolah Menengah Pertama,” Infinity Journal 2, no. 1 (2013), https://doi.org/10.22460/infinity.v2i1.27. 31 Emma Mayhew, “No Longer a Silent Partner: How Mentimeter Can Enhance Teaching and Learning Within Political Science,” Journal of Political Science Education 15, no. 4 (2019), https://doi.org/10.1080/15512169.2018.1538882. 32 Pei Miin Wong and Melor Md Yunus, “Enhancing Writing Vocabulary Using Mentimeter,” International Journal of Learning, Teaching and Educational Research 19, no. 3 (2020), https://doi.org/10.26803/ijlter.19.3.7. 33 Angela Bayu Pertama Sari, “The Impacts of Mentimeter-Based Activities on EFL Students’ Engagement In Indonesia,” LLT Journal: A Journal on Language and Language Teaching 24, no. 1 (2021), https://doi.org/10.24071/llt.v24i1.3025. g f y J ( ) p g y mma Mayhew, “No Longer a Silent Partner: How Mentimeter Can Enhance Teaching and Learning With cal Science,” Journal of Political Science Education 15, no. 4 (2019), https://doi.org/10.1080/15512169.2018.153888 Sekolah Menengah Pertama, Infinity Journal 2, no. 1 (2013), https://doi.org/10.22460/infinity.v2i1.27. 31 Emma Mayhew, “No Longer a Silent Partner: How Mentimeter Can Enhance Teaching and Learning Within Political Science,” Journal of Political Science Education 15, no. 4 (2019), https://doi.org/10.1080/15512169.2018.1538882. 32 Pei Miin Wong and Melor Md Yunus, “Enhancing Writing Vocabulary Using Mentimeter,” International Journal of Learning, Teaching and Educational Research 19, no. 3 (2020), https://doi.org/10.26803/ijlter.19.3.7. 33 Angela Bayu Pertama Sari, “The Impacts of Mentimeter-Based Activities on EFL Students’ Engagement In Indonesia,” LLT Journal: A Journal on Language and Language Teaching 24, no. 1 (2021), Political Science, Journal of Political Science Education 15, no. 4 (2019), https://doi.org/10.1080/15512169.2018.1538882. 32 Pei Miin Wong and Melor Md Yunus, “Enhancing Writing Vocabulary Using Mentimeter,” International Journal of Learning, Teaching and Educational Research 19, no. 3 (2020), https://doi.org/10.26803/ijlter.19.3.7. 33 Angela Bayu Pertama Sari, “The Impacts of Mentimeter-Based Activities on EFL Students’ Engagement In Indonesia,” LLT Journal: A Journal on Language and Language Teaching 24, no. 1 (2021), https://doi.org/10.24071/llt.v24i1.3025. Method In this scientific study, the method used is a qualitative method, Scientific research that produces descriptive scientific study information in the form of written or spoken words and human actions where the researcher is the primary tool and the focus is comprehensive research. This type of scientific study is descriptive qualitative in Sharf learning. This type is used in this study with the aim of describing and explaining scientific information about the use of Mentimeter as an effective innovative medium in learning in Sharf courses at Kalijaga Islamic State University. The selected scientific study design is a qualitative descriptive study. In this study, the use of Mentimeter in Sharf learning as an innovative communicative medium is explained and described in detail. The application of the design is carried out by collecting, processing, and presenting research information objectively. The subjects of scientific Vol. 13, No. 2, Desember 2022 \| 55 Vol. 13, No. 2, Desember 2022 \| 5 \| 55 research are 12 students and Sharf lecturers in Arabic language education at Kalijaga Islamic State University. Meanwhile, the source of data in scientific studies is lecturers who teach courses and students who take part in Sharf courses. The chosen research information collection technique is observation during three meetings. In the initial two meetings, observations were carried out in a non-participatory manner where researchers were directly involved in learning. Meanwhile, the third meeting of observations was carried out in a participatory manner. In addition, this study also utilizes interviews with students and lecturers who teach the course. Another selected data collection is documentation. Analysis of research information carried out is condensation, data display, as well as verification and drawing conclusions to achieve the results of scientific studies. Scientific studies are carried out at Kalijaga Islamic State University Yogyakarta in the Arabic language education study program, especially in the Sharf course. The research began with identifying the employ of Mentimeter as an online learning media in Sharf courses. The social situation that is the object of this scientific study is in the form of learning in the Sharf course, both with Mentimeter media and not with media. The information collection, review, and inference are carried out as needed. Sharf Learning in Arabic Language Education at Sunan Kalijaga State Islamic University Sharf Learning in Arabic Language Education at Sunan Kalijaga State Islamic University In the Sharf credits course, there are 4 credits. However, it is divided into two meetings with 2 credits at each meeting. Each credit takes 50 minutes. So for each meeting, the time passed is 100 minutes. The learning that took place in the Sharf course at Sunan Kalijaga Islamic State University was carried out by the method of presentation and question and answer conducted by students and lecturers. Learning is carried out with zoom meeting media as a meeting room. Learning begins with student presentations on relevant material and continues with questions and answers about the material. If the speaker has not answered the question correctly, the lecturer will straighten out the answer. In learning the Sharf course at Kalijaga Islamic State University conventionally without the Mentimeter media, information was found that students' enthusiasm for learning was still lacking. This is supported by data from observations made which illustrate that students' enthusiasm for learning Sharf is still relatively weak. Based on information from observations made during two meetings, the first meeting conducted online through the zoom meeting media illustrates the unenthusiasm of students in learning as evidenced by a large number of students who are still on the road, many students who are not on-cam, the absence of students who ask questions, criticize, \| Vol. 13, No. 2, Desember 2022 56 and refute opinions that are not in accordance with scientific studies. Lectures only last for 60 minutes which should be 100 minutes. In the second meeting, learning was carried out still using zoom meetings. The learning carried out is still conventional with presentation and question and answer methods. At the meeting, none of the students attended the lecture on-cam (turning on the camera) during the lesson. Only the lecturer turns on the camera during the lecture. There were two students who made presentations, while the students who asked questions were only one person. In addition, lectures only last for an hour which should run for 100 minutes. Mentimeter as a Learning Media Mentimeter is a web developed by a Swedish company based in Stockholm that is used for presentations that want feedback from participants in a short period of time. This web was originally created by Johnny Warstorm who is an entrepreneur as a solution to problems that exist at meetings that are not conducive. Therefore Johnny created a medium that can give rise to dynamic interactions between presenters and participants. In Mentimeter, various results of interactions between people can be displayed with polls, ratings, or expressing opinions. An example of the Mentimeter view can be seen in figure 1. Figure 1. Mentimeter View Figure 1. Mentimeter View In learning Sharf in the Arabic Language Education study program at Kalijaga Islamic State University, Mentimeter is designed as a medium that can provoke students to express their opinions in several related materials. The questions posed in Mentimeter on Sharf's learning on Ibdal material are: a. What do you know about Ibdal? b. What is the meaning of I’lal? c. Mention 1 ibdal rule in Sharf science that you know! Sharf learning with Mentimeter as media is done offline. Learning is carried out for 100 minutes according to the expected time in one meeting in 2 credits. The learning begins with a Vol. 13, No. 2, Desember 2022 \| 57 Vol. 13, No. 2, Desember 2022 \| 57 \| 57 presentation made by students without Mentimeter media which is then continued with a question and answer between the presenter students and the participants. The speakers presented a number of one student. The enthusiasm of students began to be seen from the beginning of learning, which raised one question from other students. Sharf learning is continued with presentations made by the teacher using Mentimeter media. The use of Mentimeter media begins with basic questions about the material presented by students. This aroused students' enthusiasm for learning as evidenced by the eight answers given by seven of all students who answered the first question posed in the Mentimeter. It can be seen in figure 2. Figure 2. Student Answers on Question 1 Figure 2. Student Answers on Question 1 While using Mentimeter in learning, teachers intersperse presentations with explanations of the material, then continue with other questions. This is done so that learning is not monotonous only using asking questions to students so that learning will be more varied. Mentimeter as a Learning Media In the second question, the enthusiasm of the students has not diminished. All students tried to answer the questions asked in Mentimeter. This is evidenced by as many as 12 answers put forward by seven of all students which can be seen in figure 3. Figure 3. Student Answers on Question 3 Figure 3. Student Answers on Question 3 In the second question, the enthusiasm of the students has not diminished. All students tried to answer the questions asked in Mentimeter. However, because there was little time left, the \| Vol. 13, No. 2, Desember 2022 58 teacher dismissed the submission of answers, and only five answers from four students were recorded. Figure 4 below shows the answers. Figure 4. Student Answers on Question 3 Figure 4. Student Answers on Question 3 Discussion Mentimeter has been widely used in learning and their success in increasing the enthusiasm of participants has been found in many studies. The event also occurred in Sharf's learning. The use of a Mentimeter during Sharf learning can increase students' enthusiasm for learning. Mentimeter, which replaced Zoom Meeting as a medium, succeeded in making it an effective innovative medium during teaching and learning activities. The following table shows the increase in student enthusiasm. Table 1. Learning Enthusiasm Indicators No. Student Activities 1st Meeting 2nd Meeting 3rd Meeting 1. Set up a notebook Enough Enough Good 2. Follow and pay attention to teacher delivery Less Less Very Good 3. Follow learning focusly Less Less Very Good 4. Asking, criticizing, and debunking Enough Good Good 5 Expressing an opinion Less Less Very Good Table 1. Learning Enthusiasm Indicators Based on what can be seen in the table above, it can be concluded that the student's enthusiasm for learning increased from the first to the third lesson by using the "Mentimeter" as a learning tool. The table above shows that at the first to second meetings before Mentimeter media has not been used in Sharf learning, the indicators are dominated by fewer categories. At the third meeting, the enthusiasm of the students was dominated by the excellent category after the media Mentimeter was used in Sharf's learning. This increase can occur because in Mentimeter there are questions that attract students to be able to answer them and encourage them to raise opinions. It makes students think critically so that they can focus during learning. However, due to limited time, \| 59 Vol. 13, No. 2, Desember 2022 \| 59 Vol. 13, No. 2, Desember 2022 \| 59 the use of Mentimeter in Sharf learning has not been able to run optimally and not all students have answered the questions asked in Mentimeter. References Abdillah, L. A., Alwi, Simarmata M. H., Bisyri J., Nasrullah M., and Asmeati , N. Aplikasi Teknologi Informasi: Konsep Dan Penerapan. Medan: Yayasan Kita Menulis, 2020. Abdillah, L. A., Alwi, Simarmata M. H., Bisyri J., Nasrullah M., and Asmeati , N. Aplikasi Teknologi Informasi: Konsep Dan Penerapan. Medan: Yayasan Kita Menulis, 2020. Anjarini, Titi. “Strategi, Model, Media Dan Teknologi Pembelajaran Di Sekolah Dasar.” ELSE : Jurnal Pendidikan Dan Pembelajaran Sekolah Dasar Universitas Muhammadiyah Purworejo 1, no. Strategi, Model, Media dan Teknologi Pembelajaran di Sekolah Dasar (2017). Anshori, Sodiq. “Pemanfaatan Teknologi Informasi Dan Komunikasi Sebagai Media Pembelajaran.” Civic-Culture: Jurnal Ilmu Pendidikan PKn Dan Sosial Budaya 2, no. 1 (2019). Ardiansyah, M A M. “Inovasi Pemanfaatan Teknologi Informasi Dan Komunikasi Sebagai Media Pembelajaran Efektif.” Semnas Ristek (Seminar Nasional … 9924 (2021). Dolong, H. M. Jufri. “Teknik Analisis Dalam Komponen Pembelajaran.” Jurnal UIN Alauddin 5, no. 2 (2016). Elisabeth, Duma Megaria. “Kajian Terhadap Peranan Teknologi Informasi Dalam Perkembangan Audit Komputerisasi (Studi Kajian Teoritis).” METHOMIKA: Jurnal Manajemen Informatika & Komputerisasi Akuntansi 3, no. 1 (2019). Audit Komputerisasi (Studi Kajian Teoritis).” METHOMIKA: Jurnal Manajemen Informatika & Komputerisasi Akuntansi 3, no. 1 (2019). Audit Komputerisasi (Studi Kajian Teoritis).” METHOMIKA: Jurnal Manajemen Informatika & Komputerisasi Akuntansi 3, no. 1 (2019). Fujiawati, Fuja Siti. “Pemahaman Konsep Kurikulum Dan Pembelajaran Dengan Peta Konsep Bagi Mahasiswa Pendidikan Seni.” Jurnal Pendidikan Dan Kajian Seni 1, no. 1 (2016). Hanifah Salsabila, Unik, Lailli Irna Sari, Khusna Haibati Lathif, Ayu Puji Lestari, and Asyharinur Ayuning. “Peran Teknologi Dalam Pembelajaran Di Masa Pandemi Covid-19.” Al- Mutharahah: Jurnal Penelitian Dan Kajian Sosial Keagamaan 17, no. 2 (2020). https://doi.org/10.46781/al-mutharahah.v17i2.138. Hartanto, Wiwin. “Penggunaan E-Learning Sebagai Media Pembelajaran.” Jurnal Pendidikan Ek i 10 1 (2016) Fujiawati, Fuja Siti. “Pemahaman Konsep Kurikulum Dan Pembelajaran Dengan Peta Konsep Bagi Mahasiswa Pendidikan Seni.” Jurnal Pendidikan Dan Kajian Seni 1, no. 1 (2016). Fujiawati, Fuja Siti. Pemahaman Konsep Kurikulum Dan Pembelajaran Dengan Peta Konsep Bagi Mahasiswa Pendidikan Seni.” Jurnal Pendidikan Dan Kajian Seni 1, no. 1 (2016). Hanifah Salsabila, Unik, Lailli Irna Sari, Khusna Haibati Lathif, Ayu Puji Lestari, and Asyharinur Ayuning. “Peran Teknologi Dalam Pembelajaran Di Masa Pandemi Covid-19.” Al- Mutharahah: Jurnal Penelitian Dan Kajian Sosial Keagamaan 17, no. 2 (2020). https://doi.org/10.46781/al-mutharahah.v17i2.138. H t t Wi i “P E L i S b i M di P b l j ” J l P didik Hanifah Salsabila, Unik, Lailli Irna Sari, Khusna Haibati Lathif, Ayu Puji Lestari, and Asyharinur Ayuning. Conclusion Mentimeter is able to be an innovative medium in Sharf learning. Learning using Mentimeter will bring out student attention so that students will focus during learning. It can increase student enthusiasm for learning which makes it carry out teaching and learning activities optimally. However, due to limited time, the use of Mentimeter in Sharf learning has not been able to run optimally and not all students have answered the questions asked in Mentimeter because of less time. Therefore, future research should be able to examine other similar media that can be used more briefly so that learning will be more effective and efficient. References “Peran Teknologi Dalam Pembelajaran Di Masa Pandemi Covid-19.” Al- Mutharahah: Jurnal Penelitian Dan Kajian Sosial Keagamaan 17, no. 2 (2020). https://doi.org/10.46781/al-mutharahah.v17i2.138. Hartanto, Wiwin. “Penggunaan E-Learning Sebagai Media Pembelajaran.” Jurnal Pendidikan Ekonomi 10, no. 1 (2016). 60 \| Vol. 13, No. 2, Desember 2022 Hutagaol, Kartini. “Pembelajaran Kontekstual Untuk Meningkatkan Kemampuan Representasi Matematis Siswa Sekolah Menengah Pertama.” Infinity Journal 2, no. 1 (2013). https://doi.org/10.22460/infinity.v2i1.27. Jatmika, Herka Maya. “Pemanfaatan Media Visual Dalam Menunjang Pembelajaran Pendidikan Jasmani Di Sekolah Dasar.” Jurnal Pendidikan Jasmani Indonesia 3, no. 1 (2005). Junaidi, Junaidi. “Peran Media Pembelajaran Dalam Proses Belajar Mengajar.” Diklat Review : Jurnal Manajemen Pendidikan Dan Pelatihan 3, no. 1 (2019). https://doi.org/10.35446/diklatreview.v3i1.349. Khuroidah, Linda. “The Transformation of Arabic Learning Language Majors in High School.” Studi Arab 13, no. 2 (2022). https://doi.org/10.35891/sa.v13i2.3393. Maadi, Alan Suud. “Digitalisasi Manajemen Pendidikan Islam Dan Ekonomi Syariah Di Perguruan Tinggi.” FIKROTUNA 7, no. 1 (2018). https://doi.org/10.32806/jf.v7i1.3185. Mayhew, Emma. “No Longer a Silent Partner: How Mentimeter Can Enhance Teaching and Learning Within Political Science.” Journal of Political Science Education 15, no. 4 (2019). https://doi.org/10.1080/15512169.2018.1538882. Mubarok, Ramdanil. “Dinamika Lembaga Pendidikan Dasar Dalam Pengelolaan Pembelajaran Daring.” Pedagogi: Jurnal Ilmu Pendidikan 21, no. 1 (2021). https://doi.org/10.24036/pedagogi.v21i1.1033. Ngafifi, Muhamad. “Kemajuan Teknologi Dan Pola Hidup Manusia Dalam Perspektif Sosial Budaya.” Jurnal Pembangunan Pendidikan: Fondasi Dan Aplikasi 2, no. 1 (2014). https://doi.org/10.21831/jppfa.v2i1.2616. Nurfalah, Erfin. “Optimalisasi E-Learning Berbasis Virtual Class Dengan Google Classroom Sebagai Media Pembelajaran Fisika.” Physics Education Research Journal 1, no. 1 (2019). https://doi.org/10.21580/perj.2019.1.1.3977. Nurrita, Teni. “Pengembangan Media Pembelajaran Untuk Meningkatkan Hasil Belajar Siswa.” MISYKAT: Jurnal Ilmu-Ilmu Al-Quran, Hadist, Syari’ah Dan Tarbiyah 3, no. 1 (2018). https://doi.org/10.33511/misykat.v3n1.171. Pichardo, J. I., E. F. López-Medina, O. Mancha-Cáceres, I. González-Enríquez, A. Hernández- Melián, and M Blázquez-Rodríguez. “A Brief Review of Mentimeter – a Student Response System.” Journal of Applied Learning & Teaching 1, no. 1 (2017). https://doi.org/10.37074/jalt.2018.1.1.5. Pribadi, R Benny A. “Media Dan Teknologi Dalam Pembelajaran.” Jakarta: Kencana, 2017. Rohmah, Annisa Nidaur. “Belajar Dan Pembelajaran (Pendidikan Dasar).” CENDEKIA Media Komunikasi Penelitian Dan Pengembangan Pendidikan Islam 09, no. 02 (2017). Saddam Husein, Samad Umarella M. Sahrawi Saimima. “Urgensi Media Dalam Proses Vol. 13, No. 2, Desember 2022 \| 61 Pembelajaran.” Al-Iltizam: Jurnal Pendidikan Agama Islam 3, no. 2 (2018). https://doi.org/10.33477/alt.v3i2.605. Pembelajaran.” Al-Iltizam: Jurnal Pendidikan Agama Islam 3, no. 2 (2018). https://doi.org/10.33477/alt.v3i2.605. Salasa, Nasir, and Hasan Syaiful Rizal. “Penerapan Permainan Al-Asrar Al-Mutasalsilah Dalam Pembelajaran Maharah Istima’ Di SMP Darussalam 2 Watukosek Gempol.” Studi Arab 13, no. 1 (2022). https://doi.org/10.35891/sa.v13i1.3131. References Samsugi, Selamet, Andi Nurkholis, Berlintina Permatasari, Ady Candra Nugroho, and Aldi Bagus Prasetyo. “Internet of Things Untuk Peningkatan Pengetahuan Teknologi Bagi Siswa.” Journal of Technology and Social for Community Service (JTSCS) 2, no. 2 (2020). Sari, Angela Bayu Pertama. “The Impacts of Mentimeter-Based Activities on EFL Students’ Engagement In Indonesia.” LLT Journal: A Journal on Language and Language Teaching 24, no. 1 (2021). https://doi.org/10.24071/llt.v24i1.3025. Skoyles, Alison, and Erin Bloxsidge. “Have You Voted? Teaching OSCOLA with Mentimeter.” Legal Information Management 17, no. 4 (2017). https://doi.org/10.1017/s1472669617000457. Sundari, Nina. “Penggunaan Media Gambar Dalam Meningkatkan Keaktifan Siswa Dalam Pembelajaran Pengetahuan Sosial Di Sekolah Dasar.” EduHumaniora \| Jurnal Pendidikan Dasar Kampus Cibiru 5, no. 1 (2016). https://doi.org/10.17509/eh.v5i1.2836. Susilana, Rudi, and Cepi Riyana. Media Pembelajaran: Hakikat, Pengembangan, Pemanfaatan, Dan Penilaian. Bandung: CV Wacana Prima, 2008. Sutera dkk. “Analisis Sikap Siswa Dalam Proses Pembelajaran Dengan Pendekatan Saintifik Pada Kurikulum 2013 Tema Sejarah Peradaban Indonesia Kelas v Di Sekolah Dasar Negeri 28 Dangin Puri.” E-Journal PGSD Universitas Pendidikan Ganesha Jurusan PGSD Volume: 3 No: 1 Tahun 2015, 2015. Vallely, Kat Sarah Anne, and Poppy Gibson. “Engaging Students on Their Devices with Mentimeter.” Compass: Journal of Learning and Teaching 11, no. 2 (2018). https://doi.org/10.21100/compass.v11i2.843. Wijaya, Mu’alim, and Siti Maisuroh. “Pengembangan Bahan Ajar Kosakata Dengan Media Kartu Di MIN 1 Probolinggo.” Studi Arab 13, no. 1 (2022). https://doi.org/10.35891/sa.v13i1.3137. Wong, Pei Miin, and Melor Md Yunus. “Enhancing Writing Vocabulary Using Mentimeter.” International Journal of Learning, Teaching and Educational Research 19, no. 3 (2020). https://doi.org/10.26803/ijlter.19.3.7.
https://openalex.org/W2037082039	https://europepmc.org/articles/pmc3166140?pdf=render	English	null	A Small Mammal Community in a Forest Fragment, Vegetation Corridor and Coffee Matrix System in the Brazilian Atlantic Forest	PloS one	2,011	cc-by	4,974	Abstract The objective of our work was to verify the value of the vegetation corridor in the conservation of small mammals in fragmented tropical landscapes, using a model system in the southeastern Minas Gerais. We evaluated and compared the composition and structure of small mammals in a vegetation corridor, forest fragments and a coffee matrix. A total of 15 species were recorded, and the highest species richness was observed in the vegetation corridor (13 species), followed by the forest fragments (10) and the coffee matrix (6). The absolute abundance was similar between the vegetation corridor and fragments (F = 22.94; p = 0.064), and the greatest differences occurred between the vegetation corridor and the matrix (F = 22.94; p = 0.001) and the forest fragments and the matrix (F = 22.94; p = 0.007). Six species showed significant habitat preference possibly related to the sensitivity of the species to the forest disturbance. Marmosops incanus was the species most sensitive to disturbance; Akodon montensis, Cerradomys subflavus, Gracilinanus microtarsus and Rhipidomys sp. displayed little sensitivity to disturbance, with a high relative abundance in the vegetation corridor. Calomys sp. was the species least affected by habitat disturbance, displaying a high relative abundance in the coffee matrix. Although the vegetation corridors are narrow (4 m width), our results support the hypothesis in which they work as a forest extension, share most species with the forest fragment and support species richness and abundance closer to forest fragments than to the coffee matrix. Our work highlights the importance and cost-effectiveness of these corridors to biodiversity management in the fragmented Atlantic Forest landscapes and at the regional level. Editor: Adina Maya Merenlender, University of California, Berkeley, United States of America Received September 17, 2010; Accepted July 14, 2011; Published August 31, 2011 cha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Rocha et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Rocha et al. This is an open-access article distributed under the terms of the Creative Commons Attr unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. A Small Mammal Community in a Forest Fragment, Vegetation Corridor and Coffee Matrix System in the Brazilian Atlantic Forest Mariana Ferreira Rocha, Marcelo Passamani, Ju´ lio Louzada Mariana Ferreira Rocha, Marcelo Passamani, Ju´ lio Louzada Setor de Ecologia, Departamento de Biologia, Universidade Federal de Lavras, Lavras, Brazil Mariana Ferreira Rocha, Marcelo Passamani, Ju´ lio Louzada Setor de Ecologia, Departamento de Biologia, Universidade Federal de Lavras, Lavras, Brazil Abstract Funding: This work was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG), (http://www.fapemig.br/) and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel (CAPES), (http://www.capes.gov.br/) awarded to Dr. Rocha. The funders had no role in the 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: marianafrocha@hotmail.com * E-mail: marianafrocha@hotmail.com * E-mail: marianafrocha@hotmail.com August 2011 \| Volume 6 \| Issue 8 \| e23312 Small Mammal Community in Vegetation Corridors to Koeppen, with an annual mean temperature of 19.9uC and annual mean precipitation of 1.597 mm [23,24]. Introduction Forest corridors are important components of landscape structure and function, especially where less permeable matrices predominate [11,14]. By allowing species to disperse and colonize fragments, vegetation corridors help maintain the richness, composition and abundance of small mammals in agricultural landscapes [12,15,16,17,18,19], increasing the functional connec- tivity in fragmented landscapes [11,12,14]. Vegetation corridors also increase the movement of individuals between fragments [19,20,21] and may serve as a habitat for many species of mammals [12,15,18,22]. The deforestation of the Brazilian Atlantic Forest over the last two centuries has resulted in a highly fragmented landscape [1]. Except for a few large (.10.000 ha) governmental preserves, most of the 8% remaining Atlantic Forest is composed of small isolated patches of secondary forests (,80 ha) immersed in agricultural or occasionally urban matrices [2]. In the last two decades, studies of the Atlantic Forest fragments have demon- strated that landscape changes have drastic ecological conse- quences on small mammals communities, reducing the incidence of forest specialists and increasing generalist species in the smallest fragments [3,4,5,6]. Most of the landscape in the south and southwest of Minas Gerais, Brazil, is composed of small forest fragments immersed in an agriculturally diversified matrix. Although vegetation corridors occur frequently, they are far less studied than the remaining fragments. In this study, we evaluated the conservation value of thin forest strips (corridors) resulting from the tree colonization of linear ditches. These ditches were made by the slave workforce during the nineteenth century to isolate pastures and farm borders. We evaluated the compo- sition and structure of the small mammal community in forest fragments, a vegetation corridor and a coffee matrix in a model system. Small mammal species displaying low colonization and dispersion abilities in relation to their surrounding matrix environment are more vulnerable to the deleterious effects of fragmentation, reducing their persistence in fragmented land- scapes [4,7,8,9,10]. Therefore, the persistence of small mammals in fragmented landscapes is associated with the functional connectivity of the landscape [4,7,8,9], provided by structural components such as hedgerows, forest strips, riparian corridors and forested ‘‘terra firme’’ corridors [11,12,13]. August 2011 \| Volume 6 \| Issue 8 \| e23312 PLoS ONE \| www.plosone.org 1 Small Mammal Community in Vegetation Corridors Study area and sample sites The vegetation is classified as semi-deciduous seasonal forest, with floristic influences of Cerrado [25,26]. The most important families in the forest fragments and the vegetation corridor included Fabaceae, Myrtaceae, Lauraceae, Rubiaceae, Annona- ceae, Euphorbiaceae and Meliaceae. Floristically, the fragments were dominated by Protium spruceanum, Copaifera langsdorffii, Myrcia The study area consists of two forest fragments and a vegetation corridor immersed in a coffee matrix, located in the municipality of Santo Antoˆnio do Amparo, Minas Gerais, Brazil (20u53957.10S, 44u50911.50W, Figure 1). The climate of this region is classified as Cwa (humid climate with dry winter and hot summer), according Figure 1. (A) Location of the municipality of Santo Antoˆ nio do Amparo, Minas Gerais, Brazil. (B) Location of the stu design of the treatments and transects. doi:10.1371/journal.pone.0023312.g001 Figure 1. (A) Location of the municipality of Santo Antoˆ nio do Amparo, Minas Gerais, Brazil. (B) design of the treatments and transects. doi:10.1371/journal.pone.0023312.g001 Figure 1. (A) Location of the municipality of Santo Antoˆ nio do Amparo, Minas Gerais, Brazil. (B) Location of the study area. (C) Schematic design of the treatments and transects. doi:10.1371/journal.pone.0023312.g001 August 2011 \| Volume 6 \| Issue 8 \| e23312 August 2011 \| Volume 6 \| Issue 8 \| e23312 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org 2 Small Mammal Community in Vegetation Corridors Figure 2. Rarefaction curve of the observed richness of small mammals in treatments. doi:10.1371/journal.pone.0023312.g002 Small Figure 2. Rarefaction curve of the observed richness of small mammals in treatments. doi:10.1371/journal.pone.0023312.g002 Small Mammal Community in Vegetation Corrido Figure 2. Rarefaction curve of the observed richness of small mammals in treatments. doi:10.1371/journal.pone.0023312.g002 splendens, Tapirira obtusa and Magnolia ovate, and Pera glabrata, Copaifera langsdorffii, Casearia arborea, Protium widgrenii and Tapirira obtuse dominated the corridors [26]. The composition and abundance of woody species in the corridor were more similar to the interior than the edge of the fragments, and the diversity index values for the fragment and corridor (3.75 and 3.74, respectively) were among the largest found for other forest remnants in the region studied. splendens, Tapirira obtusa and Magnolia ovate, and Pera glabrata, Copaifera langsdorffii, Casearia arborea, Protium widgrenii and Tapirira obtuse dominated the corridors [26]. Study area and sample sites The composition and abundance of woody species in the corridor were more similar to the interior than the edge of the fragments, and the diversity index values for the fragment and corridor (3.75 and 3.74, respectively) were among the largest found for other forest remnants in the region studied. depth, 4 m in width and 3.2 km in length, with a main axis linking two fragments of forest [26] fenced by a matrix consisting primarily of coffee plantations (Coffea arabica L.) that are 2 m in height and to a smaller degree by pastures. This system of fragments connected by corridors (Figure 1) allows the evaluation of how small mammals use the landscape. Small Mammal Community in Vegetation Corridors Figure 4. Mean and total standard deviation of small mammals in the forest fragment (FRAGM), corridor (CORR) and coffee matrix (MATR). doi:10.1371/journal.pone.0023312.g004 medium and large (45.0616.0616.0 cm) wire mesh traps. Only small Sherman traps were used in the understory vegetation. Ten day sampling periods were performed monthly between December 2008 and May 2009, totaling 60 days of sampling at each site and an effort of 2.880 trap-nights in the fragments, 3.600 in the vegetation corridor and 3,600 in the matrix, with the total sampling effort of 10.080 trap-nights. We marked the captured individuals in one ear with a numbered tag (National Band and Tag Co.) and released them in the same place of capture to undertake capture/recapture data. We collected voucher speci- mens of all species, which were determined by specialists (Y.Leite, L.P. Costa, R.C. Duda, and J.A. de Oliveira) and were hostened to the collection of the Laboratory of Ecology and Mammals Conservation of the Universidade Federal de Lavras. All procedures regarding capture and tagging of animals were conducted under the legal approval and consent of the Brazilian Federal Authority (IBAMA process number 14083-1) and by following the guidelines of the American Society of Mammalogists [27]. Figure 4. Mean and total standard deviation of small mammals in the forest fragment (FRAGM), corridor (CORR) and coffee matrix (MATR). doi:10.1371/journal.pone.0023312.g004 Sample design and data collection The forest fragments studied are 26 ha and 48 ha in size and were the few remnants remaining after the large scale introduction of coffee crops in the region. The vegetation corridor has historic and cultural importance because it occurs in ditches or excavations built by slaves in the late nineteenth century to divide pieces of property and was formed through the natural tree colonization of these ditches [26]. Their dimensions are from 1.5 to 2.5 m in The small mammals were sampled in two forest fragments, one vegetation corridor and one coffee matrix, referred to here as treatments. The peculiar features of each treatment, including shape and size, prevented us from acquiring an equal sample size from each treatment. Thus, in each forest fragment, two parallel transects 100 m in length and 50 m apart were marked. In the vegetation corridor, five transects 100 m in length were placed in Figure 3. MDS analysis of the 14 sampled transects regarding species composition of small mammals in the fragment-corridor- matrix system. doi:10.1371/journal.pone.0023312.g003 Figure 3. MDS analysis of the 14 sampled transects regarding species composition of small mammals in the fragment-corridor- matrix system. doi:10.1371/journal.pone.0023312.g003 August 2011 \| Volume 6 \| Issue 8 \| e23312 August 2011 \| Volume 6 \| Issue 8 \| e23312 PLoS ONE \| www.plosone.org 3 Small Mammal Community in Vegetation Corridors Data analyses The total abundance, or sum of the individuals captured in every species, and the abundance per species, or number of individuals captured in each species, were calculated on a transect basis. lines approximately 250 m from each other. In the coffee matrix, five transects 100 m in length were installed perpendicular to the corridor transects. Thus, 14 transects in total were marked. This sampling design enabled us to evaluate which species were present in each treatment and to determine any species exclusive to certain treatments. The observed richness of the species was compared among the sampled treatments by contrasting the individual-based rarefaction curve (Mao Tau estimation) generated using EstimateS 8.0 software [28] with 1.000 randomizations. In each transect, six sampling stations were marked with a distance of 20 m from each other. Two traps were placed in each station, one on the ground (lower strata) and another in the understory vegetation at 1–2 m above the ground (middle strata). The ground trap alternated between the large (43.0612.5 614.5 cm) and small (25.069.068.0 cm) Sherman traps and To compare the community composition and structure between the forest fragments, corridor and coffee matrix, multidimensional- scaling (MDS) ordinations were used with qualitative (composition) and quantitative (structure) data in a similarity matrix obtained using Bray-Curtis similarity matrices. Analysis of similarity (ANOSIM) were used to assess the differences between sampling Figure 5. MDS analysis of the 14 transects sampled regarding species abundance of small mammals in the fragment-corridor- matrix system. doi:10.1371/journal.pone.0023312.g005 Figure 5. MDS analysis of the 14 transects sampled regarding species abundance of small mammals in the fragment-corridor- matrix system. doi:10.1371/journal.pone.0023312.g005 PLoS ONE \| www.plosone.org August 2011 \| Volume 6 \| Issue 8 \| e23312 4 Small Mammal Community in Vegetation Corridors treatments. These analyses were also used in other studies that evaluate community structure in different landscapes [29,30]. re 6. Abundance (mean and standard deviation) and cator species values in each treatment. 0.1371/journal.pone.0023312.g006 The indicator species analysis was used for species that presented at least 15 captured individuals in all samples and verifies the fidelity of species in the treatments through their abundance and relative frequency, the results of which are described by the observed indication value [31]. Significance was calculated by the Monte Carlo permutation test, using p = 0.05. Richness and composition We obtained 978 captures of 444 individuals belonging to 15 species, including seven marsupials (Marmosops incanus, Gracilinanus microtarsus, Monodelphis iheringi, Monodelphis americana, Caluromys philander, Didelphis albiventris and Didelphis aurita) and eight rodents (Akodon montensis, Rhipidomys sp., Cerradomys subflavus, Euryoryzomys russatus, Calomys sp., Nectomys squamipes, Oligoryzomys nigripes and Oxymycterus delator). The rarefaction curve indicates that in a standardized sampling effort of 60 individuals, contrasting differences exist between the richness of treatments (Figure 2). The coffee matrix presented the least richness (6 species), stabilizing with approximately 30 individuals and indicating few active species in this system. The corridor richness (13) was greater than that of the fragment (10), and the curve did not stabilize, suggesting that the corridors harbor a larger number of species than the other sampled systems. Few species were shared among the sampled treatments. Only three (A. montensis, M. incanus and C. subfavus) were common in all, and four occurred in only one. N. squamipes and O. delator were restricted to the fragment, and M. americana and D. aurita were exclusive to the corridor. The MDS analysis illustrated the low similarity in species composition among treatments, whith the dimensional separation of the three land uses (Figure 3) in three distinct compositional and structural groups. The ANOSIM confirmed these results, showing that the species composition differed significantly among the treatments (R global = 0.663, p = 0.001). The dissimilarity was greater between the corridor and the matrix (R = 0.762, p = 0.008) than between these and the fragment (R = 0.609, p = 0.008). The species composition was rather dissimilar between the fragment and the matrix (R = 0.916, p = 0.008). PLoS ONE \| www.plosone.org Small Mammal Community in Vegetation Corridors the marsupial G. microtarsus (52), which was not captured in the fragments. In the matrix, the most abundant species were Calomys sp. (15), C. subflavus (13) and G. microtarsus (10). The remaining species were less abundant with less than 10 individuals captured in all treatments. Simultaneously, the corridors provide continuity to the vegetation cover and usually are more similar to the vegetation structure in the fragment interior than that of the fragment edge [26]. This may explain the presence of species in this system shared only with the forest fragments and completely absent in the coffee matrix. The superposition of the characteristics of two distinct environ- ments was also mentioned as a predictive factor of increasing mammal richness in the corridor in a temperate forest [32]. The indicator species analysis indicated that six out of the seven most abundant species had a significant preference for one of the three sampled treatments. M. incanus significantly preferred the fragment, A. montensis, C. subflavus, G. microtarsus and Rhipidomys sp. preferred the corridor, and Calomys sp. preferred the matrix. E. russatus indicated no significant preference for any site, presenting instead similar abundance and relative frequency values in the fragment and corridor (Figures 6 and 7). The lower degree of richness in the coffee matrix resulted from the low structural complexity of these sites in comparison with the corridor and fragments because small mammal species richness decreases in structurally simpler habitats [33,34]. Most small mammals species were unable to occupy this site and are sensitive to landscape alteration. The persistence of small mammals in fragmented landscapes is strongly associated with their tolerance of open habitat surrounding fragments [4,5,7,35]. Disturbance-sensitive species and abundance analysis An average of 51.2 individuals were captured in the corridor, followed by 34.5 individuals in the fragment and 9.6 individuals in the matrix, indicating a statistically significant difference (F = 22.94, p = 0.001). However, the mean abundance was similar between the corridor and the fragment (F = 22.94; p = 0.064) and was significantly smaller in the matrix (F = 22.94; p = 0.007 between the matrix and fragment; F = 22.94; p = 0.001 between the matrix and the corridor) (Figure 4). The MDS analysis illustrated differences between the three treatments (Figure 5), spatially separated as independent groups according the ANOSIM (R global = 0.782; p = 0.001). These results enhance the structural differences in small mammal communities active in the corridor, fragment and matrix (ANOSIM comparison: corridor6fragment: R = 0.913, p = 0.008; corridor6matrix: R = 0.78, p = 0.008; fragment6matrix: R = 0.956, p = 0.008). Figure 6. Abundance (mean and standard deviation) and indicator species values in each treatment. doi:10.1371/journal.pone.0023312.g006 The most abundant species in the fragment were M. incanus (50 individuals), A. montensis (32), Rhipidomys sp. (17) and E. russatus (12). In the corridor, these were also the most abundant rodents (77, 43 and 19 individuals, respectively), together with C. subflavus (23) and August 2011 \| Volume 6 \| Issue 8 \| e23312 PLoS ONE \| www.plosone.org 5 References 1. Dean W (1995) With broadax and firebrand: the Destruction of the Brazilian Atlantic Forest University of California Press, London, England. 1. Dean W (1995) With broadax and firebrand: the Destruction of the Brazilian Atlantic Forest University of California Press, London, England. tropical landscapes: a field test on Amazonian dung beetles. Journal of Applied Ecology 47: 779–788. tropical landscapes: a field test on Amazonian dung beetles. Journal of Applied Ecology 47: 779–788. 14. Hilty JA, Lidicker WZ, Merenlender AM (2006) Corridor ecology: the science and practice of linking landscapes for biodiversity conservation Island Press, Connecticut Avenue, Washington. 2. Ribeiro MC, Metzger JP, Martensen AC, Ponzoni FJ, Hirota MM (2009) The Brazilian Atlantic Forest: How much is left, and how is the remaining forest distributed? Implications for conservation. Biological Conservation 142: 1141–1153. 15. Bolger DT, Scott TA, Rotenberry JT (2001) Use of corridor-like landscape structures by bird and small mammal species. Biological Conservation 102: 213–224. 3. Fonseca GAB, Kierullf MCM (1989) Biology and natural history of Brazilian atlantic florest small mammals. Bulletin Florida State Museum Biology Science 34(3): 99–152. 16. Darveau M, Labbe P, Beauchesne P, Belanger L, Huot J (2001) The use of riparian forest strips by small mammals in a boreal balsam fir forest. Forest Ecology and Management 143: 95–104. 4. Pardini R (2004) Effects of forest fragmentation on small mammals in an Atlantic Forest landscape. Biodiversity and Conservation 13: 2567–2586. 17. Downes SJ, Handasyde KA, Elgar MA (1997) The Use of Corridors by Mammals in Fragmented Australian Eucalypt Forests. Conserv Biol 11(3): 718–726. 5. Passamani M, Fernandez FAS (2011) Abundance and richness of small mammals in fragmented Atlantic forest of southeastern Brazil. Journal of Natural History 45(9): 553–565. 18. Lima MG, Gascon C (1999) The conservation value of linear forest remnants in central Amazonia. Biological Conservation. pp 241–247. 6. Pu¨ttker T, Meyer-Lucht Y, Sommer S (2008) Fragmentation effects on population density of three rodent species in secondary Atlantic Rainforest, Brazil. Studies on Neotropical Fauna and Environment 43(1): 11–18. 19. Mesquita AO (2009) Comunidades de pequenos mamı´feros em fragmentos florestais conectados por corredores de vegetac¸a˜o no sul de Minas Gerais. Dissertation, Universidade Federal de Lavras. 7. Castro EV, Fernandez FAS (2004) Determinants of differential extinction vulnerabilities of small mammals in Atlantic forest fragments in Brazil. Biological Conservation 119: 73–80. 20. Hobbs RJ (1992) The role of corridors in conservation: solution or badwagon? Trends Ecology Evolutions 7(11): 389–392. 8. Acknowledgments We thank the NKG Fazendas Brasileiras S.A. for logistic help and the Brazilian Institute of Environment and Renewable Natural Resource (IBAMA) for the license to collect specimens. To specialists for helping identifying small mammal species; Jos Barlow for her critical reading of the manuscript and L.F.S. Magnago and V. Korasaki to make the Figure 1 and 7; A. Rufino, M.G. Yankous, D.G. Rocha and F.S. Machado for help during field work. Because Calomys sp. significantly prefers the matrix and shows decreasing abundance in the forest fragment, it follows that this species has more affinity for open sites, reflecting similar results for the species [19] and the genera [12,36]. Results illustrate that habitat disturbance in fragmented forest landscapes does not affect this species. E. russatus displayed no significant preference for the fragment or the corridor. This species exhibits sensitivity to habitat disturbance, and results indicate that the corridor may function as an extension of its habitat, decreasing the isolation imposed by the coffee matrix, a site the species could not use. Small Mammal Community in Vegetation Corridors small mammals [19]. Detour results for small mammals support these results and highlight the high conservation value of this structure in a fragmented landscape. Because these corridors occur in south and southwest Minas Gerais [26] and considering this region’s intense fragmentation featuring small fragments with native vegetation immersed in agricultural lands, the results point to the necessity of specific policies that will permit the conservation of these structures on a regional scale. species in the fragment are more abundant in the corridor. The similar total abundance in the corridor and the fragments enhances the efficiency of this element in the fragmented landscape for conservation of small mammals, given that many individuals use the corridor as a habitat or for dispersion [12,18,19]. Even though A. montensis, C. subflavus and G. microtarsus displayed a significant preference for the corridor, these species occupied different treatments, corroborating available data that show that they are generally resilient to alterations of the Atlantic Forest landscape [5,6,11,18,34,35]. Rhipidomys sp. seems to favor the corridor because it was unable to use the matrix. In an adjacent area exhibiting similar results, this species was common and abundant in the corridors and able to move among them [19]. M. incanus was significantly more abundant in the fragments than in the corridor and matrix, and as an indicator species, this marsupial reveals its sensitivity to the fragmentation of the landscape. These results reflect other studies of the Atlantic Forest indicating that this species is negatively affected by changes to the natural landscape [4,12,36,37,38]. The findings also indicate that the vegetation corridors function as an extension of the fragment for many species of small mammals because most fragment-inhabiting species were also present in the vegetation corridor and because the most abundant species in the fragment are more abundant in the corridor. This highlights the importance of these landscape elements in the conservation of small mammals in fragmented tropical landscapes. Author Contributions Conceived and designed the experiments: MFR MP. Performed the experiments: MFR MP JL. Analyzed the data: MFR MP JL. Contributed reagents/materials/analysis tools: MFR MP JL. Wrote the paper: MFR MP JL. Conceived and designed the experiments: MFR MP. Performed the experiments: MFR MP JL. Analyzed the data: MFR MP JL. Contributed reagents/materials/analysis tools: MFR MP JL. Wrote the paper: MFR MP JL. In this region, this kind of vegetation corridor was of importance for communities of dung beetles [39], vegetation [26] and other In this region, this kind of vegetation corridor was of importance for communities of dung beetles [39], vegetation [26] and other Discussion The corridor sampled in this study has a narrow, linear shape and a high proportion of edge to interior, allowing its occupation by species such as A. montensis, O. nigripes, and Calomys sp., which were mostly associated with open sites of the nearby matrix [4,12]. Although the corridor, fragment and matrix present different compositions, the corridor was more similar to the fragments than to the matrix, indicating that this structure acts as an extension of the fragment for many small mammal species. The most abundant Figure 7. Indicator species. From left to right, (1) Marmosops incanus, (2) Akodon montensis, (3) Cerradomys subflavus, (4) Rhipidomys sp., (5) Gracilinanus microtarsus and (6) Calomys sp. doi:10.1371/journal.pone.0023312.g007 Figure 7. Indicator species. From left to right, (1) Marmosops incanus, (2) Akodon montensis, (3) Cerradomys subflavus, (4) Rhipidomys sp., (5) Gracilinanus microtarsus and (6) Calomys sp. doi:10.1371/journal.pone.0023312.g007 August 2011 \| Volume 6 \| Issue 8 \| e23312 August 2011 \| Volume 6 \| Issue 8 \| e23312 PLoS ONE \| www.plosone.org 6 Small Mammal Community in Vegetation Corridors References Passamani M, Ribeiro D (2009) Small mammals in a fragment and adjacent matrix in southeastern Brazil. Brazilian Journal of Biology 69(2): 631–637. 21. Uezu A, Metzger JP, Vielliard JME (2005) Effects of structural and functional connectivity and patch size on the abundance of seven Atlantic Forest bird species. Biological Conservation 123: 507–519. 9. Pires AS, Lira PK, Fernandez FAS, Schittini GM, Oliveira LC (2002) Frequency of movements of small mammals among Atlantic Coastal Forest fragments in Brazil. Biological Conservation 108: 229–237. 22. Less AC, Peres CA (2008) Conservation value of remmant riparian forest corridors of varying quality of birds and mammals. Conservation biology 22: 439–449. g 10. Passamani M, Fernandez FAS (2011) Movements of small mammals among Atlantic Forest fragments in Espı´rito Santo, Southeastern Brazil. Mammalia 75: 83–86. 23. Brasil (1992) Ministe´rio da Agricultura. Departamento Nacional de Meteor- ologia. Normas Climatolo´gicas (1961–1990). Brası´lia. 132 p. 11. Pardini R, De Souza SM, Braga-Neto R, Metzger JP (2005) The role of Forest structure, fragment size and corridors in maintaining small mammal abundance and diversity in Atlantic Forest landscape. Biological Conservation 124: 253–266. 24. Ometto JC (1981) Bioclimatologia vegetal. Sa˜o Paulo: Agronoˆmica Ceres. 480 p. 25. INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATI´STICA – IBGE (1983) Folhas SF.23/24 Rio de Janeiro/Vito´ria: geologia, geomorfologia, pedologia, vegetac¸a˜o e uso potencial da terra. Projeto Radambrasil, Rio de Janeiro. 12. Naxara LRC (2008) Importaˆncia dos corredores ripa´rios para a fauna – pequenos mamı´feros em manchas de floresta, matriz do entorno e elementos lineares em uma paisagem fragmentada da Mata Atlaˆntica. Dissertation, Universidade de Sa˜o Paulo. 26. Castro GC (2008) Ecologia da vegetac¸a˜o de corredores ecolo´gicos naturais origina´rios de valos de divisa em Minas Gerais. Doctoral Tesis, Universidade Federal de Lavras. 13. Barlow J, Louzada J, Parry L, Hernandez MIM, Hawes J, et al. (2010) Improving the design and management of forest strips in human-dominated PLoS ONE \| www.plosone.org August 2011 \| Volume 6 \| Issue 8 \| e23312 August 2011 \| Volume 6 \| Issue 8 \| e23312 7 PLoS ONE \| www.plosone.org 34. Umetsu F, Metzger JP, Pardini R (2008) Importance of estimating matrix quality for modeling species distribution in complex tropical landscapes: a test with Atlantic forest small mammals. Ecography 31: 359–370. Small Mammal Community in Vegetation Corridors Small Mammal Community in Vegetation Corridors Small Mammal Community in Vegetation Corridors 27. Gannon WL, Sikes RS (2007) Guidelines of the American Society of Mammalogists for the use of wild mammals in research. Journal of Mammalogy 88: 809–823. 34. Umetsu F, Metzger JP, Pardini R (2008) Importance of estimating matrix quality for modeling species distribution in complex tropical landscapes: a test with Atlantic forest small mammals. Ecography 31: 359–370. 28. Colwell RK EstimateS: statistical estimation of species richness and shared species from samples. 35. Gascon C, Lovejoy TE, Bierregard-Ju´nior RO, Malcolm JR, Stouer PC, et al. (1999) Matrix habitat and species richness in tropical forest remnants. Biological Conservation 91: 223–229. 29. Hawes J, Barlow J, Gardner TA, Peres CA (2008) The value of forest strips for understorey birds in an Amazonian plantation landscape. Biological Conserva- tion 41: 2262–2278. 36. Pu¨ttker T, Pardini R, Meyer-Lucht Y, Sommer S (2008b) Responses of five small mammal species to micro-scale variations in vegetation structure in secondary Atlantic Forest remnants, Brazil. BMC Ecology 8: 9. 30. Pardini R, Faria D, Accacio GM, Laps RR, Mariano-Neto E, et al. (2009) The challenge of maintaining Atlantic forest biodiversity: A multi-taxa conservation assessment of specialist and generalist species in an agro-forestry mosaic in southern Bahia. Biological Conservation 142: 1178–1190. 37. Umetsu F, Pardini R (2007) Small mammals in a mosaic of forest remnants and anthropogenic habitats - evaluating matrix quality in an Atlantic Forest landscape. Landscape Ecology 22: 517–530. g 31. Dufreˆne M, Legendre P (1997) Species assemblages and indicator species: the need for a flexible asymmetrical approach. Ecological Monogographys 67(3): 345–366. 38. Forero-Medina G, Vieira MV (2009) Perception of a fragmented landscape by neotropical marsupials: effects of body mass and environmental variables. Journal of Tropical Ecology 25: 53–62. 32. Lomolino MV, Perault DR (2000) Assemblegy and disassembly of mammal communities in a fragmented temperate rain forest. Ecology 81(6): 1517–1532. 39. Me´ndez HAG (2007) Influeˆncia do corredor de vegetac¸a˜o na riqueza e abundaˆncia de Scarabaeinae (Insecta: Coleoptera) e de parasito´ides (Insecta: Hymenoptera) em um agrocossistema de cafeeiro. Dissertation, Universidade Federal de Lavras. 33. Grelle CEV (2003) Forest Structure and Vertical Stratification of Small Mammals in a Secondary Atlantic Forest, Southeastern Brazil. Studies on Neotropical Fauna and Environment 38(2): 81–85. 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https://openalex.org/W4210627009	https://zenodo.org/records/6778649/files/fnagi-13-834697.pdf	English	null	Does Soluble TREM2 Protect Against Alzheimer's Disease?	Frontiers in aging neuroscience	2,022	cc-by	5,933	Does Soluble TREM2 Protect Against Alzheimer’s Disease? Guy C. Brown 1* and Peter St George-Hyslop 2,3 1 Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom, 2 Department of Medicine, University of Cambridge, Cambridge, United Kingdom, 3 Department of Medicine, University of Toronto, Toronto, ON, Canada Guy C. Brown 1* and Peter St George-Hyslop 2,3 1 Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom, 2 Department of Medicine, University of Cambridge, Cambridge, United Kingdom, 3 Department of Medicine, University of Toronto, Toronto, ON, Canada 1 Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom, 2 Department of Medicine, University of Cambridge, Cambridge, United Kingdom, 3 Department of Medicine, University of Toronto, Toronto, ON, Canada Triggering Receptor Expressed in Myeloid Cells 2 (TREM2) is a pattern recognition receptor on myeloid cells, and is upregulated on microglia surrounding amyloid plaques in Alzheimer’s disease (AD). Rare, heterozygous mutations in TREM2 (e.g., R47H) increase AD risk several fold. TREM2 can be cleaved at the plasma membrane by metalloproteases to release the ectodomain as soluble TREM2 (sTREM2). Wild-type sTREM2 binds oligomeric amyloid beta (Aβ) and acts as an extracellular chaperone, blocking and reversing Aβ oligomerization and ﬁbrillization, and preventing Aβ-induced neuronal loss in vitro. Whereas, R47H sTREM2 increases Aβ ﬁbrillization and neurotoxicity. AD brains expressing R47H TREM2 have more ﬁbrous plaques with more neuritic pathology around these plaques, consistent with R47H sTREM2 promoting Aβ ﬁbrillization relative to WT sTREM2. Brain expression or injection of wild-type sTREM2 reduces pathology in amyloid models of AD in mice, indicating that wild-type sTREM2 is protective against amyloid pathology. Levels of sTREM2 in cerebrospinal ﬂuid (CSF) fall prior to AD, rise in early AD, and fall again in late AD. People with higher sTREM2 levels in CSF progress more slowly into and through AD than do people with lower sTREM2 levels, suggesting that sTREM2 protects against AD. However, some of these experiments can be interpreted as full-length TREM2 protecting rather than sTREM2, and to distinguish between these two possibilities, we need more experiments testing whether sTREM2 itself protects in AD and AD models, and at what stage of disease. If sTREM2 is protective, then treatments could be designed to elevate sTREM2 in AD. MINI REVIEW published: 28 January 2022 doi: 10.3389/fnagi.2021.834697 MINI REVIEW TREM2 Triggering Receptor Expressed in Myeloid Cells 2 (TREM2) is a pattern recognition receptor found on the plasma membrane of myeloid cells. When activated by ligands, such as phospholipids, lipoproteins, and amyloid beta peptide (Aβ), TREM2 induces an innate immune response, which includes phagocytosis, chemotaxis, and transcriptional changes (Keren-Shaul et al., 2017; Deczkowska et al., 2020; Kulkarni et al., 2021). TREM2 signaling is mainly via binding DAP12 (DNAX-activating protein of 12 kDa), which activates Syk tyrosine kinase (Deczkowska et al., 2020). Within the brain, TREM2 is almost uniquely expressed by microglia, and is upregulated on microglia around amyloid plaques in AD (Giraldo et al., 2013; Yuan et al., 2016; Brendel et al., 2017). Rare, heterozygous mutations of TREM2 are known to aﬀect AD risk, including the R47H Keywords: TREM2, sTREM2, microglia, Alzheimer’s disease, amyloid beta, neuroinﬂammation, neurodegeneration, neuroprotection Edited by: Haigang Ren, Soochow University, China Reviewed by: Bijay Parajuli, University of Yamanashi, Japan Correspondence: Guy C. Brown gcb3@cam.ac.uk Edited by: Haigang Ren, Soochow University, China Reviewed by: Bijay Parajuli, University of Yamanashi, Japan Correspondence: Guy C. Brown gcb3@cam.ac.uk Keywords: TREM2, sTREM2, microglia, Alzheimer’s disease, amyloid beta, neuroinﬂammation, neurodegeneration, neuroprotection Specialty section: This article was submitted to Alzheimer’s Disease and Related Dementias, a section of the journal Frontiers in Aging Neuroscience Received: 13 December 2021 Accepted: 31 December 2021 Published: 28 January 2022 Regulation of sTREM2 Shedding Conditions that increase or decrease sTREM2 shedding from full-length TREM2 are not clear, but LPS or IL-1β can induce sTREM2 release from primary mouse microglia (Zhong et al., 2019). Also, oligomeric Aβ, which can bind both full-length TREM2 and sTREM2, induced shedding of sTREM2 for TREM2- overexpressing cells (Vilalta et al., 2021), suggesting that sTREM2 shedding may be induced prior to and during AD as a result of Aβ oligomerization. CSF sTREM2 levels increase in amyloid mouse models and correlate with microglial activation (Brendel et al., 2017). Viral infection of the lungs can increase sTREM2 levels post-infection, due to IL-13 or IL-4 induced sTREM2 shedding (Wu et al., 2015). And HIV viral infection of the brain increases CSF levels of sTREM2 (Gisslén et al., 2018). sTREM2 levels in CSF are thought to be a biomarker of microglial activation, although there is limited evidence for this in vivo (Bekris et al., 2018; Rauchmann et al., 2020; Pascoal et al., 2021), and sTREM2 may itself cause microglial activation (see below). CSF sTREM2 levels rise with age in humans from about 2 ng/ml at 43 years to 6 ng/ml at 80 years of age (Henjum et al., 2016). Citation: Brown GC and St George-Hyslop P (2022) Does Soluble TREM2 Protect Against Alzheimer’s Disease? Front. Aging Neurosci. 13:834697. doi: 10.3389/fnagi.2021.834697 January 2022 \| Volume 13 \| Article 834697 Frontiers in Aging Neuroscience \| www.frontiersin.org sTREM2 and Alzheimer’s Disease Brown and St George-Hyslop mutation, which increases AD risk several fold (Guerreiro et al., 2012; Giraldo et al., 2013; Jonsson et al., 2013; Kulkarni et al., 2021). These mutations are thought to increase AD risk by reducing the protective roles of microglial TREM2, in particular by reducing microglial phagocytosis of amyloid plaques (Condello et al., 2015; Yuan et al., 2016). FIGURE 1 \| Release of sTREM2 from microglia, and activation of microglia by sTREM2. sTREM2 may be generated by ADAM10/17 or meprin β proteolysis of full-length TREM2, or from expression of an isoform lacking the transmembrane domain. γ secretase can cleave the remains of TREM2 within the membrane to degrade it. Released sTREM2 can chemoattract and activate microglia via unknown receptors. Alternative Forms of sTREM2 Alternative Forms of sTREM2 TREM2 can be expressed via alternative splicing as a soluble isoform, lacking the transmembrane form, and this alternative sTREM2 may constitute 25% of total sTREM2 in the brain (Ma et al., 2016; Del-Aguila et al., 2019). This again suggests that sTREM2 has a function, rather than being simply a degradation product of full-length TREM2. The sTREM2 generated by alternative splicing would be 219 amino acids residues long, the sTREM2 generated by ADAM10 or 17 would be 157 amino acids residues long, and the sTREM2 generated by meprin β would be 136 amino acids residues long (plus shorter forms) (Berner et al., 2020), although removal of the signal peptide would shorten all these sTREM2 forms by 18 amino acid residues. The ectodomain of TREM2 and sTREM2 is highly glycosylated at Asn20 and Asn79, so the apparent molecular weight of full- length TREM2 on electrophoresis gels is about 50 kDa when fully glycosylated, and about 25 kDa when deglycosylated (Ma et al., 2016). The apparent molecular weight of sTREM2 in CSF is 30– 35 kDa (Ma et al., 2016), implying that almost half the apparent weight of sTREM2 is sugars, and that diﬀerent glycosylation states coexist. The alternative mechanisms of sTREM2 generation are illustrated in Figure 1. sTREM2 TREM2 is a single-pass type I transmembrane protein with a small C-terminal on the cytosolic side of the plasma membrane, and an N-terminal ectodomain that includes the ligand binding site (Zhong and Chen, 2019; Yang et al., 2020). However, the ectodomain of TREM2 is shed from cells expressing full-length TREM2 into the extracellular medium, and is then known as soluble TREM2 (sTREM2) (Piccio et al., 2008; Wunderlich et al., 2013). The turnover of full-length TREM2 on macrophages is very rapid with a half-life of <1 h, because of constitutive cleavage of full-length TREM2 and shedding of sTREM2 (Thornton et al., 2017). The proteases responsible for shedding sTREM2 include A Disintegrin And Metalloproteases 10 and 17 (ADAM10 and ADAM17), and this cleavage occurs at the H157-S158 peptide bond (Schlepckow et al., 2017; Thornton et al., 2017). ADAM10 and 17 appear to be responsible for sTREM2 release induced by lipopolysaccharide (LPS), whereas the protease meprin β constitutively cleaves TREM2 (predominately at the R136-D137 peptide bond) to release sTREM2 from macrophages (Berner et al., 2020). However, it is unclear whether meprin β can generate sTREM2 in microglia. After shedding of sTREM2, the remaining part of TREM2 may be cleaved within the membrane by γ secretase (Wunderlich et al., 2013). The very rapid and inducible turnover of TREM2 to generate sTREM2 suggests either that (i) TREM2 levels need to be regulated very rapidly, or (ii) that sTREM2 has a function, and full-length TREM2 is a precursor of this functional sTREM2. FIGURE 1 \| Release of sTREM2 from microglia, and activation of microglia by sTREM2. sTREM2 may be generated by ADAM10/17 or meprin β proteolysis of full-length TREM2, or from expression of an isoform lacking the transmembrane domain. γ secretase can cleave the remains of TREM2 within the membrane to degrade it. Released sTREM2 can chemoattract and activate microglia via unknown receptors. Frontiers in Aging Neuroscience \| www.frontiersin.org sTREM2 Activates Microglia g sTREM2 treatment of macrophages induced phosphorylation of ERK1/2 (extracellular signal-regulated kinases 1 and 2) and inhibited apoptosis (Wu et al., 2015). Similarly, sTREM2 treatment of microglia in culture promoted survival by inhibiting apoptosis, apparently via activation of Akt (Zhong et al., 2017). In addition, sTREM2 induced inﬂammatory activation of cultured microglia via nuclear factor-κB, resulting in morphological activation and release of pro-inﬂammatory cytokines (Zhong et al., 2017). sTREM2 also stimulated migration and phagocytosis by primary microglia in culture (Zhong et al., 2019). Injection of sTREM2 into the brains of mice expressing the amyloid precursor protein (APP) induced activation and proliferation of microglia, plus increased expression of pro-inﬂammatory cytokines, and increased microglial phagocytosis of Aβ (Zhong et al., 2019). Injection of sTREM2 into the brains of healthy mice also induced expression of pro-inﬂammatory cytokines (Fassler et al., 2021). A fragment of sTREM2 (amino acids 51–81) was suﬃcient to activate microglia (Sheng et al., 2021). Thus, sTREM2 activates microglia, although the mechanism of this activation is unclear. sTREM2 Degradation Processes responsible for degradation and clearance of extracellular sTREM2 are unclear, although it has been found that macrophages readily take up sTREM2 (Wu et al., 2015), and sTREM2 injected into mouse brain is cleared from the brain within 3 days (Zhong et al., 2019). Membrane-attached meprin β generates sTREM2 constitutively, but inﬂammation- induced ADAM10/17 releases soluble meprin β, which can rapidly degrade sTREM2 (Berner et al., 2020). However, it is unclear whether meprin β contributes to sTREM2 production or degradation in the brain. January 2022 \| Volume 13 \| Article 834697 Frontiers in Aging Neuroscience \| www.frontiersin.org Frontiers in Aging Neuroscience \| www.frontiersin.org 2 Brown and St George-Hyslop sTREM2 and Alzheimer’s Disease FIGURE 2 \| Wild-type sTREM2 blocks Aβ pathology, but R47H TREM2 does the opposite. Aβ oligomers bind to TREM2 and induce shedding of sTREM2. Wild-type sTREM2 blocks Aβ oligomerization, ﬁbrillization and neurotoxicity. R47H sTREM2 increases Aβ oligomerization, ﬁbrillization and neurotoxicity. Thus, wild-type sTREM2 may protect against amyloid pathology, while R47H TREM2 exacerbates amyloid pathology. This might help explain why a single copy of the R47H TREM2 gene increases AD risk several fold. FIGURE 2 \| Wild-type sTREM2 blocks Aβ pathology, but R47H TREM2 does the opposite. Aβ oligomers bind to TREM2 and induce shedding of sTREM2. Wild-type sTREM2 blocks Aβ oligomerization, ﬁbrillization and neurotoxicity. R47H sTREM2 increases Aβ oligomerization, ﬁbrillization and neurotoxicity. Thus, wild-type sTREM2 may protect against amyloid pathology, while R47H TREM2 exacerbates amyloid pathology. This might help explain why a single copy of the R47H TREM2 gene increases AD risk several fold. sTREM2 Protects Against Amyloid Pathology in Mice sTREM2 injection into the brains of mice expressing APP reduced amyloid plaque load (Zhong et al., 2019). Furthermore, viral expression of sTREM2 in the APP-expressing mice, reduced plaque load and reversed deﬁcits of spatial memory and long- term potentiation (Zhong et al., 2019). Thus, sTREM2 is protective against amyloid pathology in mice, and this might be by sTREM2 aﬀecting Aβ aggregation and/or sTREM2 activating microglia to phagocytose plaques. A fragment of sTREM2 (amino acids 51–81) was suﬃcient to activate microglia, but not to bind Aβ and reduce amyloid pathology in vivo; whereas a 41–81 fragment of sTREM2 bound Aβ and reduced amyloid pathology in vivo better than full-length sTREM2 (Sheng et al., 2021). This suggests that sTREM2 protects against amyloid pathology mainly by binding Aβ. ACTIONS OF sTREM2 chaperone for Aβ, blocking its folding into aggregatable forms and refolding aggregates into soluble forms, thereby inhibiting the neurotoxicity of Aβ. In contrast, R47H sTREM2 bound less to Aβ oligomers, but increased Aβ aggregation into protoﬁbrils, and increased Aβ-induced neuronal loss in glial-neuronal cultures (Vilalta et al., 2021). Thus, R47H sTREM2 may not only loose a neuroprotective function, but also gain a neurotoxic function in the presence of Aβ, probably by folding Aβ into more toxic forms (see Figure 2). sTREM2 Activates Microglia EVIDENCE AGAINST THE HYPOTHESIS THAT sTREM2 PROTECTS One piece of evidence potentially contradicting a protective role of sTREM2 in AD, is that the H157Y mutation of TREM2 expressed in cells signiﬁcantly increased sTREM2 shedding relative to wild-type TREM2, resulting in increased sTREM2 and decreased full-length TREM2, but is associated with increased AD risk (Schlepckow et al., 2017; Thornton et al., 2017). This suggests that the increased AD risk associated with the H157Y mutation is due to decreased full-length TREM2 or increased sTREM2, contradicting the hypothesis that sTREM2 is protective against AD. However, the H157Y mutation only increased shedding by about 50%, and this was from HEK293 cells (Schlepckow et al., 2017; Thornton et al., 2017), so it may be diﬃcult to extrapolate to sTREM2 levels in human brains. Additionally, the H157Y mutation would constitute the C-terminal of sTREM2, and might aﬀect its properties, such as its interactions with Aβ. Thus, it would be important to determine whether this mutation does indeed increase CSF levels of sTREM2 in humans, and whether H157Y sTREM2 has the same protective properties as wild-type sTREM2. Neurotoxicity sTREM2 is known to bind oligomeric Aβ, with minimal binding to monomeric or ﬁbrillar Aβ (Lessard et al., 2018; Zhao et al., 2018; Zhong et al., 2018; Vilalta et al., 2021). Subsequently, it was found that sTREM2 blocked Aβ oligomerisation and ﬁbrillization at a molar ratio of 1 sTREM2 to 100 Aβ (Kober et al., 2021; Vilalta et al., 2021), and at higher molar ratios sTREM2 disaggregated Aβ oligomers and ﬁbrils (Vilalta et al., 2021). Wild-type sTREM2 also inhibited Aβ-induced permeabilization of artiﬁcial membranes, and inhibited Aβ-induced neuronal loss in glial-neuronal cultures (Vilalta et al., 2021). These results suggest that wild-type sTREM2 may act as extracellular TREM2 knockout mice, crossed with APP-expressing mice, have more ﬁbrous and less compact plaques (Condello et al., 2015; Wang et al., 2016; Yuan et al., 2016; Song et al., 2018), and while this has been attributed to less microglial phagocytosis of the plaques because of less full-length TREM2, the result might alternatively be due to sTREM2 blocking Aβ aggregation and/or sTREM2 activating microglia to phagocytose plaques. TREM2 knockout mice have increased Aβ seeding (Parhizkar et al., 2019), which again could be explained by reduced microglial phagocytosis of Aβ seeds mediated by full-length TREM2, or Frontiers in Aging Neuroscience \| www.frontiersin.org Frontiers in Aging Neuroscience \| www.frontiersin.org January 2022 \| Volume 13 \| Article 834697 3 Brown and St George-Hyslop sTREM2 and Alzheimer’s Disease and if so, indicating that sTREM2, rather than full-length TREM2 is protective against AD. However, further work is required to establish whether MS4A4A directly aﬀects sTREM2 shedding. and if so, indicating that sTREM2, rather than full-length TREM2 is protective against AD. However, further work is required to establish whether MS4A4A directly aﬀects sTREM2 shedding. reduced blocking of Aβ aggregation by sTREM2. In 5xFAD mice expressing wild-type human TREM2, sTREM2 was found bound to the amyloid plaques (Song et al., 2018), consistent with sTREM2 having a role in regulating plaques. Note that the ability of sTREM2 to block Aβ aggregation and to disaggregate Aβ, might be shared with full-length TREM2, as they both bind Aβ oligomers (Vilalta et al., 2021), but this has not been tested. Humans (and mice) with heterozygous R47H TREM2 have more ﬁbrous plaques with more neuritic pathology (Yuan et al., 2016), which again might be explained by either R47H sTREM2 promoting Aβ ﬁbrillation, or by reduced microglial phagocytosis of plaques. EVIDENCE THAT sTREM2 IS PROTECTIVE AGAINST AD IN HUMANS CSF levels of sTREM2 fall signiﬁcantly in early pre-symptomatic stages prior to AD diagnosis (when amyloid is aggregating), but rise during mild cognitive impairment (MCI) and AD (when tau is aggregating), and fall again during the dementia stages of AD (Heslegrave et al., 2016; Piccio et al., 2016; Suárez-Calvet et al., 2016, 2019; Bekris et al., 2018; Liu et al., 2018; Nordengen et al., 2019; Rauchmann et al., 2019; Ma et al., 2020). People with higher CSF levels of sTREM2 progress more slowly through MCI and AD, in terms of memory loss, clinical score and brain atrophy (Ewers et al., 2019, 2020; Edwin et al., 2020; Franzmeier et al., 2020). And this apparent protective eﬀect of sTREM2 correlated with reduced amyloid and Tau aggregation measured by PET (Ewers et al., 2020), consistent with sTREM2 reducing amyloid aggregation and pathology. Other evidence potentially contradicting the hypothesis that sTREM2 protects against AD is the ﬁnding of Schlepckow et al. (2020) that an antibody binding to the ADAM cleavage site of TREM2 prevented sTREM2 release, but reduced plaques load in an amyloid mouse model. However, the antibody used directly activated TREM2 signaling, so the reduced plaque load may result from this signaling (Schlepckow et al., 2020). Additionally, the compaction of these plaques, neuritic pathology and memory loss were not tested in this model. However, these apparent protective eﬀect of high sTREM2 has been attributed to full-length TREM2, rather than sTREM2, on the untested assumption that high sTREM2 levels indicates high TREM2 levels, as a result of constant shedding. However, if elevated sTREM2 results from elevated shedding, which is for example induced by oligomeric Aβ (Vilalta et al., 2021), then this will reduce full-length TREM2. Thus, elevated levels of sTREM2 do not necessarily indicate that levels of full-length TREM2 are elevated, and the apparent protective eﬀect of sTREM2 against AD may be more simply explained by sTREM2 itself being protective. REFERENCES TREM2 and Alzheimer’s disease risk. Sci. Transl. Med. 11:eaau2291. doi: 10.1126/scitranslmed.aau2291 Bekris, L. M., Khrestian, M., Dyne, E., Shao, Y., Pillai, J. A., Rao, S. M., et al. (2018). Soluble TREM2 and biomarkers of central and peripheral inﬂammation in neurodegenerative disease. J. Neuroimmunol. 319, 19–27. doi: 10.1016/j.jneuroim.2018.03.003 Edwin, T. H., Henjum, K., Nilsson, L. N. G., Watne, L. O., Persson, K., Eldholm, R. S., et al. (2020). A high cerebrospinal ﬂuid soluble TREM2 level is associated with slow clinical progression of Alzheimer’s disease. Alzheimers Dement. 12:e12128. doi: 10.1002/dad2.12128 Ewers, M., Biechele, G., Suárez-Calvet, M., Sacher, C., Blume, T., Morenas-Rodriguez, E., et al. (2020). Higher CSF sTREM2 and microglia activation are associated with slower rates of beta-amyloid accumulation. EMBO Mol. Med. 12:e12308. doi: 10.15252/emmm.202 012308 Berner, D. K., Wessolowski, L., Armbrust, F., Schneppenheim, J., Schlepckow, K., Koudelka, T., et al. (2020). Meprin β cleaves TREM2 and controls its phagocytic activity on macrophages. FASEB J. 34, 6675–6687. doi: 10.1096/fj.2019 02183R Brendel, M., Kleinberger, G., Probst, F., Jaworska, A., Overhoﬀ, F., Blume, T., et al. (2017). Increase of TREM2 during aging of an Alzheimer’s disease mouse model is paralleled by microglial activation and amyloidosis. Front. Aging Neurosci. 9:8. doi: 10.3389/fnagi.2017.00008 Ewers, M., Franzmeier, N., Suárez-Calvet, M., Morenas-Rodriguez, E., Caballero, M. A. A., Kleinberger, G., et al. (2019). Increased soluble TREM2 in cerebrospinal ﬂuid is associated with reduced cognitive and clinical decline in Alzheimer’s disease. Sci. Transl. Med. 11:eaav6221. doi: 10.1126/scitranslmed.aav6221 Condello, C., Yuan, P., Schain, A., and Grutzendler, J. (2015). Microglia constitute a barrier that prevents neurotoxic protoﬁbrillar Aβ42 hotspots around plaques. Nat. Commun. 6:6176. doi: 10.1038/ ncomms7176 Fassler, M., Rappaport, M. S., Cuño, C. B., and George, J. (2021). Engagement of TREM2 by a novel monoclonal antibody induces activation of microglia and improves cognitive function in Alzheimer’s disease models. J. Neuroinﬂamm. 18:19. doi: 10.1186/s12974-020-01980-5 Deczkowska, A., Weiner, A., and Amit, I. (2020). The physiology, pathology, and potential therapeutic applications of the TREM2 signaling pathway. Cell 181, 1207–1217. doi: 10.1016/j.cell.2020.05.003 Franzmeier, N., Suárez-Calvet, M., Frontzkowski, L., Moore, A., Hohman, T. J., Morenas-Rodriguez, E., et al. (2020). Higher CSF sTREM2 attenuates ApoE4- related risk for cognitive decline and neurodegeneration. Mol. Neurodegener. 15:57. doi: 10.1186/s13024-020-00407-2 Del-Aguila, J. L., Benitez, B. A., Li, Z., Dube, U., Mihindukulasuriya, K. A., Budde, J. P., et al. (2019). TREM2 brain transcript-speciﬁc studies in AD and TREM2 mutation carriers. Mol. Neurodegener. 14:18. Potential Treatment Strategies Current strategies targeting TREM2 in AD have focused on agonistic antibodies to activate TREM2 with the aim of increasing microglial phagocytosis of amyloid plaques (Wang et al., 2020; Fassler et al., 2021). These antibodies will also bind sTREM2 and potentially block the protective eﬀects of sTREM2 (Fassler et al., 2021). If sTREM2 is indeed more protective against Key Experiments to Determine Whether sTREM2 Is Protective Against AD AD than full-length TREM2, then antibodies that increased sTREM2 shedding might be beneﬁcial, or other treatments designed to activate sTREM2 shedding e.g., by activating ADAM10 and ADAM17. Blocking sTREM2 degradation (e.g., by inhibiting meprin β) might increase sTREM2 levels without decreasing full-length TREM2. sTREM2 and sTREM2 fragments injected into the brain were protective in mouse models of AD (Zhong et al., 2019; Sheng et al., 2021), but may be diﬃcult to deliver practically in humans. However, viral vectors expressing sTREM2 in the brain were protective in these mouse models of AD, and thus might be protective in humans with AD (Zhong et al., 2019). Some of evidence indicating that sTREM2 is protective against AD, may alternatively be interpreted as full-length TREM2 is protective. Thus, there is a need for experiments that distinguish between these possibilities, or directly show that sTREM2 is protective. The most direct way to show that is to add or express sTREM2 independent of full-length TREM2 and test whether this is protective in AD models. This has been done for a mouse amyloid model and found to be protective (Zhong et al., 2019), but this was relatively acute model, and it would be important to test this in other models, particularly more chronic and AD-relevant models. Within such models, it would be important to test whether sTREM2 can block Aβ aggregation, or disaggregate preformed plaques or oligomers. It would also be useful to know whether Aβ oligomers in AD CSF are signiﬁcantly bound to sTREM2, and whether physiological levels of sTREM2 can disaggregate Aβ aggregation in CSF. Further, it would be worth knowing whether the diﬀerent types of sTREM2 behave diﬀerently, including sTREM2 generated by ADAM and meprin β, or by alternative splicing, or H157Y and R62H sTREM2. Is TREM2 or sTREM2 Protective in Alzheimer’s Disease? g p GWAS studies of gene variants that aﬀect the CSF levels of sTREM2 identiﬁed the membrane-spanning 4-domains superfamily A (MS4A) gene cluster as key determinants of sTREM2 levels in CSF (Piccio et al., 2016; Deming et al., 2019; Hou et al., 2019). This gene region had previously been linked to AD risk (Naj et al., 2011). For example, rs1582763 increased brain expression of MS4A4A and MS4A6A genes, increased sTREM2 levels in CSF, reduced AD risk and increased age of AD diagnosis. While rs6591561 resulted in a loss-of-function MS4A4A, reduced CSF sTREM2 levels, increased AD risk and reduced age at AD onset (Deming et al., 2019). MS4A4A and TREM2 were found to colocalize at the plasma membrane, and overexpression of MS4A4A increased sTREM2 levels, whilst silencing of MS4A4A reduced sTREM2 levels (Deming et al., 2019). This suggests that MS4A4A may aﬀect AD risk by promoting sTREM2 shedding, It appears that either TREM2 or sTREM2 are protective in Alzheimer’s disease, but which? TREM2 is thought to be protective by (i) recruiting and activating microglia into a protective state around amyloid plaques, and (ii) compacting amyloid plaques by phagocytosis of Aβ, preventing the plaques inducing neuritic pathology (Condello et al., 2015; Yuan et al., 2016; Keren-Shaul et al., 2017). Whereas, sTREM2 is thought to be protective by: (i) stimulating microglial recruitment, activation and phagocytosis of Aβ, and/or (ii) blocking and reversing Aβ aggregation, preventing neurotoxicity (Zhong et al., 2019; Vilalta et al., 2021). Thus, the putative protective eﬀects of TREM2 and sTREM2 are complimentary rather than antagonistic, and potentially both may be protective against Alzheimer’s disease. However, it is still important to verify that TREM2 and/or sTREM2 are in fact protective. January 2022 \| Volume 13 \| Article 834697 Frontiers in Aging Neuroscience \| www.frontiersin.org Frontiers in Aging Neuroscience \| www.frontiersin.org 4 sTREM2 and Alzheimer’s Disease Brown and St George-Hyslop AUTHOR CONTRIBUTIONS GB wrote the article. PG-H reviewed and adjusted the article. Both authors were responsible for its content. FUNDING This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under Grant Agreement No. 115976. This Joint Undertaking receives support from the European Union’s Horizon 2020 Research and Innovation Programme and EFPIA. PHStGH also received funding from the Canadian Institutes of Health Research (Foundation Grant and Canadian Consortium on Neurodegeneration in Aging Grant), Wellcome Trust Collaborative Award 203249/Z/16/Z, US Alzheimer’s Society Zenith Grant ZEN-18-529769, and the Alzheimer’s Society of Ontario Chair in Alzheimer’s Disease Research. REFERENCES Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region. EMBO Mol. Med. 12:e11227. doi: 10.15252/emmm.201911227 Hou, X. H., Bi, Y. L., Tan, M. S., Xu, W., Li, J. Q., Shen, X. N., et al. (2019). Genome-wide association study identiﬁes Alzheimer’s risk variant in MS4A6A inﬂuencing cerebrospinal ﬂuid sTREM2 levels. Neurobiol. Aging 84, 241.e13–241.e20. doi: 10.1016/j.neurobiolaging.2019.05.008 Sheng, X., Yao, Y., Huang, R., Xu, Y., Zhu, Y., Chen, L., et al. (2021). Identiﬁcation of the minimal active soluble TREM2 sequence for modulating microglial phenotypes and amyloid pathology. J. 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Sequential proteolytic processing of the triggering receptor expressed on myeloid cells-2 (TREM2) protein by ectodomain shedding and γ-secretase-dependent intramembranous cleavage. J. Biol. Chem. 288, 33027–33036. doi: 10.1074/jbc.M113. REFERENCES 517540 Parhizkar, S., Arzberger, T., Brendel, M., Kleinberger, G., Deussing, M., Focke, C., et al. (2019). Loss of TREM2 function increases amyloid seeding but reduces plaque-associated ApoE. Nat. Neurosci. 22, 191–204. doi: 10.1038/s41593-018-0296-9 Yang, J., Fu, Z., Zhang, X., Xiong, M., Meng, L., and Zhang, Z. (2020). TREM2 ectodomain and its soluble form in Alzheimer’s disease. J. Neuroinﬂamm. 17:204. doi: 10.1186/s12974-020-01878-2 Pascoal, T. A., Benedet, A. L., Ashton, N. J., Kang, M. S., Therriault, J., Chamoun, M., et al. (2021). Microglial activation and tau propagate jointly across braak stages. Nat. Med. 27, 1592–1599. doi: 10.1038/s41591-021-01456-w Piccio, L., Buonsanti, C., Cella, M., Tassi, I., Schmidt, R. E., Fenoglio, C., et al. (2008). Identiﬁcation of soluble TREM-2 in the cerebrospinal ﬂuid and its association with multiple sclerosis and CNS inﬂammation. Brain 131, 3081–3091. doi: 10.1093/brain/awn217 Yuan, P., Condello, C., Keene, C. D., Wang, Y., Bird, T. D., Paul, S. M., et al. (2016). TREM2 haplodeﬁciency in mice and humans impairs the microglia barrier function leading to decreased amyloid compaction and severe axonal dystrophy. Neuron 90, 724–739. doi: 10.1016/j.neuron.2016.05.003 Frontiers in Aging Neuroscience \| www.frontiersin.org Frontiers in Aging Neuroscience \| www.frontiersin.org January 2022 \| Volume 13 \| Article 834697 6 Brown and St George-Hyslop sTREM2 and Alzheimer’s Disease Zhao, Y., Wu, X., Li, X., Jiang, L. L., Gui, X., Liu, Y., et al. (2018). TREM2 is a receptor for beta-amyloid that mediates microglial function. Neuron 97, 1023–1031. doi: 10.1016/j.neuron.2018.01.031 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. Zhong, L., and Chen, X. F. (2019). The emerging roles and therapeutic potential of soluble TREM2 in Alzheimer’s disease. Front. Aging Neurosci. 11:328. doi: 10.3389/fnagi.2019.00328 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. Zhong, L., Chen, X. F., Wang, T., Wang, Z., Liao, C., Wang, Z., et al. (2017). Soluble TREM2 induces inﬂammatory responses and enhances microglial survival. J. Exp. Med. 214, 597–607. doi: 10.1084/jem.201 60844 Zhong, L., Wang, Z., Wang, D., Wang, Z., Martens, Y. A., Wu, L., et al. (2018). Frontiers in Aging Neuroscience \| www.frontiersin.org January 2022 \| Volume 13 \| Article 834697 REFERENCES Amyloid-beta modulates microglial responses by binding to the triggering receptor expressed on myeloid cells 2 (TREM2). Mol. Neurodegener. 13:15. doi: 10.1186/s13024-018-0247-7 Copyright © 2022 Brown and St George-Hyslop. 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. Zhong, L., Xu, Y., Zhuo, R., Wang, T., Wang, K., Huang, R., et al. (2019). Soluble TREM2 ameliorates pathological phenotypes by modulating microglial functions in an Alzheimer’s disease model. Nat. Commun. 10:1365. doi: 10.1038/s41467-019-09118-9 January 2022 \| Volume 13 \| Article 834697 Frontiers in Aging Neuroscience \| www.frontiersin.org 7
https://openalex.org/W2315582328	https://www.nature.com/articles/ncomms11104.pdf	English	null	Laboratory observations of slow earthquakes and the spectrum of tectonic fault slip modes	Nature communications	2,016	cc-by	5,820	ARTICLE Received 4 Nov 2015 \| Accepted 19 Feb 2016 \| Published 31 Mar 2016 Received 4 Nov 2015 \| Accepted 19 Feb 2016 \| Published 31 Mar 2016 1 Department of Geosciences, The Pennsylvania State University, 522 Deike Building, University Park, Pennsylvania 16802, USA. 2 Dipartimento di Scienze della Terra, Sapienza Universita` di Roma, Piazzale Aldo Moro 5, 00185 Rome Italy. Correspondence and requests for materials should be addressed to J.R.L. (email: jleeman@psu.edu). Results M h Mechanical behaviour. In our experiments, gouge layers initially exhibited stable sliding, followed by the emergence of repeating slow stick–slip events (Figs 1 and 2a). The slow-slip events arose gradually, over an interval of up to 1.5 mm, and then increased in amplitude over as few as 10–20 slip events before reaching a mechanical steady state, characterized by relatively uniform recurrence intervals and friction drops, up to the maximum imposed displacements of Z50 mm. For our layers, which were 3-mm-thick prior to shear, this corresponds to shear strains of 30–50. Each slow-slip event began with a gradual acceleration and culminated in a slip event and stress drop (Fig. 1). q p Despite their relevance to natural fault zones and slow earthquakes, detailed laboratory observations of repetitive slow- slip transients are few and do not include systematic studies. These behaviours have been reported in some experimental work12,14,15, but have been interpreted and modelled in the context of speciﬁc fault rheologies, using so-called ‘designer’ friction laws. In one form of these laws, slow stick–slip is produced by an increase in frictional resistance with slip velocity, such that instability is quenched during acceleration14,15,19. Other explanations for slow earthquakes have focused on processes that may arrest slip acceleration during earthquake nucleation, including dilatancy hardening20,21, transitional frictional behaviour as a function of slip22 or slip rate, and fault zone heterogeneity. Some numerical simulations successfully predict complex slip behaviour, including oscillatory behaviour and the emergence of periodic slow slip20,23. Two-dimensional (2D) numerical models also show promise in reproducing natural events, with fewer free parameters than multiple state variable models24. Stick–slip events. Our experimental results are consistent with theory, numerical experimentation20,23 and with existing lab data for stick–slip11. We document a spectrum of stick–slip behaviours in experiments conducted over a range of normal stresses (Fig. 2a). At low normal stress (6 MPa) and close to the stability transition described by equation (1), slip events have systematically longer duration and smaller stress drops than their higher normal stress counterparts (Fig. 2c). Details of the friction records for slow events show that slip begins gradually, well before the peak strength is reached and then accelerates during the stress drop (Fig. 2b). The maximum slip velocities for slow-slip events are in the range of 50–100 mm s 1, and slip speed increases systematically with increasing normal stresses, which leads to increasingly unstable behaviour (equation (1)). ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms11104 S low earthquakes are a mode of self-sustained fault rupture in which slip accelerates but does not reach rates sufﬁcient to radiate high-frequency seismic energy1,2. Seismic and geodetic observations reveal that slow-slip and the related phenomena of low-frequency earthquakes and non-volcanic tremor deﬁne a spectrum of slip behaviours that unfold over timescales ranging from seconds to months2–6. Slow earthquakes can be large, in some cases equivalent to M7 þ earthquakes, and they may play a role in stress transfer and thus triggering of damaging regular earthquakes7. Slow earthquakes have also been observed as precursors to regular earthquakes and thus they may provide insight into the processes of earthquake nucleation8,9. Although geophysical observations have resolved ﬁne details of slow earthquake slip and propagation rates of tectonic fault tremor6,8–10, the fundamental and controlling mechanics of these phenomena remain enigmatic. relevant values of normal stress and sliding rates. This allows a detailed investigation of the frictional dynamics of slow slip, which provides a robust and generalized framework to apply to tectonic fault zones. Here we describe laboratory experiments that reproduce the full spectrum of fault slip behaviours under geophysically relevant conditions of normal stress and fault composition, and which illuminate their underlying physics. Our experiments are designed to explore the full range of slip stability, as described by the stability parameter k ¼ k/kc, from k41 (inherently stable slip) to dynamic stick–slip (koo1). Consistent with previous works14,18,23 near the stability boundary, kE1, we observe complex slip patterns that precede slow slip. We document a systematic and robust relationship between departure from the stability threshold, slip velocity and duration of repetitive failure events. Our experimental results, to the best of our knowledge, are the ﬁrst complete and systematic study to investigate the full spectrum of slip behaviours from slow to fast events, as observed for tectonic faults. Regular earthquakes have long been understood in terms of stick–slip failure dictated by frictional and elastic properties of the Earth’s crust11. Laboratory studies have provided key insights into the physics of fault failure and its dynamics, both for repeating earthquake-like stick–slip failure and for more complex slip behaviours. For example, previous works have reported a range of observations including transient slip, oscillatory sliding behaviour and dynamic rupture at sub-Raleigh and supershear propagation speeds12–18. Results M h For the lowest values of normal stress that produced repeating transient slip events, we measured peak slip velocities of only a few 10’s of mm s 1, on the order of the driving velocity. For a normal stress of 14 MPa, we observed audible fast stick–slip events with slip velocities 42 mm s 1. To date, the origin of slow earthquakes has been explored largely via seismic or geodetic data or through numerical experiments with only sparse, isolated laboratory observations to probe the underlying mechanics. Although theoretical models can explain the emergence of slow-slip transients under certain conditions or for speciﬁc frictional rheologies20,21,23, a fundamental mechanical explanation for these events remains elusive. Yet, slow modes of fault rupture are observed in a variety of tectonic and geologic settings, and with a wide range of durations, raising the question as to whether they arise from a universal mechanism6,25. ARTICLE Transient and oscillatory behaviour have been interpreted as analogues for premonitory slip prior to earthquakes or transient aseismic slip12,18,19. Laboratory observations of slow earthquakes and the spectrum of tectonic fault slip modes J.R. Leeman1, D.M. Saffer1, M.M. Scuderi1,2 & C. Marone1 Slow earthquakes represent an important conundrum in earthquake physics. While regular earthquakes are catastrophic events with rupture velocities governed by elastic wave speed, the processes that underlie slow fault slip phenomena, including recent discoveries of tremor, slow-slip and low-frequency earthquakes, are less understood. Theoretical models and sparse laboratory observations have provided insights, but the physics of slow fault rupture remain enigmatic. Here we report on laboratory observations that illuminate the mechanics of slow-slip phenomena. We show that a spectrum of slow-slip behaviours arises near the threshold between stable and unstable failure, and is governed by frictional dynamics via the interplay of fault frictional properties, effective normal stress and the elastic stiffness of the surrounding material. This generalizable frictional mechanism may act in concert with other hypothesized processes that damp dynamic ruptures, and is consistent with the broad range of geologic environments where slow earthquakes are observed. 1 Department of Geosciences, The Pennsylvania State University, 522 Deike Building, University Park, Pennsylvania 16802, USA. 2 Dipartimento di Scienze della Terra, Sapienza Universita` di Roma, Piazzale Aldo Moro 5, 00185 Rome Italy. Correspondence and requests for materials should be addressed to J.R.L. (email: jleeman@psu.edu). 1 NATURE COMMUNICATIONS \| 7:11104 \| DOI: 10.1038/ncomms11104 \| www.nature.com/naturecommunications Discussion Th h Stick–slip amplitude increases gradually over a few millimetres before reaching steady state. The lower right inset shows details of fault slip events, note the gradual acceleration at the start of each failure event. The lower left inset shows the double direct shear conﬁguration and locations of displacement transducers. Spikes at 13 and 22 mm displacement are due to brief pauses in shearing to reset displacement transducers. Taken together, our direct (Fig. 3a, Supplementary Fig. 2) and independent (Fig. 3b) measurements of kc0 and k0 (Supplementary Figs 1 and 2) show that stick–slip event velocity and duration vary systematically as a function of distance from the stability threshold. The slowest events occur for kE1, with progressively faster events for lower values of k (Fig. 3d,e). The peak slip velocity and stick–slip duration for all events, measured after reaching a steady state (Fig. 3c, shaded area), deﬁne a complete spectrum of slip behaviours between stable sliding and fast stick–slip (Fig. 3d,e). For ko0.7, slip velocities of several mm s 1 were associated with audible failure events (Fig. 3d). For values of k approaching 1, the duration of slow-slip is in the order of seconds (not producing any audible emissions in the range of human hearing), with lower peak slip velocities (Fig. 3d,e). The amplitude of the stick–slip events is systematically lower for the slow events (Fig. 2), consistent with seismic and geodetic observations for tectonic faults4,6,30. To investigate the mechanics of slow stick–slip events, we carefully measured both the elastic loading stiffness k and the critical stiffness kc in each of our experiments. We measured k directly from the loading curves of stick–slip events and from unload/reload cycles (Supplementary Fig. 1). Stiffness increases with shear displacement up to 15 mm, and then reaches an approximately constant value (Fig. 3; Supplementary Fig. 1). The increase in stiffness with shearing is consistent with shear-enhanced compaction and granular comminution during the ﬁrst few millimetres of slip29. As noted above, we measure kc directly from the parameters in Equation (1) using velocity step experiments (Fig. 3a, Supplementary Fig. 2), and also empirically using the value of k0 at the observed transition between unstable and stable slip (black line, Fig. 3b). Discussion Th h The short duration, audible high slip velocity events are manifestations of dynamic instability and represent laboratory analogues of regular, fast earthquakes11. Likewise, we posit that the observed spectrum of slow to fast stick–slip events in our experiments are representative of the spectrum of slip behaviours observed on tectonic faults, including repeating slow-slip events and low-frequency earthquakes4,6. Near the stability transition, we also document complex and chaotic behaviours including period doubling and transient variations in stick–slip amplitude with long-period modulation (Fig. 2a), consistent with theoretical predictions 23. Although many fault zones are rich in phyllosilicate minerals, which have been shown to exhibit both rate-weakening and rate-strengthening behaviour under conditions comparable to those expected in situ in the seismogenic crust24,26,27, we focus on quartz gouge to investigate the systematics of frictional failure, because it is a well-studied material that is common in natural faults, and is thought to play a key role in controlling their slip behaviour27,28. Quartz gouge also exhibits frictional properties that enable us to probe the stability boundary using geophysically NATURE COMMUNICATIONS \| 7:11104 \| DOI: 10.1038/ncomms11104 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms11104 0.70 0.65 0.60 0.69 0.68 0.67 Δ 0.70 0.68 0.66 0.64 0.55 0.50 0.45 0.40 5 9.5 10.0 10.5 0 5 10 15 Time (s) 20 25 30 0 100 200 300 p4342 Disp. (μm) 11.0 LPD (mm) Shear stress Gouge layers Plastic membrane Onset of rapid acceleration Side shield Normal stress On-board DCDT 10 15 20 25 30 Load point displacement-LPD (mm) Friction Figure 1 \| Experimental run plot. Friction data for one experiment (p4342) at a normal stress of 12 MPa and shearing rate of 10 mm s 1. The upper inset shows spontaneous emergence of unstable slow slip. Stick–slip amplitude increases gradually over a few millimetres before reaching steady state. The lower right inset shows details of fault slip events, note the gradual acceleration at the start of each failure event. The lower left inset shows the double direct shear conﬁguration and locations of displacement transducers. Spikes at 13 and 22 mm displacement are due to brief pauses in shearing to reset displacement transducers. Friction Figure 1 \| Experimental run plot. Friction data for one experiment (p4342) at a normal stress of 12 MPa and shearing rate of 10 mm s 1. The upper inset shows spontaneous emergence of unstable slow slip. NATURE COMMUNICATIONS \| 7:11104 \| DOI: 10.1038/ncomms11104 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms11104 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms11104 Friction Friction Normalized friction Increasing effective stiffness 9 10 Fast stick–slip a b c 0.025 Slow stick–slip 11 12 13 14 14 MPa 0.69 0.68 0.67 0 1 2 3 4 5 6 0 20 40 60 80 Velocity (μm s–1) p4351 p4350 p4342 p4348 p4347 p4346 p4345 p4344 p4343 13 MPa 12 MPa 11 MPa 10 MPa 9 MPa 8 MPa 14 MPa 10 MPa 6 MPa 0.0 0.5 1.0 1.5 Time (s) 6 MPa p4343 7 MPa 6 MPa 15 16 Load point displacement (mm) Figure 2 \| Spectrum of fault slip behaviour. (a) Friction data for experiments (p43XX run numbers) at different effective shear-loading stiffness k0 ¼ k/sn0. Friction data are offset vertically for clarity. The emergence of slow stick–slip occurs at lower shear displacement, and stick–slip amplitude increases, for higher normal stress experiments. The spikes in friction at 13–15 mm are due to frictional aging caused by brief pauses in shearing to reset displacement transducers. (b) Details of friction (solid line) and velocity (dashed) during a stick–slip event with a peak slip velocity of E80 mm s 1, only a few times that of the background loading velocity of 10 mm s 1. (c) Stick–slip events have systematically longer duration at lower normal stresses. Slip accelerates more slowly and event durations are correspondingly longer than at higher normal stress. Friction Normalized friction b c 0.69 0.68 0.67 0 1 2 3 4 5 6 0 20 40 60 80 Velocity (μm s–1) 14 MPa 10 MPa 6 MPa 0.0 0.5 1.0 1.5 Time (s) 6 MPa p4343 b Friction Increasing effective stiffness 9 10 Fast stick–slip a 0.025 Slow stick–slip 11 12 13 14 14 MPa p4351 p4350 p4342 p4348 p4347 p4346 p4345 p4344 p4343 13 MPa 12 MPa 11 MPa 10 MPa 9 MPa 8 MPa 7 MPa 6 MPa 15 16 Load point displacement (mm) a Increasing effective stiffness Increasing effective stiffness Normalized friction 14 MPa 10 MPa 6 MPa 0.0 0.5 1.0 1.5 Time (s) Figure 2 \| Spectrum of fault slip behaviour. (a) Friction data for experiments (p43XX run numbers) at different effective shear-loading stiffness k0 ¼ k/sn0. Friction data are offset vertically for clarity. The emergence of slow stick–slip occurs at lower shear displacement, and stick–slip amplitude increases, for higher normal stress experiments. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms11104 The spikes in friction at 13–15 mm are due to frictional aging caused by brief pauses in shearing to reset displacement transducers. (b) Details of friction (solid line) and velocity (dashed) during a stick–slip event with a peak slip velocity of E80 mm s 1, only a few times that of the background loading velocity of 10 mm s 1. (c) Stick–slip events have systematically longer duration at lower normal stresses. Slip accelerates more slowly and event durations are correspondingly longer than at higher normal stress. provide a uniﬁed view of the spectrum of tectonic fault slip behaviours. In total, our results illuminate the key ingredients required for slow earthquakes. Relative to areas where regular earthquakes occur, kc must remain sufﬁciently small that it does not greatly exceed the local fault stiffness k. This can occur for speciﬁc frictional properties—small (b a) or large Dc—as may be the case at the upper and lower edges of the seismogenic zone or in areas of complicated fault zone architecture20. This condition would also be favoured by low effective normal stress, as has been suggested in a wide range of settings8,9,31–34. In addition, we suggest that the mode of fault slip should evolve as tectonic faults accumulate shear strain, or through the earthquake cycle, due to progressive changes in fault stiffness and frictional constitutive properties32,34. Finally, because fault stiffness is proportional to the ratio of shear modulus to rupture nucleation patch size, we expect that regions of large, coherent creep slip, which effectively reduce k, would favour nucleation of slow earthquakes. Methods E i t Experimental apparatus. Experiments were performed in a servo-controlled biaxial shearing apparatus using the double direct shear conﬁguration (Fig. 1). Displacements on the normal and shearing axes were measured by Direct Current Displacement Transducers (DCDTs), referenced at the load frame and ram nose. The displacement of the shearing block was measured with DCDTs referenced at the end-platen and the top and bottom of the shearing block (Fig. 1). Loads applied to the sample were measured with strain gauge load cells. All transducers are calibrated with instruments and methods traceable to NIST. Sample preparation. Samples were prepared using steel or titanium side blocks and steel or acrylic central shearing blocks (Supplementary Table 1). The forcing blocks were grooved 0.8 mm deep at 1 mm spacing to eliminate shear at the boundary. We used Min-U-Sil 40 powdered silica (US Silica Co.) to simulate granular fault gouge. The product is 99.5% SiO2, with traces of metal oxides, and has a median grain diameter of 10.5 mm. Samples were constructed as 3-mm-thick layers, and with 10 10 cm frictional contact area. Layers were prepared and sheared under 100% relative humidity at room temperature. Our results support previous hypotheses about the role of transitional frictional behaviour in driving complex fault slip behaviours20,23,31–33. It is likely that transitional frictional behaviour may act in concert with additional processes acting locally within a fault zone to produce the observed spectrum of slip behaviours. A wide range of key natural factors, such as compliant and evolving damage zones, low effective normal stress associated with elevated pore ﬂuid pressure and fault evolution are all captured by the stability parameter k ¼ k/kc. Ultimately, our results suggest that slow earthquakes and transient fault slip behaviours arise from the same governing frictional dynamics as normal earthquakes, and Testing procedure. After samples were placed in the testing machine, a constant normal stress was applied and maintained constant using force-feedback servo control. Samples were allowed to compact and accommodate grain rearrangement before shearing began. Shear was induced by imposing a displacement rate on the central forcing block (Fig. 1), using a feedback servo control. The displacement rate was maintained constant at 10 mm s 1 for the majority of our experiments (Supplementary Table 1), and velocity step tests were used to determine the friction rate parameters (a b) and Dc. Discussion Th h The empirically deﬁned threshold stiffness increases with displacement and reaches a steady value of E7 10 4 mm 1 at a displacement of B16 mm, equivalent to a shear strain of B5–6 (Fig. 3b). Direct measurements of kc yield similar values (6–7 10 4 mm 1; Supplementary Fig. 2), and also show that kc increases dramatically within the ﬁrst B10 mm of shear displacement. This is due to the combined effects of increasingly velocity-weakening friction (Fig. 3a) and decreasing critical slip distance Dc with shear strain (Supplementary Fig. 2). The evolution of (b a) is consistent with inferred shear localization and with the observation that unstable slip emerges after a ﬁnite shear strain (Fig. 1). The shear displacement needed for the emergence of slow slip decreases with increasing sn0 (Fig. 2a), consistent with enhancement of shear localization and fabric development at higher sn0. Our data show that the full spectrum of stick–slip behaviours can occur over a relatively narrow range of conditions near the stability phase boundary, and further that the mode—and slip velocity—of unstable sliding vary predictably as a function of departure from this threshold. Although the 1D spring-slider model is simpliﬁed relative to the geometry and rheology of natural fault systems, the predicted stability regimes are remarkably consistent with our laboratory experimental data. It is also consistent with theoretical models that incorporate more complex 2D fault geometries and elastic interactions20, suggesting that to ﬁrst order, the mechanics and dynamics of these systems are captured by this relatively simple and elegant model15,18,23,29,31. NATURE COMMUNICATIONS \| 7:11104 \| DOI: 10.1038/ncomms11104 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms11104 0.004 a b c p4309 Velocity strengthening Velocity weakening P4381 P4382 0.002 0.000 (a–b) Stiffness, k′ (μm–1)×1,000 = k kc –1 –0.002 –0.004 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 1.2 1.0 0.8 0.6 0.4 0.2 0.0 20 25 30 Load point displacement (mm) 35 40 45 50 4.0 3.6 3.2 2.8 2.4 2.0 1.6 Peak slip velocity (mm s–1) 1.2 0.8 0.4 0.0 0 10 20 30 40 50 Stable Unstable kc 0 was altered by using a compliant central forcing block and by changing and from the elastic loading portion of stick–slip events (Suppleme b c d e p4309 Velocity strengthening Velocity weakening P4381 P4382 0.002 0.000 (a–b) Stiffness, k′ (μm–1)×1,000 Peak slip velocity (mm s–1) = k kc –1 –0.002 –0.004 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 1.2 1.0 0.8 0.6 0.4 0.2 0.0 4 1.0 0.8 0.6 0.4 0.2 0.0 0.7 0.8 0.9 1.0 1.1 3 2 1 0 0.7 0.8 0.9 1.0 1.1 20 Audible Audible Silent Silent Stable Stable Slip duration (s) 25 30 Load point displacement (mm) 35 40 45 50 4.0 3.6 3.2 2.8 2.4 2.0 1.6 Peak slip velocity (mm s–1) 1.2 0.8 0.4 0.0 0 10 20 30 40 50 Stable Unstable kc 3 \| Stick–slip event properties. (a) The friction rate parameter (b a) transitions from velocity strengthening to velocity weakening ment. (b) Data from 29 experiments showing effective friction stiffness k0 ¼ k/sn0 as a function of shear displacement for stable nd stick–slip events (red dots). The heavy black line deﬁnes the evolution of kc0 based on the distinction between stable sliding and a for unstable slip events shown in b are colour coded by peak slip velocity and shown as a function of shear displacement. Stick–s . The 40–50 mm interval marked by the grey box denotes data used to compile stick–slip properties. (d) Stick–slip event velocity and ction of normalized critical stiffness k ¼ k/kc. Black dots show data from events in the displacement interval 40–50 mm for eight exp ow mean values ±1 s.d. for each experiment. b c d Peak slip velocity (mm s–1) 4 3 2 1 0 0.7 0.8 0.9 1.0 1.1 Audible Silent Stable e 1.0 0.8 0.6 0.4 0.2 0.0 0.7 0.8 0.9 1.0 1.1 Audible Silent Stable Slip duration (s) d e Figure 3 \| Stick–slip event properties. Methods E i t We used a range of shear-loading stiffnesses k given by the summation, in series, of the apparatus stiffness, the stiffness of the loading blocks and the stiffness of the layers of fault gouge. The effective loading stiffness of the testing machine 4 NATURE COMMUNICATIONS \| 7:11104 \| DOI: 10.1038/ncomms11104 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms11104 ARTICLE and from the elastic loading portion of stick–slip events (Supplementary Fig. 1). Rate-and-state friction parameters were determined (Supplementary Fig. 2) using an iterative singular value decomposition technique. References 1. Linde, A. T., Gladwin, M. T., Johnston, M., Gwyther, R. L. & Bilham, R. G. A slow earthquake sequence on the San Andreas fault. Nature 383, 65–68 (1996). Linde, A. T., Gladwin, M. T., Johnston, M., Gwyther, R. L. & B A l h k h S A d f l 32. Bilek, S. L. & Lay, T. Rigidity variations with depth along interplate megathrust faults in subduction zones. Nature 400, 443–446 (1999). 33. Kitajima, H. & Saffer, D. M. Elevated pore pressure and anomalously low stress in regions of low frequency earthquakes along the Nankai Trough subduction megathrust. Geophys. Res. Lett. 39 (2012). 2. Obara, K. Nonvolcanic deep tremor associated with subduction in southwest Japan. Science 296, 1679–1681 (2002). 3. Rogers, G. & Dragert, H. Episodic tremor and slip on the Cascadia subduction zone: the chatter of silent slip. Science 300, 1942–1943 (2003). g p y ( ) 34. Winberry, J. P., Anandakrishnan, S., Alley, R. B., Wiens, D. A. & Pratt, M. J. 34. Winberry, J. P., Anandakrishnan, S., Alley, R. B., Wiens, D. A. & Pratt, M. J. Tidal pacing, skipped slips and the slowdown of Whillans Ice Stream, Antarctica. J. Glaciol. 60, 795–807 (2014). Tidal pacing, skipped slips and the slowdown of Whillans Ice Stream, Antarctica. J. Glaciol. 60, 795–807 (2014). 4. Ide, S., Beroza, G. C., Shelly, D. R. & Uchide, T. A scaling law for slow earthquakes. Nature 447, 76–79 (2007). 5. Shelly, D. R., Beroza, G. C. & Ide, S. Non-volcanic tremor and low-frequency earthquake swarms. Nature 446, 305–307 (2007). Author contributions Experiments were conducted by J.R.L and M.M.S. Data analysis was completed by J.R.L., D.M.S. and C.J.M. All authors contributed to the experimental design and writing. Experiments were conducted by J.R.L and M.M.S. Data analysis was completed by J.R.L., D.M.S. and C.J.M. All authors contributed to the experimental design and writing. 12. Voisin, C., Grasso, J.-R., Larose, E. & Renard, F. Evolution of seismic signals and slip patterns along subduction zones: Insights from a friction lab scale experiment. Geophys. Res. Lett. 35, L08302–L08305 (2008). 13. Shimamoto, T. Transition between frictional slip and ductile ﬂow for halite shear zones at room temperature. Science 231, 711–714 (1986). Acknowledgements 6. Peng, Z. & Gomberg, J. An integrated perspective of the continuum between earthquakes and slow-slip phenomena. Nat. Geosci. 3, 599–607 (2010). We thank Steve Swavely for help in the laboratory and Paul Johnson and Cristiano Collettini for discussions regarding this work. This material is based on work supported by the National Science Foundation under Grants: DGE1255832 to J.R.L., EAR1045825, EAR1520760 to CM, OCE0752114, OCE1347344 to C.M. and D.M.S. and European Union Horizon 2020 research and innovation program under the Marie Sklodowska- Curie No. 656676 FEAT to M.M.S. Any opinions, ﬁndings and conclusions or recom- mendations expressed in this material are those of the author(s) and do not necessarily reﬂect the views of the National Science Foundation. The work was also supported by funds from the GDL Foundation and Shell Oil Company. 7. Kato, A. et al. Propagation of slow slip leading up to the 2011 Mw 9.0 Tohoku-Oki earthquake. Science 335, 705–708 (2012). q 8. Houston, H. Low friction and fault weakening revealed by rising sensitivity of tremor to tidal stress. Nat. Geosci. 8, 409–415 (2015). 9. Hawthorne, J. C. & Rubin, A. M. Tidal modulation of slow slip in Cascadia. J. Geophys. Res. 115, B09406–B09415 (2010). p y 10. Shelly, D. R. Migrating tremors illuminate complex deformation beneath the seismogenic San Andreas fault. Nature 463, 648–652 (2010). 11. Brace, W. F. & Byerlee, J. D. Stick–slip as a mechanism for earthquakes. Science 153, 990–992 (1966). ARTICLE Positive values of (b a) indicate velocity-weakening friction and are a prerequisite for instability and earthquake nucleation. Within the velocity-weakening regime, if the condition in equation (1) is satisﬁed (that is, stiffness of the loading system, k, is less than the critical stiffness; kokc), instability occurs because the fault weakening rate, kc, exceeds the rate of elastic unloading, leading to a force imbalance. For stiffer systems (that is, k4kc), in which elastic unloading outpaces frictional weakening, sliding is stable. For convenience, we normalize the stiffness and critical stiffness by the normal stress, appending a prime symbol to denote this; k0 ¼ k/sn0 and kc0 ¼ kc/sn0 25. Ide, S., Shelly, D. R. & Beroza, G. C. Mechanism of deep low frequency earthquakes: further evidence that deep non-volcanic tremor is generated by shear slip on the plate interface. Geophys. Res. Lett. 34, L03308 ð2007Þ: ð Þ 26. Saffer, D. M., Frye, K. M., Marone, C. & Mair, K. Laboratory results indicating complex and potentially unstable frictional behaviour of smectite clay. Geophys. Res. Lett. 28, 2297–2300 (2001). 27. Hartog, den, S. A. M. & Spiers, C. J. Inﬂuence of subduction zone conditions and gouge composition on frictional slip stability of megathrust faults. Tectonophysics 600, 75–90 (2013). p y We selected values of k and normal stress for our experiments to span the stability boundary for our fault gouge. To achieve this, we made careful measurements of the evolution of k and kc with shear strain (Supplementary Fig. 1). For a given set of frictional properties, deﬁned by (b a) and Dc, the ratio k/sn0 deﬁnes an effective system stiffness, k0 (mm 1), that governs sliding stability. In our experiments, the testing machine, sample assembly and gouge layer together determine the system stiffness. We varied k using different forcing block materials (Supplementary Table 1) and k0 via the normal stress. 28. Ikari, M. J., Saffer, D. M. & Marone, C. Effect of hydration state on the frictional properties of montmorillonite-based fault gouge. J. Geophys. Res. 112, B06423–12 (2007). 29. Marone, C. Laboratory-derived friction laws and their application to seismic faulting. Annu. Rev. Earth Planet. Sci. 26, 643–696 (1998). 30. Brodsky, E. E. & Mori, J. Creep events slip less than ordinary earthquakes. Geophys. Res. Lett. 34 (2007). 31. Kodaira, S. et al. High pore ﬂuid pressure may cause silent slip in the nankai trough. Science 304, 1295–1298 (2004). ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms11104 Frictional stability. In the context of frictional stability, the criterion for unstable stick–slip in a simpliﬁed 1D system is deﬁned by the interaction between loading system stiffness k and a rheologic critical stiffness of the fault, kc: 22. Ikari, M. J., Marone, C., Saffer, D. M. & Kopf, A. J. Slip weakening as a mechanism for slow earthquakes. Nat. Geosci. 6, 468–472 (2013). 22. Ikari, M. J., Marone, C., Saffer, D. M. & Kopf, A. J. Slip weakening as a h f l h k ( ) mechanism for slow earthquakes. Nat. Geosci. 6, 468–472 (2013 23. Gu, J. C., Rice, J. R., Ruina, A. L. & Tse, S. T. Slip motion and stability of a single 23. Gu, J. C., Rice, J. R., Ruina, A. L. & Tse, S. T. Slip motion and stability of a single degree of freedom elastic system with rate and state dependent friction. J. Mech. Phys. Solids 32, 167–196 (1984). degree of freedom elastic system with rate and state dependent friction. J. Mech. Phys. Solids 32, 167–196 (1984). ð1Þ kokc ¼ sn0 b a ð Þ=Dc ð1Þ 24. Hartog, den, S. A. M., Niemeijer, A. R. & Spiers, C. J. New constraints on megathrust slip stability under subduction zone P–T conditions. Earth Planet. Sci. Lett. 353-354, 240–252 (2012). where (b a) is the friction rate parameter and Dc is the critical slip distance29. Negative rate parameters, (b a)o0, indicate velocity-strengthening behaviour, which is inherently stable. Positive values of (b a) indicate velocity-weakening friction and are a prerequisite for instability and earthquake nucleation. Within the velocity-weakening regime, if the condition in equation (1) is satisﬁed (that is, stiffness of the loading system, k, is less than the critical stiffness; kokc), instability occurs because the fault weakening rate, kc, exceeds the rate of elastic unloading, leading to a force imbalance. For stiffer systems (that is, k4kc), in which elastic unloading outpaces frictional weakening, sliding is stable. For convenience, we normalize the stiffness and critical stiffness by the normal stress, appending a prime symbol to denote this; k0 ¼ k/sn0 and kc0 ¼ kc/sn0 where (b a) is the friction rate parameter and Dc is the critical slip distance29. Negative rate parameters, (b a)o0, indicate velocity-strengthening behaviour, which is inherently stable. Additional information shear zones at room temperature. Science 231, 711–714 (1986 14. Baumberger, T., Heslot, F. & Perrin, B. Crossover from creep to inertial motion in friction dynamics. Nature 367, 544–546 (1994). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications y 15. Kaproth, B. M. & Marone, C. Slow earthquakes, preseismic velocity changes, and the origin of slow frictional stick–slip. Science 341, 1229–1232 (2013). Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms11104 (a) The friction rate parameter (b a) transitions from velocity strengthening to velocity weakening at B5–7 mm displacement. (b) Data from 29 experiments showing effective friction stiffness k0 ¼ k/sn0 as a function of shear displacement for stable sliding (black dots) and stick–slip events (red dots). The heavy black line deﬁnes the evolution of kc0 based on the distinction between stable sliding and stick–slip. (c) Data for unstable slip events shown in b are colour coded by peak slip velocity and shown as a function of shear displacement. Stick–slip is slowest for kB1. The 40–50 mm interval marked by the grey box denotes data used to compile stick–slip properties. (d) Stick–slip event velocity and (e) duration as a function of normalized critical stiffness k ¼ k/kc. Black dots show data from events in the displacement interval 40–50 mm for eight experiments; red dots show mean values ±1 s.d. for each experiment. k0 ¼ k/sn0 was altered by using a compliant central forcing block and by changing the applied normal stresses (Fig. 2a). We measured k in experiments using a least- squares linear ﬁt to friction versus shear displacement for the interval m ¼ 0.3 0.4 and from the elastic loading portion of stick–slip events (Supplementary Fig. 1). Rate-and-state friction parameters were determined (Supplementary Fig. 2) using an iterative singular value decomposition technique. 5 NATURE COMMUNICATIONS \| 7:11104 \| DOI: 10.1038/ncomms11104 \| www.nature.com/naturecommunications Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. 16. Ben-David, O., Rubinstein, S. M. & Fineberg, J. Slip-stick and the evolution of frictional strength. Nature 463, 76–79 (2010). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 17. Passele`gue, F. X., Schubnel, A., Nielsen, S., Bhat, H. S. & Madariaga, R. From sub-Rayleigh to supershear ruptures during stick–slip experiments on crustal rocks. Science 340, 1208–1211 (2013). How to cite this article: Leeman, J. R. et al. Laboratory observations of slow earthquakes and the spectrum of tectonic fault slip modes. Nat. Commun. 7:11104 doi: 10.1038/ncomms11104 (2016). 18. Scholz, C., Molnar, P. & Johnson, T. Detailed studies of frictional sliding of granite and implications for the earthquake mechanism. J. Geophys. Res. 77, 6392–6406 (1972). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 19. Rubin, A. M. Designer friction laws for bimodal slow slip propagation speeds. Geochem. Geophys. Geosyst. 12 (2011). 20. Liu, Y. & Rice, J. R. Spontaneous and triggered aseismic deformation transients in a subduction fault model. J. Geophys. Res. 112 (2007). 21. Segall, P., Rubin, A. M., Bradley, A. M. & Rice, J. R. Dilatant strengthening as a mechanism for slow slip events. J. Geophys. Res. 115, B12305–B12337 (2010). 6 NATURE COMMUNICATIONS \| 7:11104 \| DOI: 10.1038/ncomms11104 \| www.nature.com/naturecommunications
https://openalex.org/W4382932398	https://www.nature.com/articles/s41598-023-37861-z.pdf	English	null	Survival analysis of a stochastic impulsive single-species population model with migration driven by environmental toxicant	Scientific reports	2,023	cc-by	14,778	www.nature.com/scientificreports www.nature.com/scientificreports Survival analysis of a stochastic impulsive single‑species population model with migration driven by environmental toxicant Xiangjun Dai 1,2, Jianjun Jiao 1,3* & Qi Quan 1 OPEN Xiangjun Dai 1,2, Jianjun Jiao 1,3* & Qi Quan 1 Considering the influence of environmental toxicant on population migration between patches, we propose and study a stochastic impulsive single-species population model with migration driven by environmental toxicant in this paper. We first discuss the existence and uniqueness of global positive solutions of the model by constructing the Lyapunov function. Then, we obtain sufficient conditions for extinction, stochastic persistence and persistence in the mean of the single-species population. Finally, we present some numerical simulations to illustrate our results. These results provide insights for the conservation and management of species in polluted environments. Due to differences in the geographical environment and the influence of human activities, the habitats of many species are broken up into isolated patches, which may lead to the extinction of species within the patch. There- fore, the study of population migration between patches plays a very important role in the conservation and man- agement of species, and many scholars have analysed the effects of migration on stability, permanence, extinction, and other dynamic properties by establishing mathematical models (see1–14). For example, Feng et al.9 proposed and studied a predator-prey model with predator population migration dependent on prey. Kang et al.10 consid- ered the situation that predators migrate towards patches with more concentrated predator-prey interactions in the model. Specifically, some scholars proposed single-species population models with migrations between the non-nature reserve and the nature reserve to study the survival and extinction of single-species populations. For example, Zou and Wang11 proposed and studied the following deterministic single-species diffusion model.        dx1(t) dt = x1(t)(r −ax1(t)) + D H (x2(t) −x1(t)), dx2(t) dt = x2(t)(r −ax2(t)) + D h (x1(t) −x2(t)), where r > 0 and a > 0 stand for the population growth rate and the intra-specific competition coefficient of population. D > 0 is the diffusion coefficient. H and h are sizes of the non-nature reserve and the nature reserve. And then, the extinction and permanence in the mean of single species under fluctuated environments were also studied by Zou et al.12,13 and Dieu et al.14. Based on the model in12, Wei and Wang15 established the following stochastic single-species model with migrations between two patches. www.nature.com/scientificreports/ In this paper, we assume that toxins are emitted in regular pulses, a common example being the use of pesticides, and propose a deterministic single-species population model with migration driven by environmental toxicant as follows: (1)                                dx1(t) dt = x1(t)[r1e −δco(t) −a1x1(t)] + d21x2(t) −d12(1 + ρc2 e(t) 1 + αc2e(t))x1(t), dx2(t) dt = x2(t)[r2e −a2x2(t)] + d12(1 + ρc2 e(t) 1 + αc2e(t))x1(t) −d21x2(t), dco(t) dt = fce(t) −(g + m)co(t), dce(t) dt = −hce(t),                          t = nγ , x1(t) = 0, x2(t) = 0, co(t) = 0, ce(t) = b, t = nγ , n ∈Z+. (1)   x1(t) = 0, x2(t) = 0, co(t) = 0, ce(t) = b, t = nγ , n ∈Z+. Here xi(t) denotes the density of population in patch i. ce(t) and co(t) represent the concentration of toxicant in the environment and organism at time t respectively. f > 0 represents the uptake rate of toxicant from the environment by the population in patch 1. (g + m)co(t) describes loss due to egestion and metabolic process at time t. b ≥0 and γ > 0 represent the pulse input amount of toxins and the pulse input period of toxicant respec- tively. hc(t) represents the total lose at time t from the system environment including processes such as biological transformation, microbial degradation, volatilization and photosynthetic degradation. δco(t) represents the lethal rate of toxins in the organism to the population in patch 1. In this paper, we adopt a Holling-III response func- tion ρd12c2 e 1+αc2e to describe the influence of toxicant concentration in patch 1 on population migration. ρd12 is described as the migration rate of the population in patch 1 to patch 2 due to the stimulation of toxicant in patch 1, and α > 0 denotes the sensitivity of population to environmental toxicant. ψ(t) = ψ(t+) −ψ(t) (ψ = x1, x2, co, ce) , ψ(t+) = lim s→0+ ψ(t + s). www.nature.com/scientificreports/ and the intensity of white noise in two patches are the same, so the results obtained in15 are not suitable for the general situation. Therefore, we need to further discuss the influence of population migration on the survival of single-species. and the intensity of white noise in two patches are the same, so the results obtained in15 are not suitable for the general situation. Therefore, we need to further discuss the influence of population migration on the survival of single-species. g p With the rapid development of human society, a large number of toxic substances and pollutants are dis- charged into the ecosystem, seriously polluting the ecological environment and threatening the survival of spe- cies. Such as heavy metal pollution, and water pollution caused by crop fertilization and pesticide application. Therefore, it is most important to investigate the survival and extinction of species in a polluted environment. In recent years, many excellent results have analyzed the effects of toxicant discharged into the environment from modern industry and modern agriculture on population by establishing models16–21. But, these models mainly discussed the effect of pollutants on the population growth rate. As we all know, many creatures in nature have good sensory organs and highly differentiated nervous systems, and they can respond to information in the environment accordingly. For example, in agricultural production, many pests will choose to escape from the pesticide-treated environment due to the stimulation of chemical pesticides, and then seek a new environment conducive to population growth, this may be one of the reasons for inducing the resurgence of pest populations and the emergence of pest resistance. Therefore, it is necessary to consider the effect of environmental toxicant on population migration. Wei el at.20,21 proposed two single-species population models with physiological effect, where the “physiological” effect is described as self-protection by organisms in highly polluted environments to reduce the effective contact between the organism and the polluted environment. However, few studies have considered the influence of environmental toxicant on population migration between patches. Survival analysis of a stochastic impulsive single‑species population model with migration driven by environmental toxicant Xiangjun Dai 1,2, Jianjun Jiao 1,3* & Qi Quan 1 OPEN dx1(t) = [x1(t)(r −ax1(t)) + (d21x2(t) −d12x1(t)) −E1x1(t)]dt + σx1(t)dB(t), dx2(t) = [x2(t)(r −ax2(t)) + (d12x1(t) −d21x2(t)) −E2x2(t)]dt + σx2(t)dB(t), where d12 ≥0 stands for the migration rate of the population from the non-nature reserve (patch 1) to the nature reserve (patch 2), d21 ≥0 stands for the migration rate of the population from the nature reserve to the non- nature reserve. Ei denotes the hunting rate in the i-th patch, and E1 ≫E2 . B(t) is standard Brownian motion. In15, authors assumed that the number of individuals of a species in the nature reserve is larger than that in the non-nature reserve, and sufficient conditions for the extinction and persistence in the mean of population were obtained. However, it is not difficult to find that the growth rate, the intra-specific competition coefficient where d12 ≥0 stands for the migration rate of the population from the non-nature reserve (patch 1) to the nature reserve (patch 2), d21 ≥0 stands for the migration rate of the population from the nature reserve to the non- nature reserve. Ei denotes the hunting rate in the i-th patch, and E1 ≫E2 . B(t) is standard Brownian motion. In15, authors assumed that the number of individuals of a species in the nature reserve is larger than that in the non-nature reserve, and sufficient conditions for the extinction and persistence in the mean of population were obtained. However, it is not difficult to find that the growth rate, the intra-specific competition coefficient 1School of Mathematical Sciences, Guizhou Normal University, Guiyang 550025, People’s Republic of China. 2School of Date science, Tongren University, Tongren 554300, People’s Republic of China. 3School of Mathematics and Statistics, Guizhou University of Finance and Economics, Guiyang 550025, People’s Republic of China. *email: jiaojianjun2018@126.com \| https://doi.org/10.1038/s41598-023-37861-z Scientific Reports \| (2023) 13:10753 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Remark 1 Because each of co(t) and ce(t) is a concentration, co(t) and ce(t) must satisfy the inequalities 0 ≤co(t) ≤1 and 0 ≤ce(t) ≤1 for t ≥0 . Therefore, throughout this article, we assume that f ≤g + m and b ≤1 −e−hγ. Remark 1 Because each of co(t) and ce(t) is a concentration, co(t) and ce(t) must satisfy the inequalities 0 ≤co(t) ≤1 and 0 ≤ce(t) ≤1 for t ≥0 . Therefore, throughout this article, we assume that f ≤g + m and b ≤1 −e−hγ. www.nature.com/scientificreports/ Modifying the deterministic model (1), we propose the follow- ing stochastic impulsive single-species population model with migration driven by environmental toxicant               dx1(t) = x1(t)[r1e + (r10 −r1e)e−µ1t −δco(t) −a1x1(t)]dt + [d21x2(t) −d12(1 + ρc2 e(t) 1 + αc2e(t))x1(t)]dt + σ1(t)x1(t)dB1(t),              (3)                                    dx1(t) = x1(t)[r1e + (r10 −r1e)e−µ1t −δco(t) −a1x1(t)]dt + [d21x2(t) −d12(1 + ρc2 e(t) 1 + αc2e(t))x1(t)]dt + σ1(t)x1(t)dB1(t), dx2(t) = x2(t)[r2e + (r20 −r2e)e−µ2t −a2x2(t)]dt + [d12(1 + ρc2 e(t) 1 + αc2e(t))x1(t), −d21x2(t)]dt + σ2(t)x2(t)dB2(t), dco(t) = fce(t) −(g + m)co(t)dt, dce(t) = −hce(t)dt,                              t = nγ , x1(t) = 0, x2(t) = 0, co(t) = 0, ce(t) = b, t = nγ , n ∈Z+. (3 dx2(t) = x2(t)[r2e + (r20 −r2e)e−µ2t −a2x2(t)]dt + [d12(1 + ρc2 e(t) 1 + αc2e(t))x1(t),       t = nγ , (3) −d21x2(t)]dt + σ2(t)x2(t)dB2(t), dco(t) = fce(t) −(g + m)co(t)dt,    x1(t) = 0, x2(t) = 0, co(t) = 0, ce(t) = b, t = nγ , n ∈Z+. Because the solutions of c0(t) and ce(t) can be solved by the third and fourth equations of (3), we only consider the following system Because the solutions of c0(t) and ce(t) can be solved by the third and fourth equations of (3), we only consider the following system (4)                    dx1(t) = x1(t)[r1e + (r10 −r1e)e−µ1t −δco(t) −a1x1(t)]dt + [d21x2(t) −d12(1 + ρc2 e(t) 1 + αc2e(t))x1(t)]dt + σ1(t)x1(t)dB1(t), dx2(t) = x2(t)[r2e + (r20 −r2e)e−µ2t −a2x2(t)]dt + [d12(1 + ρc2 e(t) 1 + αc2e(t))x1(t) −d21x2(t)]dt + σ2(t)x2(t)dB2(t). (4) (4) 1 + αce(t) dx2(t) = x2(t)[r2e + (r20 −r2e)e−µ2t −a2x2(t)]dt + [d12(1 + ρc2 e(t) 1 + αc2e(t))x1(t) −d21x2(t)]dt + σ2(t)x2(t)dB2(t). www.nature.com/scientificreports/ s→0+ On the other hand, the population is inevitably affected by various factors in the environment, for example, changes in temperature, climate and weather. May22 showed that the birth rates, carrying capacity, and other parameters involved in the system can be affected by environmental noise. In order to better understand the dynamic behaviors of the population models, many researchers introduced random perturbations into deter- ministic models to show richer and more complex dynamic properties24–31. Motivated by the above studies, we suppose that environmental noises mainly affect the growth rate rie of system (1) in this paper, according to the central limits theorem, we usually use an average value plus an error term satisfying the standard normal distribution to estimate a value25,26, that is, rie(t) = rie + σi dBi(t) dt , i = 1, 2., rie(t) = rie + σi dBi(t) dt , i = 1, 2., where rie is a positive constant, dBi(t) dt is the a Gaussian white noise, Bi(t) represents the standard Brownian motion defined on the complete probability space (, F, {Ft}t≥0, P) with {Ft}t≥0 satisfying the usual conditions23. σi is the intensity of the white noise. There is another possible form of modeling for rie in a randomly-varying environment, we introduce the Ornstein-Uhlenbeck process (also called as mean-reverting process)21–27, and it has the following form (2) drie(t) = µi(rie −ri(t))dt + ξidBi(t), (2) where rie, ξi and µi are positive constants, µi is the speed of reversion and ξi is the intensity of the white noise olving the stochastic Eq. (2), from studies21–27, we have https://doi.org/10.1038/s41598-023-37861-z Scientific Reports \| (2023) 13:10753 \| https://doi.org/10.1038/s41598-023-37861-z www.nature.com/scientificreports/ www.nature.com/scientificreports/ rie(t) = rie + (ri0 −rie)e−µit + σi(t)dBi(t) dt , rie(t) = rie + (ri0 −rie)e−µit + σi(t)dBi(t) dt , where ri0 = ri(0) and σi(t) = ξi √2µi √ 1 −e−2µit . (ii) The population x is said to be strongly persistent in the mean if x(t)∗> 0. (iii) The population x is said to be stochastically permanent if for any ǫ ∈(0, 1) , there exist H1 = H1(ǫ) > 0 and H2 = H2(ǫ) > 0 such that lim inf t→+∞P{\|x(t)\| > H1} ≥1 −ǫ, lim inf t→+∞P{\|x(t)\| < H2} ≥1 −ǫ. Preliminaries F h i Proof It follows from the periodicity of co(t) and ce(t) that Proof It follows from the periodicity of co(t) and ce(t) that p y o( ) e( ) lim t→+∞t−1 t 0 co(s)ds = γ −1 γ 0 co(s)ds = fb h(g + m)γ , and lim t→+∞t−1 t 0 ( ce(s))2 1 + α( ce(s))2 ds = −1 γ h γ 0 −h( ce(s))2 1 + α( ce(s))2 ds = −1 γ h γ 0 ce(s) 1 + α( ce(s))2 d ce(s) = 1 2hγ α ln (1 −e−hγ )2 + αb2 (1 −e−hγ )2 + αb2e−2hγ = η. □ This result is confirmed. This result is confirmed. Preliminaries F h i (5) Model (5) has a unique globally asymptotically stable positive γ-periodic solution ( co(t), ce(t)) , where and cm = inf{ co(t)} cM = sup{ co(t)} cM = b h and cm = be−hγ h                            co(t) = co(0)e−(g+m)(t−nγ ) + fb(e−(g+m)(t−nγ ) −e−h(t−nγ )) (h −g −m)(1 −e−hγ ) , ce(t) = be−h(t−nγ ) 1 −e−hγ , co(0) = fb(e−(g+m)γ −e−hγ ) (h −g −m)(1 −e−hγ )(1 −e−(g+m)γ ), ce(0) = b 1 −e−hγ ,                            co(t) = co(0)e−(g+m)(t−nγ ) + fb(e−(g+m)(t−nγ ) −e−h(t−nγ )) (h −g −m)(1 −e−hγ ) , ce(t) = be−h(t−nγ ) 1 −e−hγ , co(0) = fb(e−(g+m)γ −e−hγ ) (h −g −m)(1 −e−hγ )(1 −e−(g+m)γ ), ce(0) = b 1 −e−hγ , and cm o = inf t≥0{ co(t)} , cM o = sup t≥0 { co(t)} , cM e = b 1−e−hγ and cm e = be−hγ 1−e−hγ . and cm o = inf t≥0{ co(t)} , cM o = sup t≥0 { co(t)} , cM e = b 1−e−hγ and cm e = be−hγ 1−e−hγ . Lemma 2 The positive γ-periodic solution ( co(t) , ce(t) ) of model (5) satisfies Lemma 2 The positive γ-periodic solution ( co(t) , ce(t) ) of model (5) satisfies Proof It follows from the periodicity of co(t) and ce(t) that lim t→+∞t−1 t 0 co(s)ds = fb h(g + m)γ , lim t→+∞t−1 t 0 c2e(s) 1 + α c2e(s) ds = η. lim t→+∞t−1 t 0 co(s)ds = γ −1 γ 0 co(s)ds = fb h(g + m)γ , lim t→+∞t−1 t 0 co(s)ds = fb h(g + m)γ , lim t→+∞t−1 t 0 c2e(s) 1 + α c2e(s) ds = η. Main results I d d Main results In order to study the long-time behaviors of the model (4), we first discuss the existence and uniqueness of global positive solutions to the stochastic differential equation (SDE) (4). Existence and uniqueness of the positive solution for SDE (4). h l l 2 h l b l l Preliminaries F h i Preliminaries For the convenience of later discussion, some notations are defined here: R2 + ={(x1, x2)\|xi > 0, i = 1, 2.}, f (t) = t−1 t 0 f (s)ds, f ∗= lim sup t→+∞ f (t), f∗= lim inf t→+∞f (t), η = 1 2hγ α ln (1 −e−hγ )2 + αb2 (1 −e−hγ )2 + αb2e−2hγ , r1(t) = r1e + (r10 −r1e)e−µ1t −δco(t), r∗ 1 = r1e −δcm o , d12(t) =d12 1 + ρc2 e(t) 1 + αc2e(t) , r2(t) = r2e + (r20 −r2e)e−µ2t, (r1)∗= r1e −δcM o , (d12)∗=d12 1 + ρ(cm e )2 1 + α(cm e )2 , d∗ 12 = d12 1 + ρ(cM e )2 1 + α(cM e )2 , σ 2 = ξ2 1 ξ2 2 2µ1ξ2 2 + 2µ2ξ2 1 , ˆσ 2 = max ξ2 1 2µ1 , ξ2 2 2µ2 , where cm o , cM o , cm e and cM e are given in Lemma 1. where cm o , cM o , cm e and cM e are given in Lemma 1. where cm o , cM o , cm e and cM e are given in Lemma 1. Definition 1 (see31) (i) The population x is said to go to extinction if lim t→+∞x(t) = 0.h t→+∞ (ii) The population x is said to be strongly persistent in the mean if x(t)∗> 0. ( ) Th l d b h ll f f h p p g y p (iii) The population x is said to be stochastically permanent if for any ǫ ∈(0, 1) , there exist H1 = H1(ǫ) > 0 and H2 = H2(ǫ) > 0 such that lim inf t→+∞P{\|x(t)\| > H1} ≥1 −ǫ, lim inf t→+∞P{\|x(t)\| < H2} ≥1 −ǫ. Lemma 1 (see31) Consider the following model corresponding to model (3) Scientific Reports \| (2023) 13:10753 \| https://doi.org/10.1038/s41598-023-37861-z www.nature.com/scientificreports/            dco(t) dt = fce(t) −(g + m)co(t), dce(t) dt = −hce(t), t = nγ , ce(t) = b, t = nγ . (5            dco(t) dt = fce(t) −(g + m)co(t), dce(t) dt = −hce(t), t = nγ , ce(t) = b, t = nγ . From (7), we have V(x(0)) + KT ≥P(N)V(x(τN ∧T)) ≥ǫ min N −1 + ln N, 1 N −1 −ln N . V(x(0)) + KT ≥P(N)V(x(τN ∧T)) ≥ǫ min N −1 + ln N, 1 N −1 −ln N . Letting N →+∞ , leads to the contradiction: N +∞> V(x(0)) + KT ≥P(N)V(x(τN ∧T)) ≥ǫ min{N −1 + ln N, 1 N −1 −ln N} = ∞. Letting N →+∞ , leads to the contradiction: Letting N →+∞ , leads to the contradiction: +∞> V(x(0)) + KT ≥P(N)V(x(τN ∧T)) ≥ǫ min{N −1 + ln N, 1 N −1 −ln N} = ∞. +∞> V(x(0)) + KT ≥P(N)V(x(τN ∧T)) ≥ǫ min{N −1 + ln N, 1 N −1 −ln N} = ∞. □ Therefore, we obtain τ∞= +∞ , a.s. Therefore, we obtain τ∞= +∞ , a.s. Existence and uniqueness of the positive solution for SDE (4). 2 Using Itˆo′s formula, we have https://doi.org/10.1038/s41598-023-37861-z Scientific Reports \| (2023) 13:10753 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ (6) dV(x) = LV(x)dt + σ1(t)(x1 −1)dB1(t) + σ2(t)(x2 −1)dB2(t), (6) here LV(x) = r1e + (r10 −r1e)e−µ1t −δco + a1 x1 −a1x2 1 + r2e + (r20 −r2e)e−µ2t + a2 x2 −a2x2 2 − r1e + (r10 −r1e)e−µ1t −δco − r2e + (r20 −r2e)e−µ2t + d12 1 + ρc2 e 1 + αc2e + d21 + 0.5σ 2 1 (t) + 0.5σ 2 2 (t) −d12 1 + ρc2 e 1 + αc2e x1 x2 −d21 x2 x1 ≤[r1e + r10 + a1]x1 −a1x2 1 + [r2e + r20 + a2]x2 −a2x2 2 + δco(t) + d12(1 + ρ) + d21 LV(x) = r1e + (r10 −r1e)e−µ1t −δco + a1 x1 −a1x2 1 + r2e + (r20 −r2e)e−µ2t + a2 x2 −a2x2 2 − r1e + (r10 −r1e)e−µ1t −δco − r2e + (r20 −r2e)e−µ2t + d12 1 + ρc2 e 1 + αc2e + d21 + 0.5σ 2 1 (t) + 0.5σ 2 2 (t) −d12 1 + ρc2 e 1 + αc2e x1 x2 −d21 x2 x1 ≤[r1e + r10 + a1]x1 −a1x2 1 + [r2e + r20 + a2]x2 −a2x2 2 + δco(t) + d12(1 + ρ) + d2 ≤[r1e + r10 + a1]x1 −a1x2 1 + [r2e + r20 + a2]x2 −a2x2 2 + δco(t) + d12(1 + ρ) + d21 + ξ2 1 4µ1 + ξ2 2 4µ2 . + 1 4µ1 + 2 4µ2 . here exists K > 0 such that LV(x) ≤K. Obviously, there exists K > 0 such that LV(x) ≤K. Obviously, there exists K > 0 such that LV(x) ≤K. Integrating (6) on [0, τN ∧T] , and then taking expectation obtain that y, ( ) ≤ Integrating (6) on [0, τN ∧T] , and then taking expectation obtain that ( ) ≤ ng (6) on [0, τN ∧T] , and then taking expectation obtain that y Integrating (6) on [0, τN ∧T] , and then taking expect EV(x(τN ∧T)) ≤V(x(0)) + KT. (7) EV(x(τN ∧T)) ≤V(x(0)) + KT. (7) Let N = {τN ≤T}, N > N1 , then P(N) ≥ǫ . For any ω ∈N , we get that at least one of x1(τN, ω) and x2(τN, ω) equals either N or 1 N , thus Let N = {τN ≤T}, N > N1 , then P(N) ≥ǫ . Existence and uniqueness of the positive solution for SDE (4). 2 heorem 1 For any given initial value x(0) = (x1(0), x2(0)) ∈R2 +, there exists a unique global positive solution (t) = (x1(t), x2(t)) to SDE (4), and the solution x(t) will remain R2 + with probability 1. Proof Because the coefficients of the SDE (4) are locally Lipschitz continuous, there must be a unique local solu- tion x(t) in [0, τe) for any given initial value x(0) ∈R2 + , where τe denotes the explosion time. Therefore, we need to prove τe = +∞a.s. in the following. Let N0 be large enough such that x(0) remains in the interval [ 1 N0 , N0] . For every N ≥N0 , define the stopping time τN = inf t ∈[0, τe] : xi(t) /∈ 1 N , N , i = 1, 2. . Clearly, τN is increasing as N →+∞ . Letting τ∞= lim N→∞τN , thus, τ∞≤τe a.s. In the following, we only need to prove τ∞= +∞, a.s. We next employ the reduction to absurdity to prove it. If the conclusion is not true, then there are T > 0 and ǫ ∈(0, 1) such that P{τ∞< T} > ǫ . Accordingly, there is a positive integer N1 ≥N0 such that for any N ≥N1 , P{τN ≤T} ≥ǫ . Define a C2-function V : R2 + →R+ as follows: V(x1, x2) = [x1 −1 −ln x1] + [x2 −1 −ln x2]. Existence and uniqueness of the positive solution for SDE (4). 2 For any ω ∈N , we get that at least one of x1(τN, ω) and x2(τN, ω) equals either N or 1 N , thus V(x(τN ∧T)) ≥min N −1 + ln N, 1 N −1 −ln N . From (7), we have Stochastic permanence. Let K0(p) = max{2K(p), K1(p)}, we have E[(x1(t) + x2(t))p] ≤K0(p) , that is, the solution x(t) to SDE (4) is P-moment bounded. Theorem 2 If min{r1e −δcM, r2e} > 0.5ˆσ 2 , the solution x(t) of SDE (4) is stochastically permanent. Proof Define function V1(x) = x1(t) + x2(t) , t ≥0 , we can obtain that dV1(x) = [x1(t)(r1(t) −a1x1(t)) + x2(t)(r2(t) −a2x2(t))]dt + σ1(t)x1(t)dB1(t) + σ2(t)x2(t)dB2(t). Proof Define function V1(x) = x1(t) + x2(t) , t ≥0 , we can obtain that dV1(x) = [x1(t)(r1(t) −a1x1(t)) + x2(t)(r2(t) −a2x2(t))]dt + σ1(t)x1(t)dB1(t) + σ2(t)x2(t)dB2(t). Define function U(x) = 1 V1(x) , t ≥0 . Applying Itˆo ’s formula, we have dU = −U2dV1 + U3(dV1)2 dU = −U2dV1 + U3(dV1)2 (9) = −U2[x1(t)(r1(t) −a1x1(t)) + x2(t)(r2(t) −a2x2(t))]dt + U3(σ 2 1 (t)x2 1 + σ 2 2 (t)x2 2)dt −U2(σ1(t)x1(t)dB1(t) + σ2(t)x2(t)dB2(t)). (9) If min{r1e −δcM, r2e} > 0.5ˆσ 2 , we c an t a ke an ǫ > 0 sma l l enoug h such t hat ˇr = min{(r1(t))∗, (r2(t))∗} = min{r1e −δcM, r2e} > 0.5ˆσ 2 + ǫ . Moreover, we can also select a θ > 0 such that (ˇr −ǫ) −0.5(θ + 1)ˆσ 2 > 0 . Define function V2(t) = (1 + U(x))θ . An application of Itˆo ’s formula gives dV2(x) = θ(1 + U(x))θ−1dU(x) + 1 2θ(θ −1)(1 + U(x))θ−2(dU(x))2 = LV2(x)dt −θ(1 + U)θ−1U2(σ1(t)x1(t)dB1(t) + σ2(t)x2(t)dB2(t)), here ˆa = max{a1, a2} and (10 LV2(x) = θ(1 + U)θ−1{−U2{x1[r1(t) −a1x1] + x2[r2(t) −a2x2]} + U3(σ 2 1 (t)x2 1 + σ 2 2 (t)x2 2)} + 1 2θ(θ −1)(1 + U)θ−2U4(σ 2 1 (t)x2 1 + σ 2 2 (t)x2 2) ≤(1 + U)θ−2{θU3{−x1(r1e −ǫ −δcM) −x2(r2e −ǫ) + θ(1 + U)U2(a1x2 1 + a2x2 2)} + (θ(1 + U)U3 + 1 2θ(θ −1)U4)ˆσ(x2 1 + x2 2)} ≤(1 + U)θ−2 1 2θ(θ + 1)ˆσ −(ˇr −ǫ)θ U2 + (θ ˆa + θ ˆσ)U + θ ˆa (10) for t large enough. We select a ζ > 0 small enough to satisfy for t large enough. Stochastic permanence. Stochastic permanence. Lemma 3 For any given initial value x(0) ∈R2 + , there must be a K(p) > 0 such that the solution x(t) of SDE (4) satisfies lim sup t→+∞ E[(x1 + x2)p] ≤K(p), p > 1. Proof Define function V(x) = (x1 + x2)p , (p > 1) , using Itˆo ’s formula to V(x), we obtain Proof Define function V(x) = (x1 + x2)p , (p > 1) , using Itˆo ’s formula to V(x), we obtain where r = p max{r1e + r10, r2e + r20} + 1 2p(p −1) max ξ2 1 2θ1 , ξ2 2 2θ2 , a = p min{a1,a2} 2 . Thus, dV(x) = p(x1 + x2)p−1d(x1 + x2) + 1 2p(p −1)(x1 + x2)p−2(d(x1 + x2))2 ≤p(x1 + x2)p−1{x1[r1e + r10 −a1x1] + x2[r2e + r20 −a2x2]}dt + 1 2p(p −1)(x1 + x2)p−2 x2 1 ξ2 1 2θ1 + x2 2 ξ2 2 2θ2 dt + p(x1 + x2)p−1(σ1(t)x1dB1(t) + σ2(t)x2dB2(t)) ≤(x1 + x2)p{r −a(x1 + x2)}dt + p(x1 + x2)p−1(σ1(t)x1dB1(t) + σ2(t)x2dB2(t)), EV(x(t)) −V(x(0)) ≤ t 0 rEV(x(s)) −aEV p+1 p (x(s))ds, dV(x) = p(x1 + x2)p−1d(x1 + x2) + 1 2p(p −1)(x1 + x2)p−2(d(x1 + x2))2 ≤p(x1 + x2)p−1{x1[r1e + r10 −a1x1] + x2[r2e + r20 −a2x2]}dt further, Let y(t) = EV(x(t)) , from (8), we have (8) dEV(x(t)) dt ≤EV(x(t)) r −a(EV(x(t))) 1 p . dy(t) dt ≤y(t) r −ay 1 p (t) . Let y(t) = EV(x(t)) , from (8), we have dEV(x(t)) dt ≤EV(x(t)) r −a(E (8) dEV(x(t)) dt ≤EV(x(t)) r −a(EV(x(t))) 1 p . ( ) E ( ( )) f ( ) h dEV(x(t)) dt ≤EV(x(t)) r −a(EV(x(t))) 1 p . dEV(x(t)) dt ≤EV(x(t)) r −a(EV(x(t))) 1 p (8) Let y(t) = EV(x(t)) , from (8), we have https://doi.org/10.1038/s41598-023-37861-z https://doi.org/10.1038/s41598-023-37861-z Scientific Reports \| (2023) 13:10753 \| www.nature.com/scientificreports/ By the comparison theorem, we obtain lim sup t→+∞ y(t) ≤( r a)p , that is, lim sup t→+∞ E(x1(t) + x2(t))p ≤( r a)p = K(p) . This ends the proof. □ □ Remark 2 From Lemma 3, we know that there exists a T > 0 such that E[(x1(t) + x2(t))p] ≤2K(p) for t > T . On the other hand, E[(x1(t) + x2(t))p] is continuous with respect to t on the interval [0, T], then there exists a K1(p) > 0 such that E[(x1(t) + x2(t))p] ≤K1(p) for t ∈[0, T] . Extinction. h Lemma 4 The solution x(t) to SDE (4) satisfies lim sup t→+∞ ln xi(t) t ≤0, a.s., i = 1, 2. roof Define function V3(x) = ln(x1 + θx2) (θ > 0) . Applying Itˆo ’s formula for V3(x) , we have ( d ln(x1 + θx2) = x1[r1(t) −a1x1] + [d21x2 −d12(t)x1] x1 + θx2 + θx2[r2(t) −a2x2] + θ[d12(t)x1 −d21x2] x1 + θx2 −σ 2 1 (t)x2 1 + σ 2 2 (t)θ2x2 2 2(x1 + θx2)2 dt + σ1(t)x1dB1(t) + σ2(t)θx2dB2(t) x1 + θx2 ≤ (r1e + r10 + θd12(1 + ρ))x1 + (r2e + r20 + d21 θ )θx2 −a1x2 1 −θa2x2 2 x1 + θx2 −σ 2 1 (t)x2 1 + σ 2 2 (t)θ2x2 2 2(x1 + θx2)2 dt + σ1(t)x1dB1(t) + σ2(t)θx2dB2(t) x1 + θx2 ≤ r −ν(x1 + θx2) −σ 2 1 (t)x2 1 + σ 2 2 (t)θ2x2 2 2(x1 + θx2)2 dt + σ1(t)x1dB1(t) + σ2(t)θx2dB2(t) x1 + θx2 , (14) where r = max{r1e + r10 + θd12(1 + ρ), (r2e + r20 + d21 θ )} and ν = 0.5 min{a1, a2 θ } . Thus, (14) where r = max{r1e + r10 + θd12(1 + ρ), (r2e + r20 + d21 θ )} and ν = 0.5 min{a1, a2 θ } . Thus, where r = max{r1e + r10 + θd12(1 + ρ), (r2e + r20 + d21 θ )} and ν = 0.5 min{a1, a2 θ } . Thus, ( ) (15) detV(x) = etV(x)dt + etdV(x) ≤et ln(x1 + θx2) + r −ν(x1 + θx2) −σ 2 1 (t)x2 1 + σ 2 2 (t)θ2x2 2 2(x1 + θx2)2 dt + et σ1(t)x1dB1(t) + σ2(t)θx2dB2(t) x1 + θx2 . (15) ) ( ) ( ) ≤et ln(x1 + θx2) + r −ν(x1 + θx2) −σ 2 1 (t)x2 1 + σ 2 2 (t)θ2x2 2 2(x1 + θx2)2 dt + et σ1(t)x1dB1(t) + σ2(t)θx2dB2(t) x1 + θx2 . (15) Integrating both sides of inequality (15) in the interval [0, t], we have Integrating both sides of inequality (15) in the interval [0, t], we have (16) etV(x) ≤V(x(0)) + t 0 es(ln(x1(s) + θx2(s)) + r −ν(x1(s) + θx2(s)) −σ 2 1 (t)x2 1(s) + σ 2 2 (t)θx2 2(s) 2(x1(s) + θ2x2(s))2 )ds + M(t), (16) w here M(t) = t 0 es σ1(t)x1(s)dB1(s)+σ2(t)θx2(s)dB2(s) x1(s)+θx2(s) . Stochastic permanence. thus lim sup t→+∞ P{(x1(t) + x2(t)) < H1} ≤ǫ, and lim inf t→+∞P{(x1(t) + x2(t)) > H1} ≥1 −ǫ. t→+∞ We will prove in the following that for any ǫ > 0 , there is a H2(ǫ) > 0 such that lim inf t→+∞P{(x1(t) + x2(t)) ≤H2} ≥1 −ǫ. According to Lemma 3 and the Chebyshev’s inequality, this result can be easily confirmed. □ Stochastic permanence. We select a ζ > 0 small enough to satisfy (11) (ˇr −ǫ)θ −1 2θ(θ + 1)ˆσ −ζ > 0, (ˇr −ǫ)θ −1 2θ(θ + 1)ˆσ −ζ > 0, (11) By computing, we have By computing, we have (12) E[eζtV2(x)] = V2(x(0)) + E t 0 L[eζsV2(x(s))]ds, (12) where where (1 L[eζtV2(x)] = ηeζtV2(x) + eζtLV2(x) ≤eζt(1 + U)θ−2 ζ(1 + U)2 + 1 2θ(θ + 1)ˆσ −(ˇr −ǫ)θ U2 + (θ ˆa + θ ˆσ)U + θ ˆa = eζt(1 + U)θ−2 − (ˇr −ǫ)θ −1 2θ(θ + 1)ˆσ −ζ U2 + (θ ˆa + θ ˆσ + 2ζ)U + θ ˆa + ζ ≤ζeζtκ(x), (13) here (13) κ(x) = 1 ζ {(1 + U)θ−2{−[(ˇr −ǫ)θ −1 2θ(θ + 1)ˆσ −ζ]U2 + (θ ˆa + θ ˆσ + 2ζ)U + θ ˆa + ζ}}. here κ(x) = 1 ζ {(1 + U)θ−2{−[(ˇr −ǫ)θ −1 2θ(θ + 1)ˆσ −ζ]U2 + (θ ˆa + θ ˆσ + 2ζ)U + θ ˆa + ζ}}. From (11), we know that κ(x) is bounded in R2 + . Let κ1 = max   sup x∈R2+ κ(x), 1   < +∞. It follows from (12) that From (11), we know that κ(x) is bounded in R2 + . Let κ1 = max   sup x∈R2+ κ(x), 1   < +∞. It follows from (12) that https://doi.org/10.1038/s41598-023-37861-z https://doi.org/10.1038/s41598-023-37861-z Scientific Reports \| (2023) 13:10753 \| Scientific Reports \| (2023) 13:10753 \| www.nature.com/scientificreports/ E[eζtV2(x)] ≤V2(x(0)) + κ1(eζt −1) for t large enough. Further, we can obtain that for t large enough. Further, we can obtain that lim sup t→+∞ E 1 (x1 + x2)θ ≤lim sup t→+∞ E 1 + 1 (x1 + x2)θ ≤κ1. For any ǫ ∈(0, 1) , denote H1 = ǫθ/κθ 1 . By Chebyshev’s inequality (see23), we can obtain that P{(x1(t) + x2(t)) < H1} = P 1 (x1(t) + x2(t))θ > 1 Hθ 1 ≤Hθ 1 E 1 (x1(t) + x2(t))θ , For any ǫ ∈(0, 1) , denote H1 = ǫθ/κθ 1 . By Chebyshev’s inequality (see23), we can obtain that P{(x1(t) + x2(t)) < H1} = P 1 (x1(t) + x2(t))θ > 1 Hθ 1 ≤Hθ 1 E 1 (x1(t) + x2(t))θ , thus lim sup t→+∞ P{(x1(t) + x2(t)) < H1} ≤ǫ, and lim inf t→+∞P{(x1(t) + x2(t)) > H1} ≥1 −ǫ. Proof From SDE (4), we obtain that (19) d(x1 + θx2) = (r1(t) + d12(t)(θ −1))x1 + (θr2(t) + d21(1 −θ))x2 −a1x2 1 −a2θx2 2 dt + σ1(t)x1dB1(t) + θσ2(t)x2dB2(t). (19) d(x1 + θx2) = (r1(t) + d12(t)(θ −1))x1 + (θr2(t) + d21(1 −θ))x2 −a1x2 1 −a2θx2 2 dt + σ1(t)x1dB1(t) + θσ2(t)x2dB2(t). (19) d(x1 + θx2) = (r1(t) + d12(t)(θ −1))x1 + (θr2(t) + d21(1 −θ))x2 −a1x2 1 −a2θx2 2 dt + σ1(t)x1dB1(t) + θσ2(t)x2dB2(t). (19) From Lemma 1, we derive that for ǫ > 0 , there exists a T1 > 0 such that for t ≥T1, (20) (r1)∗−ǫ = r1e −δcM o −ǫ ≤r1(t) ≤r1e −δcm + ǫ = r∗ 1 + ǫ, r2e −ǫ ≤r2(t) ≤r2e + ǫ, (d12)∗−ǫ = d12 1 + ρ1(cm e )2 1 + α(cm e )2 −ǫ ≤d12(t) ≤d12 1 + ρ1(cM e )2 1 + α(cM e )2 + ǫ = d∗ 12 + ǫ. (20) (i) : If r∗ 1 = r2e , we take θ = 1 , and obtain from (19) that Case (i) : If r∗ 1 = r2e , we take θ = 1 , and obtain from (19) that Case (i) : If r∗ 1 = r2e , we take θ = 1 , and obtain from (19) that (21) d(x1 + x2) = (r1(t)x1 + r2(t)x2) −a1x2 1 −a2x2 2 dt + σ1(t)x1dB1(t) + σ2(t)x2dB2(t) ≤(r∗ 1 + ǫ)(x1 + x2)dt + σ1(t)x1dB1(t) + σ2(t)x2dB2(t). (21) d(x1 + x2) = (r1(t)x1 + r2(t)x2) −a1x2 1 −a2x2 2 dt + σ1(t)x1dB1(t) + σ2(t)x2dB2(t) ≤(r∗ 1 + ǫ)(x1 + x2)dt + σ1(t)x1dB1(t) + σ2(t)x2dB2(t). (21) Applying Itˆo′s formula, we have Applying Itˆo′s formula, we have (22) d ln(x1 + x2) ≤ (r∗ 1 + ǫ) −0.5σ 2 1 (t)x2 1 + σ 2 2 (t)x2 2 (x1 + x2)2 dt + σ1(t)x1dB1(t) + σ2(t)x2dB2(t) x1 + x2 , t ≥T1. By Cauchy inequality, we can obtain that By Cauchy inequality, we can obtain that σ 2 1 (t) x2 1 (x1 + x2)2 + σ 2 2 (t) x2 2 (x1 + x2)2 1 σ 2 1 (t) + 1 σ 2 2 (t) ≥1. Further from (22), we have Further from (22), we have d ln(x1 + x2) ≤ (r∗ 1 + ǫ) −0.5σ 2 1 (t)σ 2 2 (t) σ 2 1 (t) + σ 2 2 (t) dt + σ1(t)x1dB1(t) + σ2(t)x2dB2(t) x1 + x2 , t ≥T1. Extinction. h Let ϕ(x) = ln(x1 + θx2) + r −ν(x1 + θx2) , we obtain that there must be a positive constant K such that ϕ(x) ≤K for x ∈R2 + . It follows from (16) and (17) that for all n > n1(ω), (18) et ln(x1 + θx2) ≤V(x(0)) + K(et −1) + 2en ln n. (18) If n −1 ≤t ≤n and n > n1(ω) , we have ln(x1 + θx2) t ≤V(x(0)) tet + K(et −1) tet + 2en ln n tet . etting t →+∞ , we can obtain that lim sup t→+∞ ln ln(x1(t)+θx2(t)) t ≤0, a.s. , this can also imply that l ln xi(t) i h k θ Letting t →+∞ , we can obtain that lim sup t→+∞ ln ln(x1(t)+θx2(t)) t ≤0, a.s. , this can also imply that lim sup t→+∞ ln ln xi(t) t ≤0, a.s., i = 1, 2. when we take θ = 1. Letting t →+∞ , we can obtain that lim sup t→+∞ ln ln(x1(t)+θx2(t)) t ≤0, a.s. , this can also imply that lim sup t→+∞ ln ln xi(t) t ≤0, a.s., i = 1, 2. when we take θ = 1. m sup →+∞ ln ln xi(t) t ≤0, a.s., i = 1, 2. when we take θ = 1. t→+∞ This completes the proof. □ →+∞ This completes the proof. □ t→+∞ This completes the proof. □ t + This completes the proof. □ □ Theorem 3 Let x(t) be a solution of SDE (4) with initial value x(0) ∈R2 + . If any of the following conditions is true, Theorem 3 Let x(t) be a solution of SDE (4) with initial value x(0) ∈R2 + . If any of the following conditions is true, (i): r∗ 1 = r2e and r∗ 1 + r2e < σ 2. (ii): r∗ 1 < r2e and (r∗ 1 + r2e −d∗ 12 −d21) + (r∗ 1 −r2e + d21 −d∗ 12)2 + 4d∗ 12d21 < σ 2. (iii): r∗ 1 > r2e and (r∗ 1 + r2e −d21 −(d12)∗) + (r∗ 1 −r2e + d21 −(d12)∗)2 + 4(d12)∗d21 < σ 2 . Then the single-species population goes to die out, that is, lim t→+∞xi(t) = 0, a.s. Extinction. h The quadratic variation of M(t) is M(t), M(t)t = t 0 e2s σ 2 1 (t)x2 1(s)+σ 2 2 (t)θ2x2 2(s) (x1(s)+θx2(s))2 ds . According to the exponential martingale inequality, for all posi- tive constants ε, β and T0 , we can obtain that M(t), M(t)t = t 0 e2s σ 2 1 (t)x2 1(s)+σ 2 2 (t)θ2x2 2(s) (x1(s)+θx2(s))2 ds . According to the exponential martingale inequality, for all p tive constants ε, β and T0 , we can obtain that P sup 0≤t≤T0 [M(t) −0.5εM(t), M(t)t] > β ≤e−εβ, and choose ε = e−n , β = 2en ln n and T0 = n , then P sup 0≤t≤n [M(t) −0.5e−nM(t), M(t)t] > 2en ln n ≤n−2. and choose ε = e−n , β = 2en ln n and T0 = n , then P sup 0≤t≤n [M(t) −0.5e−nM(t), M(t)t] > 2en ln n ≤n−2. https://doi.org/10.1038/s41598-023-37861-z https://doi.org/10.1038/s41598-023-37861-z Scientific Reports \| (2023) 13:10753 \| www.nature.com/scientificreports/ Because the series +∞ n=1 n−2 < ∞ , by Borel-Cantalli lemma, we obtain that there is a 0 ∈ with P(0) = 1 such that for e er ω ∈ a positive integer n n (ω)can be found that Because the series +∞ n=1 n−2 < ∞ , by Borel-Cantalli lemma, we obtain that there is a 0 ∈ with P(0) = 1 such h f b f d h hat for every ω ∈0 , a positive integer n1 = n1(ω) can be found that that for every ω ∈0 , a positive integer n1 = n1(ω) can be found that (17) M(t) ≤0.5e−nM(t), M(t)t + 2en ln n (17) or 0 ≤t ≤n and n ≥n1(ω) . Let ϕ(x) = ln(x1 + θx2) + r −ν(x1 + θx2) , we obtain that there must be a positive onstant K such that ϕ(x) ≤K for x ∈R2 + . It follows from (16) and (17) that for all n > n1(ω), for 0 ≤t ≤n and n ≥n1(ω) . Let ϕ(x) = ln(x1 + θx2) + r −ν(x1 + θx2) , we obtain that there must be a positive constant K such that ϕ(x) ≤K for x ∈R2 + . It follows from (16) and (17) that for all n > n1(ω), for 0 ≤t ≤n and n ≥n1(ω) . Proof From SDE (4), we obtain that And t T1 ξ2 1 ξ2 2 4θ1θ2 (1 −e−2θ1s)(1 −e−2θ2s) ξ2 1 2θ1 + ξ2 2 2θ2 ds ≤ t T1 σ 2 1 (s)σ 2 2 (s) σ 2 1 (s) + σ 2 2 (s)ds ≤ t T1 ξ2 1 ξ2 2 4θ1θ2 ξ2 1 2θ1 (1 −e−2θ1s) + ξ2 2 2θ2 (1 −e−2θ2s) ds, thus, lim t→+∞t−1 t T1 σ 2 1 (s)σ 2 2 (s) σ 2 1 (s)+σ 2 2 (s)ds = ξ2 1 ξ2 2 2θ1ξ2 2 +2θ2ξ2 1 = σ 2.fi 1 2 1 2 thus, lim t→+∞t−1 t T1 σ 2 1 (s)σ 2 2 (s) σ 2 1 (s)+σ 2 2 (s)ds = ξ2 1 ξ2 2 2θ1ξ2 2 +2θ2ξ2 1 = σ 2.fi thus, lim t→+∞t−1 t T1 σ 2 1 (s)σ 2 2 (s) σ 2 1 (s)+σ 2 2 (s)ds = ξ2 1 ξ2 2 2θ1ξ2 2 +2θ2ξ2 1 = σ 2.fi + 1 2 2 1 If r∗ 1 + r2e −σ 2 < 0 , we can take a sufficiently small ǫ ∈(0, 1) such that r∗ 1 + r2e + 2ǫ −σ 2 < 0 , from (23), we have lim t→+∞(x1(t) + x2(t)) = 0, a.s , that is, lim t→+∞xi(t) = 0, a.s., i = 1, 2. + 1 2 2 1 If r∗ 1 + r2e −σ 2 < 0 , we can take a sufficiently small ǫ ∈(0, 1) such that r∗ 1 + r2e + 2ǫ −σ 2 < 0 , from (23), we have lim t→+∞(x1(t) + x2(t)) = 0, a.s , that is, lim t→+∞xi(t) = 0, a.s., i = 1, 2. t→+∞ t→+∞ Case(ii) : If r∗ 1 < r2e , we take a θ1 > 1 , from (19), we have t→+∞ t→+∞ Case(ii) : If r∗ 1 < r2e , we take a θ1 > 1 , from (19), we have (24) d(x1 + θ1x2) ≤[((r∗ 1 + ǫ) + (d∗ 12 + ǫ)(θ1 −1))x1 + (θ1(r2e + ǫ) + d21(1 −θ1))x2 −a1x2 1 −a2θ1x2 2]dt + σ1(t)x1dB1(t) + θ1σ2(t)x2dB2(t). Proof From SDE (4), we obtain that Integrating both sides of above inequality on [T1, t] and dividing by t, we can obtain that (23) ln(x1(t) + x2(t)) t ≤ln(x1(T1) + x2(T1)) t + (r∗ 1 + ǫ)(t −T1) t −1 2t t T1 σ 2 1 (s)σ 2 2 (s) σ 2 1 (s) + σ 2 2 (s)ds + M1(t) t , (23) ln(x1(t) + x2(t)) t ≤ln(x1(T1) + x2(T1)) t + (r∗ 1 + ǫ)(t −T1) t −1 2t t T1 σ 2 1 (s)σ 2 2 (s) σ 2 1 (s) + σ 2 2 (s)ds + M1(t) t , where M1(t) = t T1 σ1(s)x1(s)dB1(s)+σ2(s)x2(s)dB2(s) x1(s)+x2(s) . Let N(t) = T1 0 σ1(s)x1(s)dB1(s)+σ2(s)x2(s)dB2(s) x1(s)+x2(s) + M1(t) , then the quadratic variation of N(t) is Scientific Reports \| (2023) 13:10753 \| https://doi.org/10.1038/s41598-023-37861-z www.nature.com/scientificreports/ www.nature.com/scientificreports/ N(t), N(t)t = t 0 σ 2 1 (t)x2 1(s) + σ 2 2 (s)x2 2(s) (x1(s) + x2(s))2 ds ≤max{ ξ2 1 2θ1 , ξ2 2 2θ2 }t. According to the strong law of large number, we have lim t→+∞ N(t) t = 0 , thus, lim t→+∞ M(t) t = 0 . And According to the strong law of large number, we have lim t→+∞ N(t) t = 0 , thus, lim t→+∞ M(t) t = 0 . And According to the strong law of large number, we have lim t→+∞ N(t) t = 0 , thus, lim t→+∞ M(t) t = 0 . Proof From SDE (4), we obtain that (24) 1) be the solution of the following equations Let (θ1, 1) be the solution of the following equations (r∗ 1 + ǫ) + (θ1 −1)(d∗ 12 + ǫ) = 1, θ1(r2e + ǫ) + (1 −θ1)d21 = θ11, and and (25)        θ1 −1 = 1 −(r∗ 1 + ǫ) d∗ 12 + ǫ > 0, 1 −θ1 = θ1 1 −(r2e + ǫ) d21 < 0, (25) which implies that r∗ 1 + ǫ < 1 < r2e + ǫ . Denote p = 1 −(r∗ 1 + ǫ) > 0 , q = r∗ 1 −r2e < 0 . From (25), we have (26) f (p) = p2 + (q + d∗ 12 + d21 + ǫ)p + (d∗ 12 + ǫ)q = 0, which implies that r∗ 1 + ǫ < 1 < r2e + ǫ . Denote p = 1 −(r∗ 1 + ǫ) > 0 , q = r∗ 1 −r2e < 0 . From (25), we have (26) f (p) = p2 + (q + d∗ 12 + d21 + ǫ)p + (d∗ 12 + ǫ)q = 0, which implies that r∗ 1 + ǫ < 1 < r2e + ǫ . Denote p = 1 −(r∗ 1 + ǫ) > 0 , q = r∗ 1 −r2e < 0 . From (25), we have (26) f (p) = p2 + (q + d∗ 12 + d21 + ǫ)p + (d∗ 12 + ǫ)q = 0, (26) it is easy to calculate that the quadratic equation (26) has two real roots: it is easy to calculate that the quadratic equation (26) has two real roots: p1 = −(q + d∗ 12 + d21 + ǫ) + (q + d21 −d∗ 12 −ǫ)2 + 4d21(d∗ 12 + ǫ) 2 > 0, p2 = −(q + d∗ 12 + d21 + ǫ) − (q + d21 −d∗ 12 −ǫ)2 + 4d21(d∗ 12 + ǫ) 2 < 0. And because f (−q) = −d21q > 0 , it is easy to see that 0 < p1 < −q , further,    θ1 = p1 d∗ 12 + ǫ + 1 > 1, 1 = p1 + (r∗ 1 + ǫ) < r2e + ǫ. From (24), we obtain that From (24), we obtain that (27) d(x1 + θ1x2) ≤1(x1 + θ1x2)dt + σ1(t)x1dB1(t) + θ1σ2(t)x2dB2(t). Proof From SDE (4), we obtain that There exist two real roots to quadratic equation g(p) = 0, moreover, p4 < −q < p3 < 0 , thus, (30) g(p) = p2 + (q + (d12)∗+ d21 −ǫ)p + ((d12)∗−ǫ)q = 0, p3 = −(q + d21 + (d12)∗−ǫ) + (q + d21 −(d12)∗+ ǫ)2 + 4((d12)∗−ǫ)d21 2 < 0, p4 = −(q + d21 + (d12)∗−ǫ) − (q + d21 −(d12)∗+ ǫ)2 + 4((d12)∗−ǫ)d21 2 < 0,  θ2 = p3 + 1 < 1 p 1 ( ) where p = 2 −(r∗ 1 + ǫ) < 0 , q = r∗ 1 −r2e > 0 . There exist two real roots to quadratic equation g(p) = 0, (30) g(p) = p2 + (q + (d12)∗+ d21 −ǫ)p + ((d12)∗−ǫ)q = 0, p3 = −(q + d21 + (d12)∗−ǫ) + (q + d21 −(d12)∗+ ǫ)2 + 4((d12)∗−ǫ)d21 2 < 0, 2 (30) g(p) = p2 + (q + (d12)∗+ d21 −ǫ)p + ((d12)∗−ǫ)q = 0, (30) e p = 2 −(r∗ 1 + ǫ) < 0 , q = r∗ 1 −r2e > 0 . There exist two real roots to quadratic equation g(p) = 0, where p = 2 −(r∗ 1 + ǫ) < 0 , q = r∗ 1 −r2e > 0 . There exist two real roots to quadratic equation g(p) = 0, moreover, p4 < −q < p3 < 0 , thus, p3 = −(q + d21 + (d12)∗−ǫ) + (q + d21 −(d12)∗+ ǫ)2 + 4((d12)∗−ǫ)d21 2 < 0, p4 = −(q + d21 + (d12)∗−ǫ) − (q + d21 −(d12)∗+ ǫ)2 + 4((d12)∗−ǫ)d21 2 < 0, p3 = −(q + d21 + (d12)∗−ǫ) + (q + d21 −(d12)∗+ ǫ)2 + 4((d12)∗−ǫ)d21 2 < 0, p4 = −(q + d21 + (d12)∗−ǫ) − (q + d21 −(d12)∗+ ǫ)2 + 4((d12)∗−ǫ)d21 2 < 0, moreover, p4 < −q < p3 < 0 , thus,    θ2 = p3 (d12)∗−ǫ + 1 < 1, 2 = p3 + (r∗ 1 + ǫ) > r2e + ǫ.    θ2 = p3 (d12)∗−ǫ + 1 < 1, 2 = p3 + (r∗ 1 + ǫ) > r2e + ǫ. Similar to the proof of Case (ii), we have lim t→+∞xi(t) = 0, a.s. (i = 1, 2) if Similar to the proof of Case (ii), we have lim t→+∞xi(t) = 0, a.s. Proof From SDE (4), we obtain that (27) d(x1 + θ1x2) ≤1(x1 + θ1x2)dt + σ1(t)x1dB1(t) + θ1σ2(t)x2dB2(t). (27) If (r∗ 1 + r2e −d∗ 12 −d21) + (r∗ 1 −r2e + d21 −d∗ 12)2 + 4d∗ 12(r2e −r∗ 1) < σ 2 , we choose an ǫ small enough such that 1 < 0.5σ 2 , from (27), we also conclude that lim t→+∞xi(t) = 0, a.s., i = 1, 2. Case (iii): If r∗ 1 > r2e , we select a 0 < θ2 < 1 , from (19), we have ∗ If (r∗ 1 + r2e −d∗ 12 −d21) + (r∗ 1 −r2e + d21 −d∗ 12)2 + 4d∗ 12(r2e −r∗ 1) < σ 2 , we choose an ǫ small enough such that 1 < 0.5σ 2 , from (27), we also conclude that lim t→+∞xi(t) = 0, a.s., i = 1, 2. t→+∞ Case (iii): If r∗ 1 > r2e , we select a 0 < θ2 < 1 , from (19), we have (28) d(x1 + θ2x2) ≤[((r∗ 1 + ǫ) + ((d12)∗−ǫ)(θ2 −1))x1 + (θ2(r2e + ǫ) + d21(1 −θ2))x2 −a1x2 1 −a2θ2x2 2]dt + σ1(t)x1dB1(t) + θ2σ2(t)x2dB2(t). t→+∞ Case (iii): If r∗ 1 > r2e , we select a 0 < θ2 < 1 , from (19), we have (28) d(x1 + θ2x2) ≤[((r∗ 1 + ǫ) + ((d12)∗−ǫ)(θ2 −1))x1 + (θ2(r2e + ǫ) + d21(1 −θ2))x2 −a1x2 1 −a2θ2x2 2]dt + σ1(t)x1dB1(t) + θ2σ2(t)x2dB2(t). (28) Let (θ2, 2) be the solution of the following equations et (θ2, 2) be the solution of the following equations (r∗ 1 + ǫ) + (θ2 −1)((d12)∗−ǫ) = 2, θ2(r2e + ǫ) + (1 −θ2)d21 = θ22, and https://doi.org/10.1038/s41598-023-37861-z Scientific Reports \| (2023) 13:10753 \| www.nature.com/scientificreports/ (29)        θ2 −1 = 2 −(r∗ 1 + ǫ) (d12)∗−ǫ < 0, 1 −θ2 = θ2 2 −(r2e + ǫ) d21 > 0, (29) this implies that r2e + ǫ < 2 < r∗ 1 + ǫ . It follows from (29) that this implies that r2e + ǫ < 2 < r∗ 1 + ǫ . It follows from (29) that where p = 2 −(r∗ 1 + ǫ) < 0 , q = r∗ 1 −r2e > 0 . Proof From SDE (4), we obtain that (i = 1, 2) if (r∗ 1 + r2e −d21 −(d12)∗) + (r∗ 1 −r2e + d21 −(d12)∗)2 + 4(d12)∗d21 < σ 2. □ This proof is completed. Remark 3 From the proof of Theorem 3’(ii), we know that species goes to extinction when r∗ 1 < 0 and d∗ 12r2e + r∗ 1d21 −r∗ 1r2e < 0 , which is independent of the intensity of the noise. Remark 4 If ρ = 0 , that is, without considering the influence of environmental toxicant concentra- tion on population migration. From Theorem 3, we obtain that single-species population will be extinct if (r∗ 1 + r2e −d21 −d12) + (r∗ 1 −r2e + d21 −d12)2 + 4d12d21 < σ 2. Permanence in the mean. In this subsection, we aim to analyze the permanence in the mean of SDE (4). Permanence in the mean. In this subsection, we aim to analyze the permanence in the mean of SDE (4). Theorem 4 Let (x1(t), x2(t)) be the solution of SDE (4) with initial value x(0) ∈R2 +. If any of the following condi- tions is true, Theorem 4 Let (x1(t), x2(t)) be the solution of SDE (4) with initial value x(0) ∈R2 +. If any of the following condi- tions is true, (i): (r1)∗= r2e and (r1)∗+ r2e > ˆσ 2. (ii): (r1)∗< r2e and (r1)∗+ r2e −(d12)∗−d21 + ((r1)∗−r2e + d21 −(d12)∗)2 + 4(d12)∗d21 > ˆσ 2. (i): (r1)∗= r2e and (r1)∗+ r2e > ˆσ 2. (ii): (r1)∗< r2e and (r1)∗+ r2e −(d12)∗−d21 + ((r1)∗−r2e + d21 −(d12)∗)2 + 4(d12)∗d21 > ˆσ 2. (iii): (r1)∗> r2e and (r1)∗+ r2e −d∗ 12 −d21 + ((r1)∗−r2e + d21 −d∗ 12)2 + 4d∗ 12d21 > ˆσ 2 . Then the single- species population is strongly persistent in the mean. (iii): (r1)∗> r2e and (r1)∗+ r2e −d∗ 12 −d21 + ((r1)∗−r2e + d21 −d∗ 12)2 + 4d∗ 12d21 > ˆσ 2 . Then the single- species population is strongly persistent in the mean. (iii): (r1)∗> r2e and (r1)∗+ r2e −d∗ 12 −d21 + ((r1)∗−r2e + d21 −d∗ 12)2 + 4d∗ 12d21 > ˆσ 2 . Then the sin species population is strongly persistent in the mean. Proof Using the same proof method as Theorem 3. Let ǫ ∈(0, 1) be small enough, and r2e −ǫ > 0 , (d21)∗−ǫ > 0 roof Using the same proof method as Theorem 3. Proof From SDE (4), we obtain that Let ǫ ∈(0, 1) be small enough, and r2e −ǫ > 0 , (d21)∗−ǫ > 0 Case (i) : If (r1)∗= r2e , we take θ = 1 , and obtain from (19) that (31) d(x1 + x2) = (r1(t)x1 + r2(t)x2) −a1x2 1 −a2x2 2 dt + σ1(t)x1dB1(t) + σ2(t)x2dB2(t) ≥((r2e −ǫ)(x1 + x2) −a(x1 + x2)2)dt + σ1(t)x1dB1(t) + σ2(t)x2dB2(t). (31) d(x1 + x2) = (r1(t)x1 + r2(t)x2) −a1x2 1 −a2x2 2 dt + σ1(t)x1dB1(t) + σ2(t)x2dB2(t) ≥((r2e −ǫ)(x1 + x2) −a(x1 + x2)2)dt + σ1(t)x1dB1(t) + σ2(t)x2dB2(t). (31) Applying Itˆo′s formula, we have Applying Itˆo′s formula, we have d ln(x1 + x2) ≥((r2e −ǫ) −0.5ˆσ 2 −a(x1 + x2))dt + σ1(t)x1dB1(t) + σ2(t)x2dB2(t) x1 + x2 , t ≥T1, where ˆσ 2 = max{ ξ1 2θ1 , ξ2 2θ2 } and a = max{a1,a2} 2 . And, ˆfi ˆ (32) ax1 + x2 ≥−ln(x1(t) + x2(t)) t + ln(x1(T1) + x2(T1)) t + ((r2e −ǫ) −0.5ˆσ 2)(t −T1) t + M(t) t . If (r1)∗+ r2e > ˆσ , we select a sufficiently small ǫ such that (r1)∗+ r2e −2ǫ > ˆσ , from Lemma 4 and (32), we obtain that (32) ax1 + x2 ≥−ln(x1(t) + x2(t)) t + ln(x1(T1) + x2(T1)) t + ((r2e −ǫ) −0.5ˆσ 2)(t −T1) t + M(t) t . If (r1)∗+ r2e > ˆσ , we select a sufficiently small ǫ such that (r1)∗+ r2e −2ǫ > ˆσ , from Lemma 4 and (32), we obtain that t t t t If (r1)∗+ r2e > ˆσ , we select a sufficiently small ǫ such that (r1)∗+ r2e −2ǫ > ˆσ , from Lemma 4 and (32), we obtain that https://doi.org/10.1038/s41598-023-37861-z https://doi.org/10.1038/s41598-023-37861-z Scientific Reports \| (2023) 13:10753 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ x1(t) + x2(t)∗≥(r2e −ǫ) −0.5ˆσ 2 a > 0. x1(t) + x2(t)∗≥(r2e −ǫ) −0.5ˆσ 2 a > 0. x1(t) + x2(t)∗≥(r2e −ǫ) −0.5ˆσ 2 a > 0. Case(ii) : If (r1)∗< r2e , we take a θ3 > 1 , from (19), we have (33) d(x1 + θ3x2) ≥[(((r1)∗−ǫ) + ((d12)∗−ǫ)(θ3 −1))x1 + (θ3(r2e −ǫ) + d21(1 −θ3))x2 −a1x2 1 −a2θ3x2 2]dt + σ1(t)x1dB1(t) + θ3σ2(t)x2dB2(t). Proof From SDE (4), we obtain that (33) Let (θ3, 4) be the solution of the following equations ((r1)∗−ǫ) + (θ3 −1)((d12)∗−ǫ) = 3, θ3(r2e −ǫ) + (1 −θ3)d21 = θ33, further, (34)        θ3 −1 = 3 −((r1)∗−ǫ) (d12)∗−ǫ > 0, 1 −θ3 = θ3 3 −(r2e −ǫ) d21 < 0, (34) this implies that (r1)∗−ǫ < 3 < r2e −ǫ . Denote u = 3 −((r1)∗−ǫ) > 0 , v = (r1)∗−r2e < 0 . From (34), we have this implies that (r1)∗−ǫ < 3 < r2e −ǫ . Denote u = 3 −((r1)∗−ǫ) > 0 , v = (r1)∗−r2e < 0 . From (34), we have (35) h(u) = u2 + (v + (d12)∗−ǫ + d21)u + ((d12)∗−ǫ)v = 0, h(u) = u2 + (v + (d12)∗−ǫ + d21)u + ((d12)∗−ǫ)v = 0, (35) there exist two real roots to quadratic equation (35), there exist two real roots to quadratic equation (35), there exist two real roots to quadratic equation (35), u1 = −(v + d21 + (d12)∗−ǫ) + (v + d21 −(d12)∗+ ǫ)2 + 4d21((d12)∗−ǫ) 2 > 0, u2 = −(v + d21 + (d12)∗−ǫ) − (v + d21 −(d12)∗+ ǫ)2 + 4d21((d12)∗−ǫ) 2 < 0. Since h(−v) = −d21v > 0 , thus 0 < u1 < −v , further, Since h(−v) = −d21v > 0 , thus 0 < u1 < −v , further,    θ3 = u1 (d12)∗−ǫ + 1 > 1, 3 = u1 + ((r1)∗−ǫ) < r2e −ǫ. From (33), we have om (33), we have From (33), we have (36) d(x1 + θ3x2) ≥[3(x1 + θ3x2) −max{a1, a2 θ3 }(x1 + θ3x2)2]dt + σ1(t)x1dB1(t) + θ3σ2(t)x2dB2(t). When ((r1)∗+ r2e −d21 −(d12)∗) + ((r1)∗−r2e + d21 −(d12)∗)2 + 4(d12)∗d21 > ˆσ 2 , we can select an ǫ small enough such that condition 3 > 0.5ˆσ 2 holds. We conclude from (36) that (36) d(x1 + θ3x2) ≥[3(x1 + θ3x2) −max{a1, a2 θ3 }(x1 + θ3x2)2]dt + σ1(t)x1dB1(t) + θ3σ2(t)x2dB2(t). x1(t) + θ3x2(t)∗≥3 −0.5ˆσ 2 max{a1, a2 θ3 } > 0, a.s. When ((r1)∗+ r2e −d21 −(d12)∗) + ((r1)∗−r2e + d21 −(d12)∗)2 + 4(d12)∗d21 > ˆσ 2 , we can select an ǫ small enough such that condition 3 > 0.5ˆσ 2 holds. We conclude from (36) that x1(t) + θ3x2(t)∗≥3 −0.5ˆσ 2 max{a1, a2 θ3 } > 0, a.s. Proof From SDE (4), we obtain that x1(t) + θ3x2(t)∗≥3 −0.5ˆσ 2 max{a1, a2 θ3 } > 0, a.s. Case (iii): If (r1)∗> r2e , the following proof is similar to Theorem 3, we omit it. The proof of Theorem 4 is competed. □ □ Remark 5 If ρ = 0 , when (r1)∗+ r2e −d12 −d21 + ((r1)∗−r2e + d12 + d21)2 + 4d12(r2e −(r1)∗) > ˆσ 2, Remark 5 If ρ = 0 , when Remark 5 If ρ = 0 , when the population x is strongly permanent in the mean. (r1)∗+ r2e −d12 −d21 + ((r1)∗−r2e + d12 + d21)2 + 4d12(r2e −(r1)∗) > ˆσ 2, the population x is strongly permanent in the mean. the population x is strongly permanent in the mean. Theorem 5 If r1e − δfb h(g+m)γ −d12(1 + ρη) > 0 and r2e −d21 > 0, we have x1(t)∗≥ r1e − δfb h(g+m)γ −d12(1 + ρη) a1 , x2(t)∗≥r2e −d21 a2 , a.s. Theorem 5 If r1e − δfb h(g+m)γ −d12(1 + ρη) > 0 and r2e −d21 > 0, we have 5 If r1e h(g+m)γ d12(1 + ρη) > 0 and r2e d21 > 0, we have x1(t)∗≥ r1e − δfb h(g+m)γ −d12(1 + ρη) a1 , x2(t)∗≥r2e −d21 a2 , a.s. a1 Proof From (4), we have Proof From (4), we have dx1 ≥x1 r1e + (r10 −r1e)e−µ1t −δco(t) −d12 1 + ρc2 e(t) 1 + αc2e(t) −a1x1 dt + σ1(t)x1dB1(t), dx2 ≥x2[r2e + (r20 −r2e)e−µ2t −d21 −a2x2]dt + σ2(t)x2dB2(t). https://doi.org/10.1038/s41598-023-37861-z Scientific Reports \| (2023) 13:10753 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ (37) ln x1(t) −ln x1(0) t ≥t−1 t 0 r1(s) −d12(s)ds −a1t−1 t 0 x1(s)ds + t−1 t 0 σ1(s)dB1(s), (38) ln x2(t) −ln x2(0) t ≥t−1 t 0 r2(s) −d21ds −a2t−1 t 0 x2(s)ds + t−1 t 0 σ2(s)dB2(s). (37) (38) According to Lemma 4, (37) and (38), we obtain that According to Lemma 4, (37) and (38), we obtain that According to Lemma 4, (37) and (38), we obtain that x1(t)∗≥ r1e − δfb h(g+m)γ −d12(1 + ρη) a1 , x2(t)∗≥r2e −d21 a2 , a.s. □ This ends the proof of Theorem 5. This ends the proof of Theorem 5. This ends the proof of Theorem 5. □ Stochastic single‑species population model for migration between two non‑polluted patches. Numerical simulation and discussions In this section, we give some numerical simulations to demonstrate the analytical results for the SDE model (3) presented in the previous sections by applying the positive preserving truncated Euler-Maruyama method (PPTEMM) given in32,33. g We give some parameters as: (40) r1e = 0.3, r10 = 0.6, δ = 0.8, a1 = 0.1, r2e = 0.15, r20 = 0.3, a2 = 0.5, d12 = 0.5, d21 = 0.6, α = 0.2, h = 0.3, ρ = 1.2, f = 0.5, g = 0.3, m = 0.2, (40) and initial value (x1(0), x2(0), co(0), ce(0)) = (0.8, 0.5, 0.2, 0.6) . And then we take different values of ξi , µi , γ and b to show the influence of the intensity of white noise ξi , the speed of reversion µi , the pulse input cycle of toxi- cant γ and the toxicant input amount each time b on the dynamics of the SDE model (3). We first take µ1 = 0.1 , µ2 = 0.1 , γ = 1 , b = 0.1 . If we choose ξ1 = 0.4 and ξ2 = 0.4 , by calculation, we have r∗ 1 −r2e < −0.1132 < 0 and (r∗ 1 +r2e −d21 −(d12)∗)+ (r∗ 1 −r2e + d21 −(d12)∗)2 + 4(d12)∗d21 −σ 2 < −0.2091 < 0 , which satisfy condition (ii) in Theorem 3. From Theorem 3, we can obtain that species will be extinct as shown in Fig. 1a. If we choose ξ1 = 0.1 , ξ2 = 0.1 , after calculating, we obtain that (r1)∗−r2e < 0 and (r1)∗+ r2e −(d12)∗−d∗ 21 + ((r1)∗−r2e −(d12)∗+ d21)2 + 4(d12)∗d21 −ˆσ 2 > 0.1323 > 0, it implies by Theorem 4 that system (3) is strongly permanent in the mean, see Fig. 1b. We can easily find that higher intensity of white noise ξi may lead to the extinction of species by comparing Fig. 1a,b. In the following, we will show the influence of speed of reversion µi (i = 1, 2) on the population dynam- ics of SDE model (3). We take µ1 = 0.01 , ξ1 = 0.1 , µ2 = 0.01 , ξ2 = 0.1 , γ = 1 , b = 0.1 , and derive that (r∗ 1 +r2e −d21 −(d12)∗)+ (r∗ 1 −r2e + d21 −(d12)∗)2 + 4(d12)∗d21 −σ 2 < −0.0591 < 0 . Proof From SDE (4), we obtain that If there is no polluted patch, the model (3) will degenerate into the following stochastic single- species population migration model. (39) dx1(t) = x1(t)(r1e + (r10 −r1e)e−µ1t −a1x1(t)) + d21x2(t) −d12x1(t) dt + σ1(t)x1(t)dB1(t), dx2(t) = x2(t)(r2e + (r20 −r2e)e−µ2t −a2x2(t)) + d12x1(t) −d21x2(t) dt + σ2(t)x2(t)dB2(t). (39) From Theorems 3 and 4, we can also get the following results for system (39). From Theorems 3 and 4, we can also get the following results for system (39). Corollary 3.1 (i) Species in system (39) will be extinct if (ii) Species in system (39) is permanent in the mean if r1e + r2e −d12 −d21 + (r1e −r2e + d21 −d12)2 + 4d12d21 < σ 2. r1e + r2e −d12 −d21 + (r1e −r2e + d21 −d12)2 + 4d12d21 > ˆσ 2. Corollary 3.1 (i) Species in system (39) will be extinct if p y f (ii) Species in system (39) is permanent in the mean if r1e + r2e −d12 −d21 + (r1e −r2e + d21 −d12)2 + 4d12d21 < σ 2. d d d d 2 d d ˆ2 ystem (39) is permanent in the mean if r1e + r2e −d12 −d21 + (r1e −r2e + d21 −d12)2 + 4d12d21 < σ 2. r1e + r2e −d12 −d21 + (r1e −r2e + d21 −d12)2 + 4d12d21 < σ 2. r1e + r2e −d12 −d21 + (r1e −r2e + d21 −d12)2 + 4d12d21 > ˆσ 2. Numerical simulation and discussions (a): µ1 = 0.1 , ξ1 = 0.4 , µ2 = 0.1 , ξ2 = 0.4 , γ = 1 , b = 0.1 ; (b): µ1 = 0.1 , ξ1 = 0.1 , µ2 = 0.1 , ξ2 = 0.1 , γ = 1 , b = 0.1 ; (c): µ1 = 0.01 , ξ1 = 0.1 , µ2 = 0.01 , ξ2 = 0.1 , γ = 1 , b = 0.1 ; (d): µ1 = 0.1 , ξ1 = 0.1 , µ2 = 0.1 , ξ2 = 0.1 , γ = 0.8 , b = 0.2. 100 150 200 250 time x1(t) x2(t) a 0 50 100 150 200 250 300 350 400 time 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Population size x1(t) x2(t) b 0 100 200 300 400 500 600 700 time 0 0.2 0.4 0.6 0.8 1 1.2 Population size x1(t) x2(t) c Figure 2. Time series of SDE model (3) with parameters given in (41) for different migration rate. (a) d12 = d21 = 0 ; (b) d12 = 0.4 , d21 = 0.2 , ρ = 1.2 ; (c) d12 = 0.2 , d21 = 0.8 , ρ = 0.4. www.nature.com/scientificreports/ 0 10 20 30 40 50 60 70 80 90 100 time 0 0.5 1 1.5 2 2.5 3 Population size x1(t) x2(t) a 0 100 200 300 400 500 600 time 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Population size x1(t) x2(t) b 0 100 200 300 400 500 600 time 0 0.5 1 1.5 2 2.5 3 3.5 Population size x1(t) x2(t) c 0 5 1 0 0 1 0 5 0 time 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Population size x1(t) x2(t) d Figure 1. Time series of SDE model (3) with parameters given in (40) for different µi, ξi, γ , b . Numerical simulation and discussions (a): µ1 = 0.1 , ξ1 = 0.4 , µ2 = 0.1 , ξ2 = 0.4 , γ = 1 , b = 0.1 ; (b): µ1 = 0.1 , ξ1 = 0.1 , µ2 = 0.1 , ξ2 = 0.1 , γ = 1 , b = 0.1 ; (c): µ1 = 0.01 , ξ1 = 0.1 , µ2 = 0.01 , ξ2 = 0.1 , γ = 1 , b = 0.1 ; (d): µ1 = 0.1 , ξ1 = 0.1 , µ2 = 0.1 , ξ2 = 0.1 , γ = 0.8 , b = 0.2. 0 100 200 300 400 500 600 time 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Population size x1(t) x2(t) b 0 10 20 30 40 50 60 70 80 90 100 time 0 0.5 1 1.5 2 2.5 3 Population size x1(t) x2(t) a Population size 0 100 200 300 400 500 600 time 0 0.5 1 1.5 2 2.5 3 3.5 Population size x1(t) x2(t) c d c 0 5 1 0 0 1 0 5 0 time 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Population size x1(t) x2(t) d Population size Figure 1. Time series of SDE model (3) with parameters given in (40) for different µi, ξi, γ , b . (a): µ1 = 0.1 , ξ1 = 0.4 , µ2 = 0.1 , ξ2 = 0.4 , γ = 1 , b = 0.1 ; (b): µ1 = 0.1 , ξ1 = 0.1 , µ2 = 0.1 , ξ2 = 0.1 , γ = 1 , b = 0.1 ; (c): µ1 = 0.01 , ξ1 = 0.1 , µ2 = 0.01 , ξ2 = 0.1 , γ = 1 , b = 0.1 ; (d): µ1 = 0.1 , ξ1 = 0.1 , µ2 = 0.1 , ξ2 = 0.1 , γ = 0.8 , b = 0.2. 0 50 100 150 200 250 time 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Population size x1(t) x2(t) a 0 50 100 150 200 250 300 350 400 time 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Population size x1(t) x2(t) b 0 100 200 300 400 500 600 700 time 0 0.2 0.4 0.6 0.8 1 1.2 Population size x1(t) x2(t) c Figure 2. Numerical simulation and discussions It follows from Theo- rem 3 that the single-species dies out (see Fig. 1c). From Fig. 1b,c, we can find that a small speed of reversion µi can give rise to extinction of species. Finally, we will show the influence of γ and b on species survival, and take µ1 = 0.1 , ξ1 = 0.1 , µ2 = 0.1 , ξ2 = 0.1 , γ = 0.8 , b = 0.2 , simple calculation obtain that r∗ 1 −r2e = −0.5112 < 0 and (r∗ 1 +r2e −d21 −(d12)∗)+ (r∗ 1 −r2e + d21 −(d12)∗)2 + 4(d12)∗d21 −σ 2 = −0.0482 < 0 , which satisfy the condition (ii) of the Theorem 3, hence, population xi goes to extinction (see Fig. 1d). Comparing Fig. 1b,d, we can observe that species may tend to survive when increasing the toxins input period γ or decreasing the toxins input amount b.f the condition (ii) of the Theorem 3, hence, population xi goes to extinction (see Fig. 1d). Comparing Fig. 1b,d, we can observe that species may tend to survive when increasing the toxins input period γ or decreasing the toxins input amount b.f p On the other hand, population migration both patches will also affect the survival of the single-species. We choose parameters as p On the other hand, population migration both patches will also affect the survival of the single-species. We choose parameters as Scientific Reports \| (2023) 13:10753 \| https://doi.org/10.1038/s41598-023-37861-z /scientificreports/ 0 10 20 30 40 50 60 70 80 90 100 time 0 0.5 1 1.5 2 2.5 3 Population size x1(t) x2(t) a 0 100 200 300 400 500 600 time 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Population size x1(t) x2(t) b 0 100 200 300 400 500 600 time 0 0.5 1 1.5 2 2.5 3 3.5 Population size x1(t) x2(t) c 0 5 1 0 0 1 0 5 0 time 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Population size x1(t) x2(t) d Figure 1. Time series of SDE model (3) with parameters given in (40) for different µi, ξi, γ , b . Numerical simulation and discussions Time series of SDE model (3) with parameters given in (41) for different migration rate. (a) d12 = d21 = 0 ; (b) d12 = 0.4 , d21 = 0.2 , ρ = 1.2 ; (c) d12 = 0.2 , d21 = 0.8 , ρ = 0.4. 0 50 100 150 200 250 300 350 400 time 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Population size x1(t) x2(t) b 0 100 200 300 400 500 600 700 time 0 0.2 0.4 0.6 0.8 1 1.2 Population size x1(t) x2(t) c 0 50 100 150 200 250 time 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Population size x1(t) x2(t) a a Population size Population size Figure 2. Time series of SDE model (3) with parameters given in (41) for different migration rate. (a) d12 = d21 = 0 ; (b) d12 = 0.4 , d21 = 0.2 , ρ = 1.2 ; (c) d12 = 0.2 , d21 = 0.8 , ρ = 0.4. (41) r1e = 0.25, r10 = 0.4, δ = 0.8, a1 = 0.1, r2e = 0.2, r20 = 0.1, a2 = 0.6, α = 0.1, h = 0.3, γ = 1, b = 0.12, ξ1 = 0.1, µ1 = 0.2, ξ2 = 0.1, µ2 = 0.2, (41) and initial value (x1(0), x2(0), co(0), ce(0)) = (0.8, 0.5, 0.2, 0.6) . If d12 = d21 = 0 , that is, there is no mutual migra- tion between populations of two patches. According to the theoretical results of Ref.21, we know that population xi is permanent in the mean if ri(t) −0.5σi(t) > 0 , and population xi goes to extinction if ri(t) −0.5σi(t) < 0 . and initial value (x1(0), x2(0), co(0), ce(0)) = (0.8, 0.5, 0.2, 0.6) . If d12 = d21 = 0 , that is, there is no mutual migra- tion between populations of two patches. According to the theoretical results of Ref.21, we know that population xi is permanent in the mean if ri(t) −0.5σi(t) > 0 , and population xi goes to extinction if ri(t) −0.5σi(t) < 0 . After calculation, we have r1(t) −0.5σ1(t) = r1e −δb hγ −1 2 ξ2 1 2µ1 = −0.0825 < 0 and r2(t) −0.5σ2(t) = and initial value (x1(0), x2(0), co(0), ce(0)) = (0.8, 0.5, 0.2, 0.6) . Numerical simulation and discussions If d12 = d21 = 0 , that is, there is no mutual migra- tion between populations of two patches. According to the theoretical results of Ref.21, we know that population xi is permanent in the mean if ri(t) −0.5σi(t) > 0 , and population xi goes to extinction if ri(t) −0.5σi(t) < 0 . After calculation, we have r1(t) −0.5σ1(t) = r1e −δb hγ −1 2 ξ2 1 2µ1 = −0.0825 < 0 and r2(t) −0.5σ2(t) = r2e −1 2 ξ2 2 2µ2 = 0.1875 > 0 , population x1 in patch 1 will be extinct and population x2 in patch 2 is strongly per- sistent in the mean, see Fig. 2a. Moreover, when we take d12 = 0.4 , d21 = 0.2 , ρ = 1.2 , and calculate that (r1)∗−r2e = −0.2722 < 0 and (r1)∗+ r2e −(d12)∗−d21 + ((r1)∗−r2e −(d12)∗+ d21)2 + 4(d12)∗d21 After calculation, we have r1(t) −0.5σ1(t) = r1e −δb hγ −1 2 ξ2 1 2µ1 = −0.0825 < 0 and r2(t) −0.5σ2(t) = r2e −1 2 ξ2 2 2µ2 = 0.1875 > 0 , population x1 in patch 1 will be extinct and population x2 in patch 2 is strongly per- sistent in the mean, see Fig. 2a. Moreover, when we take d12 = 0.4 , d21 = 0.2 , ρ = 1.2 , and calculate that (r1)∗−r2e = −0.2722 < 0 and (r1)∗+ r2e −(d12)∗−d21 + ((r1)∗−r2e −(d12)∗+ d21)2 + 4(d12)∗d21 After calculation, we have r1(t) −0.5σ1(t) = r1e −δb hγ −1 2 ξ2 1 2µ1 = −0.0825 < 0 and r2(t) −0.5σ2(t) = r2e −1 2 ξ2 2 2µ2 = 0.1875 > 0 , population x1 in patch 1 will be extinct and population x2 in patch 2 is strongly per- sistent in the mean, see Fig. 2a. Moreover, when we take d12 = 0.4 , d21 = 0.2 , ρ = 1.2 , and calculate that (r1)∗−r2e = −0.2722 < 0 and (r1)∗+ r2e −(d12)∗−d21 + ((r1)∗−r2e −(d12)∗+ d21)2 + 4(d12)∗d21 https://doi.org/10.1038/s41598-023-37861-z Scientific Reports \| (2023) 13:10753 \| www.nature.com/scientificreports/ −ˆσ 2 > 0.2492 > 0 . According to Theorem 3’s (ii), we know that species is strongly permanent in the mean, as shown in Fig. 2b. Conclusions With the rapid growth of economy, a large number of toxic substances are discharged into the ecosystem, which seriously threatens the survival of species and human beings. Based on its theoretical and practical significance, stochastic population models with impulsive toxicant input and stochastic single-species population models with migration have attracted many scholars’ attention (see, e.g.,14–31). Up to our knowledge, few studies have considered the influence of environmental toxins on population migration between patches. In this paper, we propose and study a stochastic single-species population system with migration driven by environmental toxicant and impulsive toxicant input. We prove the existence and uniqueness of the global positive solution of SDE (3) by constructing the Lyapunov function, and analyze the boundedness of the p-moments of the solution. And then, we obtain sufficient conditions for population extinction, stochastic permanence and permanence in the mean. There results show that the intensity of white noise ξi , the speed of reversion µi , the pulse input period of toxicant γ , the toxicant input amount each time b and the population migration between patches play a very important role on the survival of the population, see Figs. 1 and 2. Finally, we also study the stochastic single- species population model with migration between two non-polluted patches, and give the sufficient conditions for population extinction and permanence. p p p On the other hand, there are many interesting problems that deserve further study, for example, the existence and uniqueness of the ergodic stationary probability density for system (3) (see34,35), and many more realistic but complex models should be formulated (see36). In addition, the telegraph noise can be illustrated as a switching between two or more regimes of environment, which differ by factors such as nutrition or as rain falls37,38, which is memoryless and the waiting time for the next switch has an exponential distribution, we can use a finite-state Markov chain to simulate regime switching in here. Therefore, it is interesting to introduce the telegraph noise into model (3). We shall also consider this question in our future work. Numerical simulation and discussions However, If we take d12 = 0.2 , d21 = 0.8 , ρ = 0.4 , it can be calculated that (r1)∗−r2e = −0.2659 < 0 and (r1)∗+ r2e −(d12)∗−d21 + ((r1)∗−r2e −(d12)∗+ d21)2 + 4(d12)∗d21 −ˆσ 2 < −0.0039 < 0 by Theorem 3’s (ii), which means species goes extinct, see Fig. 2c. Data availability y All data used in this study have been given within the article. All data used in this study have been given within the article. Received: 28 April 2023; Accepted: 28 June 2023 References 1. 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Additional information Correspondence and requests for materials should be addressed to J.J. Correspondence and requests for materials should be addressed to J.J. References p y , ( ) 2. May, R. M. Stability and Complexity in Model Ecosystems (Princeton University Press, 2001). https://doi.org/10.1038/s41598-023-37861-z Scientific Reports \| (2023) 13:10753 \| 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. 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https://openalex.org/W3040810843	https://pure.mpg.de/pubman/item/item_3248453_1/component/file_3248454/Front_Immunol_2020_11_813.pdf	English	null	NK Cell Development in Times of Innate Lymphoid Cell Diversity	Frontiers in immunology	2,020	cc-by	22,555	REVIEW published: 08 July 2020 doi: 10.3389/ﬁmmu.2020.00813 Edited by: Ewa Sitnicka, Lund University, Sweden Reviewed by: Gabrielle Belz, Walter and Eliza Hall Institute of Medical Research, Australia Barbara L. Kee, University of Chicago, United States Correspondence: Andreas Diefenbach andreas.diefenbach@charite.de Christoph S. N. Klose christoph.klose@charite.de Specialty section: This article was submitted to NK and Innate Lymphoid Cell Biology, a section of the journal Frontiers in Immunology NK Cell Development in Times of Innate Lymphoid Cell Diversity Vladislava Stokic-Trtica 1,2, Andreas Diefenbach 1,3,4 and Christoph S. N. Klose 1* 1 Department of Microbiology, Infectious Diseases and Immunology, Charité–Universitätsmedizin Berlin, Berlin, Germany, 2 Max-Planck Institute for Infection Biology, Berlin, Germany, 3 Berlin Institute of Health (BIH), Berlin, Germany, 4 Mucosal and Developmental Immunology, Deutsches Rheuma-Forschungszentrum, Berlin, Germany After being described in the 1970s as cytotoxic cells that do not require MHC-dependent pre-activation, natural killer (NK) cells remained the sole member of innate lymphocytes for decades until lymphoid tissue-inducer cells in the 1990s and helper-like innate lymphoid lineages from 2008 onward completed the picture of innate lymphoid cell (ILC) diversity. Since some of the ILC members, such as ILC1s and CCR6−ILC3s, share speciﬁc markers previously used to identify NK cells, these ﬁndings provoked the question of how to delineate the development of NK cell and helper-like ILCs and how to properly identify and genetically interfere with NK cells. The description of eomesodermin (EOMES) as a lineage-specifying transcription factor of NK cells provided a candidate that may serve as a selective marker for the genetic targeting and identiﬁcation of NK cells. Unlike helper-like ILCs, NK cell activation is, to a large degree, regulated by the engagement of activating and inhibitory surface receptors. NK cell research has revealed some elegant mechanisms of immunosurveillance, coined “missing-self” and “induced-self” recognition, thus complementing “non-self recognition”, which is predominantly utilized by adaptive lymphocytes and myeloid cells. Notably, the balance of activating and inhibitory signals perceived by surface receptors can be therapeutically harnessed for anti-tumor immunity mediated by NK cells. This review aims to summarize the similarities and the differences in development, function, localization, and phenotype of NK cells and helper-like ILCs, with the purpose to highlight the unique feature of NK cell development and regulation. Keywords: NK cells, innate lymphoid cells, immune recognition, immune receptor, innate lymphocytes INTRODUCTION Frontiers in Immunology Received: 30 October 2019 Accepted: 08 April 2020 Published: 08 July 2020 Citation: Stokic-Trtica V, Diefenbach A and Klose CSN (2020) NK Cell Development in Times of Innate Lymphoid Cell Diversity. Front. Immunol. 11:813. doi: 10.3389/ﬁmmu.2020.00813 Received: 30 October 2019 Accepted: 08 April 2020 Published: 08 July 2020 In the mid-70s of the last century, two groups independently reported the presence of small lymphocytes with non-MHC-restricted cytolytic activity against cells expressing tumor antigens in mice (1–4). Such “natural” killer (NK) cells, capable of cell-mediated, rapid cytotoxicity in a germline-encoded receptor-dependent fashion upon encountering of target cells, were observed in humans as well (5). NK cells remained the only subset of innate lymphocytes for two decades until an additional subset was discovered, which expressed the integrin α4β7, lymphotoxin (LT)α1β2, and lymphoid cytokine receptors. However, this newly described cell subset was giving rise to neither T-lymphocytes nor B-lymphocytes. Citation: Stokic-Trtica V, Diefenbach A and Klose CSN (2020) NK Cell Development in Times of Innate Lymphoid Cell Diversity. Front. Immunol. 11:813. doi: 10.3389/ﬁmmu.2020.00813 July 2020 \| Volume 11 \| Article 813 Frontiers in Immunology \| www.frontiersin.org NK and ILC Development Stokic-Trtica et al. B-cell lymphoma/leukemia 11B (BCL11B) for development and produce type 2 cytokines, mostly IL-5, IL-9, and IL-13, as well as other eﬀector molecules, such as amphiregulin, promoting worm expulsion and tissue remodeling (12–14, 17, 37–42). Group 3 ILCs include fetal LTi cells and can be further divided into two groups in adult mice based on CCR6 expression with diﬀerent developmental requirements and eﬀector mechanisms (43, 44). Both CCR6+ ILC3s and CCR6−ILC3s are dependent on the TF RORγt and produce IL-22 to fortify the epithelial barrier against infections, damage, and genotoxic stress (45– 51). CCR6+ ILC3s also produce IL-17 and protect from fungal infections, whereas CCR6−ILC3s down-regulate RORγt and IL- 22, up-regulate the TF T-bet. CCR6−ILC3s in addition acquire the capacity to produce IFN-γ and transform into ILC1-like cells (19, 44, 52–55). They were named lymphoid tissue-inducer (LTi) cells because they were among the ﬁrst cells to inﬁltrate lymph node anlagen during embryogenesis and hence are instrumental for the development of most secondary lymphoid tissues (6). Furthermore, from 2008 onwards, several groups reported the discovery of new types of non-T and non-B lymphocytes which, like NK cells and LTi cells, require the transcriptional regulator inhibitor of DNA-binding 2 (ID2) and the common gamma chain (γc) of the cytokine interleukins (IL)-2, 4, 7, 9, 15, and 21 for their development and/or maintenance (7–21). These cells were termed “innate lymphoid cells” (ILCs), which constitute lineages of professional cytokine-producing cells that mirror T helper cells in the utilization of transcription factors (TFs) required to establish distinct patterns of lineage-speciﬁc cytokine production and eﬀector functions. It became obvious that the diﬀerent ILC populations resemble the functional diversity found in T helper cell subsets, thus establishing a complementary innate counterpart to T helper cells (22). Helper-like ILCs were reported as tissue-resident cells enriched at barrier surfaces and underrepresented in secondary lymphoid organs (29–31). In contrast, NK cells are patrolling lymphocytes, which express CD62L to migrate from blood to lymph nodes (21, 30, 56). As patrolling cells, immune recognition by NK cells is mediated by the interaction of immunoreceptors that scan target cells for the expression of their ligands. Citation: Therefore, the development and regulation of NK cells depend on the interaction of the immunoreceptors and their ligands. Although the expression of some immunoreceptors (e.g. KLRG1, PD-1) has been reported for helper-like ILCs as well, their activity seems to be predominantly regulated by soluble factors such as cytokines and neuronal factors (21, 35, 52, 57–59). In connection with these ﬁndings of ILC diversity, a novel ILC nomenclature was proposed in 2013 and amended in 2018 (22, 23). In analogy to T cells, two principal subsets of ILCs can be distinguished: cytotoxic ILCs (i.e. conventional NK cells) and helper-like ILCs (i.e. ILC1, ILC2, and ILC3) (24, 25). The general division of NK cells and helper-like ILCs is supported by various ﬁndings. First, while there is a common progenitor to all innate lymphocytes, variably referred to as early innate lymphoid progenitor (EILP) (26) or innate lymphoid cell progenitor (ILCP) (27), a more restricted common helper- like innate lymphoid cell progenitor (CHILP) with reduced potential for helper-like ILC can only be found downstream of the bifurcation with the NK cell lineage. Second, all helper- like ILCs but not NK cells require GATA binding protein 3 (GATA-3) for their diﬀerentiation (28). Third, helper-like ILCs are remarkably tissue-resident cells, whereas NK cells are circulating cells (29–31). Finally, the use of inhibitory and activating receptors of the KIR and the Ly49 families was found in NK cells but not in ILCs. Thus, two principal lineages of innate lymphocytes exist: helper-like ILCs and cytotoxic ILCs. Frontiers in Immunology \| www.frontiersin.org Missing-Self Recognition g g In 1981, Klas Kärre formulated the missing-self hypothesis (78). Missing-self recognition was conceived as the capacity of NK cells to attack cells that fail to express suﬃcient levels of class I MHC molecules. This concept was discovered while investigating the role of class I MHC molecules in NK cell and T cell responses to tumor cells (79, 80). Given the role that the class I MHC antigen presentation pathway plays in the revelation of virally infected cells to CD8+ T cells, it is not surprising that many viruses have evolved mechanisms that interfere with this pathway, thereby binding CD8+ T cells to virus-infected cells (81). The missing-self hypothesis predicted that NK cells express inhibitory class I MHC-speciﬁc receptors and that the down- regulation of MHC-I expression on virus-infected cells or tumors would unleash NK cells from inhibition. Years after postulating “missing-self recognition,” various classes of inhibitory MHC class I-speciﬁc NK cell receptors were identiﬁed. Ly49 receptors in mice (82) and the structurally unrelated but functionally analogous KIR family of inhibitory receptors in humans (83, 84) directly interact with class I MHC molecules. Both human and mouse NK cells express the heterodimeric CD94/NKG2A receptor, which monitors class I MHC molecules by another mechanism. CD94/NKG2A recognizes a non-classical MHC class I molecule, HLA-E in humans and Qa-1b in mice, when loaded with peptides that are derived from the signal peptide of classical class I MHC proteins (85). The inhibitory receptors have an immune-receptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic domain. Upon ligand recognition, phosphorylation of the ITIM’s tyrosine residue serves as a signal for recruitment of protein tyrosine phosphatases, SHP- 1 and SHP-2, which inhibit cytotoxic activity by further dephosphorylating tyrosine residues that are critical for NK cell activation (86, 87). Upon recognition of PAMPs and MAMPs on microbes by antigen-presenting cells (APCs), phagocytosis and processing of antigens in the lysosomal compartment of these cells are triggered. T cells express strictly antigen-speciﬁc T cell receptors (TCRs) that are generated by somatic genetic recombination, thereby providing a vast repertoire of speciﬁcities. TCRs recognize self and non-self peptides presented on APCs via MHC molecules, providing “signal 1” for T cell activation. However, TCR ligation by itself is not suﬃcient for eﬃcient T cell activation. It requires a co-stimulatory signal (“signal 2”), e.g. provided by APC-expressed CD80 (B7-1) and CD86 (B7-2), ligands for the constitutively expressed CD28 receptor on T cells (68). IMMUNE RECOGNITION STRATEGIES OF NK CELLS The complexity of multicellular organisms demands essential immune recognition strategies to maintain their self-integrity in a hostile environment. Almost all organisms, from bacteria to higher animals, possess recognition systems that allow them to discriminate between self and non-self and possess eﬀector mechanisms to defend themselves from an invasion of pathogens. The immune system of vertebrates consists of two arms: innate and adaptive. Recognition of non-self molecules is broadly used by both the innate and the adaptive immune system to protect the host from infections (60). However, although NK cells are capable of directly sensing non-self molecules, their development and activation are regulated to a large extent by the recognition of self molecules. Discrimination between self and non-self is mediated by an array of stimulatory and inhibitory immunoreceptors expressed by NK cells. They either recognize non-self structures directly (Ly49H, NKG2C/CD94) or indirectly via binding immune complexes to Fc receptors. Alternatively, they interact with self MHC I (Ly49s and KIR, “missing-self” recognition) or with ligands absent on healthy cells (NKG2D and NKp30, “induced-self” recognition). The regulation of NK cells, which relies on cell surface immunoreceptor–ligand interactions, is complemented by cytokines, such as type I interferons, IL-12, IL-15, and IL-18 (61–64). In analogy to T cells, ILCs are divided into functional groups, based on TFs required for their development as well as their role in immune responses (22). NK cells are functionally important for immunity against tumors and intracellular pathogens via classical perforin-dependent, cell- mediated cytotoxicity and production of interferon-gamma (IFN-γ). ILC1s are an important source of IFN-γ and tumor necrosis factor (TNF) to trigger type 1 immune responses and limit intracellular infections. While NK cells and ILC1s are functionally both promoting type 1 immune responses, they are developmentally dependent on two evolutionary related T-box TFs: eomesodermin (EOMES) and T-box expressed in T cells (T-bet) (32). NK cells express both EOMES and T-bet, but their development is only strictly dependent on EOMES. NK cells develop in T-bet-deﬁcient mice and have a relatively mild functional defect (16, 33, 34). In contrast, ILC1s express T-bet but not EOMES and do not develop in T-bet-deﬁcient mice (21, 35, 36). ILC2s require GATA-3 and July 2020 \| Volume 11 \| Article 813 Frontiers in Immunology \| www.frontiersin.org 2 Stokic-Trtica et al. NK and ILC Development Non-self Recognition presented in the context of the non-classical MHC-I molecule HLA-E, and this recognition activates NK cells (77). The diﬀerent recognition strategies of immune cells are depicted in Figure 1. The recognition strategy of “microbial non-self” is approached diﬀerently from the innate and the adaptive immune system. The cellular components of the innate immune system express germline-encoded receptors, called pattern recognition receptors (PRRs), which come in two forms: transmembrane receptors and secreted receptors (60, 65). These molecules recognize conserved pathogen-associated molecular patterns (PAMPs) or microbe- associated molecular patterns (MAMPs). The secreted PRRs lead to the opsonization of microbes and label them for destruction either by the complement system or by phagocytosis. PRRs expressed on the cell surface of innate immune cells, such as Toll- like receptors (TLRs), lead to the activation of immune signaling pathways, which trigger inﬂammatory or antimicrobial eﬀector responses (66, 67). Missing-Self Recognition CD80 and CD86 are not expressed by unstimulated APCs but are rapidly up-regulated following the encounter of MAMPs or PAMPs, providing an additional “quality control” for T cell responses. In addition, stimulated APCs produce cytokines and IFNs, which further enhance T cell responses (“signal 3”). In the case of naive CD4+ T lymphocytes, distinct cytokines have been shown to drive the diﬀerentiation into one of three T helper (Th) subsets. IFN-γ and IL-12 are important for inducing Th1 cells, IL-4 for Th2 commitment, and TGF-β and IL-6 for Th17 cell diﬀerentiation (69–72). TLR expression was also described on NK cells, but its contribution to NK cell activation remains unclear. While the direct activation of NK cells by TLR engagement has been reported for human NK cells (73), genetic data from mice demonstrated that TLR signaling to activate NK cells was cell- extrinsic via mononuclear phagocytes (62). While recognition of “non-self” via PRRs may not be central for NK cells, they express other families of receptors to directly recognize “non- self” molecules such as Ly49H in mice or NKG2C/CD94 in humans (74–76). Ly49H is a stimulatory receptor that recognizes the MHC-like protein m157 encoded by murine cytomegalovirus (MCMV), which is expressed in infected cells and confers host protection in C57BL/6 (B6) mice. It should be noted though that, in most inbred mouse strains other than B6, m157 binds to an inhibitory Ly49 receptor, leading to immune evasion. NK cells can also recognize non-self peptides in the context of non-classical MHC I molecules, very similar to the immune recognition strategy of T cells. For example, subsets of NK cells expressing the stimulatory receptor NKG2C/CD94 were shown to recognize the UL40 antigen of human cytomegalovirus Missing-self recognition does not require viral infection or a malignant transformation of target cells. Uninfected and untransformed cells can be lysed by NK cells, as demonstrated in NK cell-mediated rejection of F1 bone marrow grafts (88) and bone marrow of β2-microglobulin-deﬁcient mice that do not express class I MHC on the cell surface (89). Since T cells are not capable of recognizing and killing cells that down- regulated class I MHC expression due to viral proteins that hijack their expression pathway, NK cells are able to compensate this immunological function via missing-self recognition of MHC- deﬁcient target cells. Frontiers in Immunology \| www.frontiersin.org Induced-Self Recognition Induced-self ligands of NK cell receptors are molecules that are absent or only at a very low level expressed on normal cells but up-regulated on infected cells, stressed cells, or tumor cells as a marker of “abnormal self.” Induced-self ligands bind to stimulatory immunoreceptors on NK cells and mediate their activation, leading to the lysis of the target cell (63, 90). The activating NK cell receptor natural killer group 2D family July 2020 \| Volume 11 \| Article 813 Frontiers in Immunology \| www.frontiersin.org 3 Stokic-Trtica et al. NK and ILC Development FIGURE 1 \| Principles of immune recognition. The immune system constantly senses the presence or absence of “self” and “non-self” molecules by stimulatory and inhibitory receptors. Activation of immune cells is triggered by the direct recognition of microbial “non-self,” “missing-self” recognition, or “induced-self” recognition (illustrations were created with BioRender.com). FIGURE 1 \| Principles of immune recognition. The immune system constantly senses the presence or absence of “self” and “non-self” molecules by stimulat nhibitory receptors. Activation of immune cells is triggered by the direct recognition of microbial “non-self,” “missing-self” recognition, or “induced-self” recog llustrations were created with BioRender.com). FIGURE 1 \| Principles of immune recognition The immune system constantly sense FIGURE 1 \| Principles of immune recognition. The immune system constantly senses the presence or absence of “self” and “non-self” molecules by stimulatory and inhibitory receptors. Activation of immune cells is triggered by the direct recognition of microbial “non-self,” “missing-self” recognition, or “induced-self” recognition (illustrations were created with BioRender.com). to class I MHC molecules, but they neither require β2- microglobulin for expression nor do they present peptides (90). Another example of “induced-self recognition” is the natural cytotoxicity receptor NKp30 which interacts with the B7-like self- ligand B7-H6, the expression of which is induced on transformed cells (97). Thus, immunosurveillance of induced-self ligands by immunoreceptors such as NKG2D and NKp30 allows the immune system to detect and eliminate cells that have undergone “stress.” These receptor–ligand pairs represent interesting targets of anti-tumor therapies. In Tables 1A and 1B, ligand–receptor (NKG2D) has served as a paradigm for understanding the recognition of induced-self antigens. NKG2D binds to several induced-self ligands. The mouse ligands include RAE1α, RAE1β, RAE1γ, RAE1δ, RAE1ε, H60 (H60a, H60b, and H60c), and MULT1 (91–96). These NKG2D ligands belong to a group of non-classical MHC I molecules and contain α1 and α2 extracellular domains with homology to class I MHC molecules. In humans, MHC class I polypeptide-related sequence A (MICA) and B (MICB) represent additional NKG2D ligands that are not present in mice (91). MICA/B possess an α3 domain homologous July 2020 \| Volume 11 \| Article 813 Frontiers in Immunology \| www.frontiersin.org Frontiers in Immunology \| www.frontiersin.org 4 NK and ILC Development Stokic-Trtica et al. TABLE 1 \| (A) Summary of ligand-receptor interactions and their effect on immune cell activation. (B) Summary of self and non-self effects on immune cells when they are present or absent. Illustrations created with BioRender.com. TABLE 1 \| (A) Summary of ligand-receptor interactions and their effect on immune cell activation. (B) Summary of self and non-self effects on immune cells when they are present or absent. Illustrations created with BioRender.com. TABLE 1 \| (A) Summary of ligand-receptor interactions and their effect on immune cell activation. TRANSCRIPTIONAL REGULATION OF PROGENITOR COMMITMENT TO ALL INNATE LYMPHOID CELL LINEAGES The existence of a common progenitor for ILCs was already hypothesized years before their discovery, mainly based on the phenotype of mice deﬁcient for ID2 (107). ID2 belongs to the family of helix–loop–helix proteins, which form heterodimers with E-proteins, thus preventing their binding to DNA and antagonizing the gene regulatory function of E-proteins during cell development (108). Since Id2−/−mice lacked all ILCs, it was hypothesized that this phenotype might be explained by the existence of a common ILC progenitor, which expresses ID2 and is developmentally dependent on it (12, 105, 109). Indeed the analysis of ID2 reporter mice [Id2Gfp/+ mice; (110)] revealed that both mature and immature ILCs expressed ID2 (17, 111, 112). An interrogation of the αLP population for ID2 expression revealed a population of ID2high cells within Lin−IL-7Rα+ CD25−integrin α4β+ 7 FLT3−αLP (21). Upon transfer and on a clonal level, this cellular subset gave rise to all three groups of ILCs, including CCR6+ ILC3, but not to conventional NK cells and was accordingly named CHILP. While the CHILP did not express any ILC lineage-deﬁning TFs, they expressed intermediate levels of GATA-3 (17). However, CHILPs were heterogeneous for the expression of the TF promyelocytic leukemia zinc ﬁnger protein (PLZF) (21). Interestingly, while PLZF+ CHILP could generate ILC1, ILC2, and CCR6−ILC3, they lacked the potential to diﬀerentiate into CCR6+ (LTi-like) ILC3s and NK cells (98). These ﬁndings might be explained by data showing that the CHILP contained subsets of CXCR5+ cells, which gave rise to CCR6+ ILC3s/LTi cells and were not contained in the PLZF+ population (113, 114). Single-cell sequencing of ILC progenitors has conﬁrmed the developmental stages of early ILC commitment and further contributed additional markers, such as programmed cell death protein 1 (PD-1), to deﬁne PLZF+ precursors (115). Therefore, we will refer to these two populations of ILC precursors as CHILP-A (ID2+ PLZF−PD- 1−) and CHILP-B (ID2+ PLZF+ PD-1+). Using reporter mice for several TFs, later studies showed some degree of heterogeneity in CHILP-A and CHILP-B which could be further subdivided into cell subsets that were committed to one ILC subset and bona ﬁde CHILP subsets that still maintained multi-ILC lineage potential (111–113). The initial steps of ILC diﬀerentiation from precursor cells take place in the fetal liver and after birth in the adult bone marrow (BM). In Figure 2, ILC progenitors and their diﬀerentiation stages into ILC subsets are presented. TRANSCRIPTIONAL REGULATION OF PROGENITOR COMMITMENT TO ALL INNATE LYMPHOID CELL LINEAGES Hematopoietic stem cells (HSCs) give rise to all blood cell progenitors, among which common lymphoid progenitors (CLPs) are precursors of all lymphocytes, belonging to both adaptive and the innate arms of the immune system. It is generally believed that the CLP diﬀerentiates into the various lymphocyte subsets by integrating environmental signals that establish characteristic transcriptional programs, usually regulated via several key TFs, that lead to a step-wise restriction of their precursor potential and to the instruction of lymphocyte subset-speciﬁc transcriptional circuitry (99–101). p p y An important conceptual advance of the last 5 years was the description of multipotent ILC progenitor cells such as α- lymphoid progenitor (α4β+ 7 αLP), EILP, CHILP, and ILCP, which have the developmental potential for ILC lineages but can no longer diﬀerentiate into adaptive lymphocytes or myeloid cells (21, 26, 27, 98). Early evidence indicated that ILC progenitors may be contained within a population with phenotypical characteristics similar to CLPs. Indeed Lin−IL-7Rα+ CXCR6− cells, which in contrast to CLPs expressed integrin α4β7 but were negative for FLT3, a receptor tyrosine kinase expressed by the CLPs (102), gave rise to all three groups of ILCs and T cells but had lost B cell potential (103). The subsequent acquisition of the chemokine receptor CXCR6 is indicative of the loss of T cell potential and Lin−IL-7Rα+ CXCR6+ integrin α4β+ 7 FLT3−cells are referred to as αLPs (103, 104). It became clear, however, that αLPs are a quite heterogeneous population of innate lymphocyte progenitors, which was further explored in subsequent work. The earliest deﬁned subset of ILC-committed progenitors downstream of the CLP (and contained within the αLP population) was characterized by high expression of the transcriptional regulator T cell factor 1 (TCF-1, encoded by the Tcf7 gene). Such TCF-1high progenitors are referred to as EILPs or ILCPs. EILPs already show a substantial expression of nuclear factor interleukin 3-regulated (NFIL3, also known as E4BP4) and thymocyte selection-associated high mobility group box protein (TOX) known to be involved in early ILC diﬀerentiation (see below) (26). While the CLP does not express ID2, a transcriptional regulator required for the diﬀerentiation A recent report attempted to challenge the view that ID2high CHILPs are progenitors to all helper-like ILCs but not to conventional NK cells (112). This study was based on the generation of a very bright reporter allele for ID2. Somewhat expectedly (26), the authors found that ID2int precursors (i.e. (B) Summary of self and non-self effects on immune cells when they are present or absent. Illustrations created with BioRender.com. present or absent. Illustrations created with BioRender.com. present or absent. Illustrations created with BioRender.com. Frontiers in Immunology \| www.frontiersin.org 5 July 2020 \| Volum July 2020 \| Volume 11 \| Article 813 5 Frontiers in Immunology \| www.frontiersin.org NK and ILC Development Stokic-Trtica et al. interactions, as well as the functional consequences for NK cell activation are summarized. of all ILCs (105), EILPs express intermediate level of ID2. The EILP gives rise to all ILC lineages (including NK cells) but lacks T and B lymphocyte or myeloid potential (26). Unlike other ILC progenitors and CLPs, EILPs were IL-7Rα low-expressing cells and developed independently of IL-7Rα signaling. Therefore this ﬁnding provoked the question on whether EILPs might constitute an alternative route to ILC development because the upstream and downstream cells are both IL-7Rα+. However, it was demonstrated in consecutive work that EILPs developed from CLP transiently down-regulating IL-7Rα expression and then diﬀerentiated into ILC progenitors with increased IL-7Rα expression (26, 106). Notably, NK cell-mediated immune regulation is tightly linked to both classical and non-classical class I MHC molecules. NK cells sense the absence of classical MHC-I (“missing-self”) but also recognize non-classical MHC-I molecules as non- self or induced-self ligands. In addition, NK cells require the recognition of self-MHC ligands not only for their activation but also for proper development, which will be discussed in the following chapters. Frontiers in Immunology \| www.frontiersin.org TRANSCRIPTIONAL REGULATION OF PROGENITOR COMMITMENT TO ALL INNATE LYMPHOID CELL LINEAGES EILP), which could be discriminated in these new reporter mice, still have NK cell diﬀerentiation potential. These results July 2020 \| Volume 11 \| Article 813 6 Stokic-Trtica et al. NK and ILC Development FIGURE 2 \| Progenitor commitment to innate lymphoid cells (ILCs). Schematic representation of progenitor populations with various differentiation potentials toward ILCs. The common lymphoid progenitor (CLP) gives rise to B-cells, T cells, and ILCs. The early innate lymphoid progenitors (26) possess the potential for NK cells, ILC1s, ILC2s, and ILC3s, whereas CHILP-A (21) and CHILP-B (98) possess the potential for ILC1s, ILC2s, and ILC3s as indicated. In square brackets are the population-deﬁning markers reported in the literature. The transcription factors required for the indicated lineage or transition from one population to another are indicated within the cells or on the arrows, respectively (illustrations created with BioRender.com). FIGURE 2 \| Progenitor commitment to innate lymphoid cells (ILCs). Schematic representation of progenitor populations with various differentiation potentials toward ILCs. The common lymphoid progenitor (CLP) gives rise to B-cells, T cells, and ILCs. The early innate lymphoid progenitors (26) possess the potential for NK cells, ILC1s, ILC2s, and ILC3s, whereas CHILP-A (21) and CHILP-B (98) possess the potential for ILC1s, ILC2s, and ILC3s as indicated. In square brackets are the population-deﬁning markers reported in the literature. The transcription factors required for the indicated lineage or transition from one population to another are indicated within the cells or on the arrows, respectively (illustrations created with BioRender.com). diﬀerentiation potential, namely, TCF1+ ID2int EILP (or ILCP), ID2high PLZF−CHILP-A, and ID2high PLZF+ CHILP-B (Figure 2). are supporting previous work which show that EILP expressed intermediate levels of ID2 and can generate all ILC subsets and NK cells (26, 106, 116). It is worth noting that during cellular diﬀerentiation processes, TFs often act as gradients rather than as binary switches. Once past the NK cell bifurcation (Figure 2), ID2 expression increases (26, 100, 112) and ID2high CHILPs have a more restricted potential. Collectively, the available data support a model of ILC diﬀerentiation downstream of CLP with three major bifurcations and consecutively restricted Single-cell sequencing data and analysis of multi-color reporter mice have demonstrated that a further subdivision of ILC progenitors is technically possible. Frontiers in Immunology \| www.frontiersin.org TRANSCRIPTIONAL REGULATION OF PROGENITOR COMMITMENT TO ALL INNATE LYMPHOID CELL LINEAGES Both pre-NKPs and rNKPs showed the potential of diﬀerentiating into NK cell receptor-positive cells in spleen and liver, albeit with diﬀerent frequencies. Since EOMES, a lineage-specifying TF for NK cells was not analyzed, these studies do not allow deﬁnite conclusions regarding a possible bipotential of NKPs for NK cells and ILC1s. The notion that the NKP population might contain committed ILC1 precursors was supported by Constantinides et al., who demonstrated heterogeneity within the pre-NKP population, with one subset expressing PLZF and other markers characteristic for ILC progenitors and another subset belonging to NKP (56). Therefore, more detailed analyses are required to delineate the separation of NK and ILC1 lineages in early precursors. Several additional TFs were recognized, which play an essential role in early commitment to the ILC fate. In addition to ID2 and PLZF, these TFs include NFIL3, TOX, and GATA-3. The phenotype of the knockout mice for these TFs was characterized by a deﬁciency or a reduction in all or almost all ILC lineages, except NK and LTi-like cells in GATA-3-deﬁcient mice. NFIL3 is important for the transition from CLPs to ILC progenitors, where the relative expression of this TF increased and its deletion led to a substantial decrease in ILC progenitor numbers (104, 117– 122). Since the down-regulation of the transcriptional regulator TOX was described in Nﬁl3−/−mice in comparison to wild- type controls, it was proposed that NFIL3 is directly regulating the expression of TOX, which then acts downstream in ILC development (104). Indeed Tox−/−mice had a similar phenotype as that of Nﬁl3−/−mice and lacked mature ILCs and ILC progenitors (123, 124). Based on these data, it was proposed that NFIL3 and TOX orchestrate the transition from CLP to EILP (106), whereas GATA-3 (28, 125) is required later in ILC development for the transition to PLZF+ ILCPs. It should be noted though that NK cells and CCR6+ ILC3s still develop in GATA-3-deﬁcient mice. Altogether these data indicate that the developmental potential for NK cell and CCR6+ ILC3s/LTi cell is consecutively lost during the transition from EILP to CHILP-B (28, 98, 114, 125, 126). While ILC progenitors are certainly present in the primary organs of hematopoiesis, the BM and fetal liver (21, 26, 98), it should be considered that ILCs may be derived from local precursors as tissue-resident cells. TRANSCRIPTIONAL REGULATION OF PROGENITOR COMMITMENT TO ALL INNATE LYMPHOID CELL LINEAGES These ﬁndings can advance the ILC ﬁeld by deﬁning diﬀerent ILC progenitors that have a more restricted diﬀerentiation potential and therefore open the perspective of ﬁnding the cues that control the July 2020 \| Volume 11 \| Article 813 Frontiers in Immunology \| www.frontiersin.org 7 NK and ILC Development Stokic-Trtica et al. and maturation stages of NK cells are described. It should be noted that the major thrust of work on NK cell development was performed before ILCs with an NK cell phenotype (ILC1, subsets of ILC3) were recognized. We will critically discuss here the conventional deﬁnition of NKPs and immature NK cells in the new framework of ILC diversity. commitment and the diﬀerentiation of CHILPs into diﬀerent ILC cell subsets. However, deﬁning progenitors by clustering based on highly expressed genes that are detected using single- cell sequencing comes with the caveat that cell clustering is not necessarily of biological relevance and diﬀerences in diﬀerentiation potential remain to be demonstrated. An NKP population, which gave rise only to mature NK cells but did not possess a potential for T or B-cell lineages, was originally identiﬁed to be within Lin−NK1.1−DX5− CD122+ cells (129). However, the frequency of such NKP diﬀerentiating into NK cells was only one in 12 in limiting dilution assays, revealing a highly heterogeneous population and a requirement for additional markers to further narrow down the true NKP, also because some T cell potential was still detectable in this population (131). Technical progress in multicolor ﬂow cytometry allowed a more accurate deﬁnition of the NKP within Lin−CD27+ CD244+ CD122−IL-7Rα+ FLT3−cells, in which 50% of the cells were giving rise to NKp46+ NK cells (132). This NKP subset was designated as a pre-NK cell precursor (pre-NKP), suggesting to be the earliest precursor of NK cells. Pre-NKPs express natural killer cell receptor 2B4 (CD244) and lack the expression of other surface markers associated with NK cells (NKp46, NKG2D, NK1.1, or inhibitory receptors such as Ly49 and CD94/NKG2A). Under NK cell-promoting culture conditions, the expression of CD122 is up-regulated, thereby giving rise to the ‘reﬁned’ NK cell precursor (rNKP), which has full NK cell potential (132). Apart from CD122, these precursors also acquire during their diﬀerentiation the IL-2 receptor γc chain, which makes NKPs responsive to IL-15, a cytokine essential for NK cell diﬀerentiation and survival (133–135). TRANSCRIPTIONAL REGULATION OF PROGENITOR COMMITMENT TO ALL INNATE LYMPHOID CELL LINEAGES In mice, fetal ILC precursors migrated to the intestine before Peyer’s patch organogenesis and accumulated at the sites where intestinal lymphoid tissue organogenesis is initiated and became a localized source of ILC populations (127). While intestinal ILC precursors were identiﬁed based on arginase expression, adult BM ILC precursors lacked arginase expression, indicating tissue adaption of the ILC precursor population (127). Since fate-labeling studies suggest that the ILC pool is generated in diﬀerent pre- and postnatal time windows (128) and ILC precursors were also detected in human blood (27), further research is required to investigate the relation among ILC precursor cells in diﬀerent compartments and their relevance in ontogeny. y p While NKG2D was already expressed by at least one subset of NKPs, the expression of NK1.1, NKp46, and CD94/NKG2A marked the immature NK cell stage (stage 2). The expression of Fc receptors and Ly49s, which provide inhibitory receptors for the NK cell education process, deﬁnes stage 3 of NK cell development (136–139). At this stage, “NK cells” display a T- bet signature, but since T-bet does not allow the distinguishing between NK cells and ILC1s, the delineation between the two lineages is diﬃcult until EOMES and DX5 are expressed in stage 4 (16, 21, 35, 36). Furthermore, surface markers such as CD69, CD51, or tumor necrosis factor-related apoptosis- inducing ligand (TRAIL), which are often used to describe immature NK cells, are phenotypic markers of ILC1s but not mature NK cells in most tissues (136, 140, 141). Therefore, Developmental Stages of NK Cell Maturation NK cells develop from a committed NK cell progenitor (NKP) in the BM, which was ﬁrst described in 2001 based on the expression of the IL-2/IL-15 receptor beta chain (CD122), a well-recognized T-bet target gene (129, 130). In Figure 3A distinct developmental July 2020 \| Volume 11 \| Article 813 Frontiers in Immunology \| www.frontiersin.org 8 Stokic-Trtica et al. NK and ILC Development FIGURE 3 \| Development of NK cells and ILC1s. (A) Developmental stages of murine conventional NK cells. Representation of markers used for the identiﬁcation of individual developmental stages. Markers highlighted in red represent those expressed mainly in the given subset and therefore important for its identiﬁcation. At each stage, the expression of the listed markers is depicted as “+”, whereas the absence of expression is indicated by “–” or alternatively “low.” (B) Tissue-speciﬁc non-conventional NK/ILC1 subsets. Representation of markers used for the identiﬁcation of non-conventional/ILC1 subsets in thymus, intestine, and liver (illustrations created with BioRender.com). FIGURE 3 \| Development of NK cells and ILC1s. (A) Developmental stages of murine conventional NK cells. Representation of markers used for the identiﬁcation of individual developmental stages. Markers highlighted in red represent those expressed mainly in the given subset and therefore important for its identiﬁcation. At each stage, the expression of the listed markers is depicted as “+”, whereas the absence of expression is indicated by “–” or alternatively “low.” (B) Tissue-speciﬁc non-conventional NK/ILC1 subsets. Representation of markers used for the identiﬁcation of non-conventional/ILC1 subsets in thymus, intestine, and liver (illustrations created with BioRender.com). the view that the T-bet+ EOMES−NKp46+ subset represents immature NK (iNK) cells is currently questionable (16, 142). cells” had an “immature phenotype” and EOMES+ NK cells had “mature” characteristics, in vivo and in vitro diﬀerentiation experiments did not show any transition between these two populations. PLZF fate mapping supported these results because EOMES+ DX5+ NK cells did not derive from PLZF-expressing progenitors, whereas EOMES−TRAIL+ populations originated from PLZF+ precursors. In addition, EOMES−TRAIL+ ILC1s did not diﬀerentiate into EOMES+ DX5+ NK cells (35, 36, 56). Moreover, Constantinides et al. demonstrated that ILC1s predominate over cNK cells during development in murine liver, while cNK cell number increases during adulthood (56). Therefore, since ILC1s and NK cells have parallel progression at an early stage during development and are phenotypically similar, further analyses have to be performed to separate iNK cells from ILC1s. Frontiers in Immunology \| www.frontiersin.org Cytokine Signals Regulating NK Cell Development p Although NK cell commitment is not dependent on IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21, which are executing their function through a common cytokine receptor γ chain, early NKPs have the capacity to respond to cytokines through the co-expression of CD122 and CD127 (146). Since mice lacking IL-2, IL-4, and IL-7 developed normal numbers of phenotypically mature NK cells with a regular capacity to exert natural cytotoxicity in vitro, produce IFN-γ, and kill tumor cells in vivo (146), IL- 15 was identiﬁed as the major γc cytokine to promote NK cell development, and it plays a dominant role in early NK cell diﬀerentiation by maintaining normal numbers of immature and mature NK cells in the BM and spleen (146, 147). Given that the close association of T-box TFs and IL-15 responsiveness via CD122 is a hallmark of many lymphocytes, including ILCs and unconventional tissue-resident T cells (148–152), IL-15 was also indispensable for the development of ILC1s and ex-ILC3s although they co-express CD127 (21, 35, 153). IL-15 activated NK cells by STAT5 signaling and promoted the expression of the anti-apoptotic protein MCL1 and, at the same time, restricted the expression of pro-apoptotic proteins such as BIM and NOXA (153, 154). p ( ) Various TFs, such as TCF-1, NFIL3, and TOX, that were already introduced to regulate early commitment to the ILC lineage are indispensable for NK cell development, likely by acting on the EILP or upstream progenitors. Mice deﬁcient for either TCF-1, NFIL3, or TOX lacked NK cells and also other ILC lineages (117, 123, 160). Since these TFs are already expressed upstream of the NKP in multipotent ILCPs such as EILP, CHILP- A, or CHILP-B, mice deﬁcient in these TFs lacked most ILC lineages. Therefore, at least some of the eﬀects likely occur already in multipotent precursors before commitment to the NK cell lineage (106, 124). Whereas, the mechanistic role for TOX after NK cell commitment is elusive, it was shown that TCF-1 restricts granzyme expression, thus protecting the developing NK cells from self-destruction (161). While NFIL3 was recognized as a TF important for the transition from CLPs to early ILC precursors, it was shown to be also up-regulated in the NKP cells. Further, its deﬁciency in mice led to decreased numbers of NK cells (121, 162). While it was proposed that NFIL3 regulates ID2 expression (117, 163), Seillet et al. Developmental Stages of NK Cell Maturation immature NK (iNK) cells is currently questionable (16, 142). T-bet+ EOMES−“NK cells” represent the major murine liver NK-like subpopulation during fetal and neonatal developmental stages. During aging, the ratio between these “T-bet+ EOMES− immature NK cells” and mature NK cells changed, which represented another argument favoring the presumption that T- bet+ EOMES−liver “NK cells” are in fact immature NK cells that can further mature. This hypothesis was supported by data demonstrating that EOMES−TRAIL+ “immature” NK cells in the adult liver gave rise to EOMES+ DX5+ NK cells (16, 142). Contradicting results were obtained in several other publications (21, 35, 36). Daussy et al. utilized EOMES reporter allele and performed extensive phenotypic proﬁling of the EOMES-positive and EOMES-negative populations. Even though EOMES−“NK July 2020 \| Volume 11 \| Article 813 Frontiers in Immunology \| www.frontiersin.org Frontiers in Immunology \| www.frontiersin.org 9 NK and ILC Development Stokic-Trtica et al. Mature bona ﬁde NK cell subsets are characterized by the following: 1) expression of EOMES and DX5 (stage 4); 2) acquisition of CD11b (stage 5); 3) the consecutive loss of CD27 (stage 6) and the up-regulation of CD43 and KLRG1 (136, 143). Developmental intermediates were identiﬁed among CD11bhi cells, designated as mature NK cell subsets (136). CD27 is a key marker of the NK cell lineage, dissecting the mature CD11b+ NK cell pool into two functionally distinct subsets (144, 145). The CD27low NK cell subset possesses a higher threshold to stimulation and appears to be tightly regulated by the expression of NK cell inhibitory receptors. The preceding subset is consisting of the CD27high NK cells that display a greater eﬀector function, exhibiting a distinct tissue distribution and responsiveness to chemokines and productively interacting with dendritic cells (144, 145). recording relatively small NKP populations given the very limited availability of multicolor ﬂow cytometry and by the separation of immature NK cells from ILC1s (as already discussed above). Therefore, although supported by data, the conclusion that certain TFs act at a certain stage of NK cell development should be taken with caution as these analyses often pre-dated the discovery of ILC1s and other ILC subsets expressing NK cell receptors such as NKp46 and NK1.1. With this in mind, we will discuss here the major TF modules that have been associated with NK cell diﬀerentiation. Developmental Stages of NK Cell Maturation Developmental defects in NK cells were reported from mice deﬁcient for the TFs PU.1 and IKAROS that are broadly expressed early in hematopoiesis before commitment to the ILC/NK lineage and therefore aﬀect multiple hematopoietic lineages, including but not limited to NK cells (155–158). Since it is controversial if PU.1 is expressed during NK cell development at all, it is unclear whether PU.1 or IKAROS are mediating eﬀects during NK cell development or whether the phenotypes might be explained by the eﬀects in upstream hematopoietic precursors (159). Cytokine Signals Regulating NK Cell Development showed that NFIL3 was inﬂuencing EOMES expression, whereas ID2 expression in the absence of NFIL3 remained the same. Moreover, ectopic expression of EOMES in Nﬁl3−/−hematopoietic progenitor cells was suﬃcient to rescue cNK cell development (162). Frontiers in Immunology \| www.frontiersin.org Transcriptional Regulation of NK Cell Development g p The conditional deletion of the Krüppel-like TF KLF2 in hematopoietic cells using VavCre resulted in the ablation of mature CD11b+ NK cells and consequently reduced the cytotoxicity toward target cells (185). It was proposed that KLF2 regulates the survival of NK cells via the regulation of IL-15 sensing and the expression of homing receptors. Reduced NK cells were also reported from mice deﬁcient in the Th1 regulator interferon regulatory factor 2 (IRF-2) (186). IRF-2 deﬁciency disturbed mainly mature splenic NK cells, whereas NK cell development in the BM was only mildly aﬀected. IRF-2 NK cells were more prone to undergo apoptosis during development independently from IL-15 (187). Several TFs with a more restricted expression during hematopoiesis played pivotal roles during the maturation of NK cells. These include EOMES, T-bet, and ZEB2. Unlike ILC1s, which only expressed and were developmentally dependent on T-bet but not EOMES, mature NK cells co-expressed both T- bet and EOMES. While mice with a conditional deletion of EOMES lacked NK cells, these cells normally diﬀerentiated in T-bet-deﬁcient mice where they were accumulating in the BM and the lymph nodes due to the altered expression of S1P5R and CXCR3. They displayed an immature phenotype characterized by the persistent expression of CD27 and the reduced CD11b, CD43, and KLRG1 levels (33, 34, 173, 174). A similar NK cell maturation phenotype was reported from mice deﬁcient in the Zinc ﬁnger-containing protein (ZEB2) (175). The notion that ZEB2 and T-bet might cooperatively regulate NK cell maturation is also supported by data showing that the overexpression of ZEB2 can partially rescue the phenotype of T-bet-deﬁcient NK cells (175). TFs regulating NK cell development involves FOXO proteins as well. However, the precise role is hard to evaluate because of data that are diﬃcult to reconcile with a model. While Wang et al. found decreased numbers of NK cells in mice with conditional deletion of FOXO1 using NKp46Cre deleter mice (188), Deng and colleagues reported increased numbers of mature, hyper- reactive NK cells using NKp46Cre and also VavCre deleter mice to genetically ablate FOXO1 (189, 190). Runt-related TFs (RUNX) are important regulators of lymphocyte development, including T cells and several ILC lineages. RUNX members 1–3 form heterodimers with the TF core-binding factor beta (CBF-β) in order to bind to regulatory DNA sequences and mediate gene transcription (191, 192). The RUNX3 isoform is highly expressed in NK cells. Transcriptional Regulation of NK Cell Development ID2 represents another transcriptional regulator that was up-regulated during NK cell development from NKPs and that was essential for the development of mature NK cells (105, 109, 162). Notably, unlike other ILC populations, NKPs and immature NK cells developed in ID2-deﬁcient mice. However, ID2 deﬁciency causes loss of terminally diﬀerentiated CD11b+ NK cells, indicating a persistent need for the sequestration of E-proteins during NK cell maturation (170, 171). In support of this notion, the genetic deletion of ID2 and ID3, which both bind E-proteins, resulted in the complete loss of NK cells. It was also proposed that ID2 regulates IL-15 receptor signaling via the suppression of SOCS3. Interestingly, both ID2 and IL-15 signaling were linked to the regulation of apoptosis in NK cells via either anti-apoptotic MCL1 or pro-apoptotic BIM (154, 170, 172). Therefore, ID2 could be a link between sensing of the vital cytokine IL-15 and cell survival. in NK cells, downstream targets of the T-box TFs are not well-deﬁned in NK cells and were largely extrapolated from studies that have investigated other cell types (180). However, the importance of EOMES in NK cell fate and in the expression of prototypic markers of NK cells is also illustrated by data showing that the overexpression of EOMES under the Tbx21 regulatory elements reprogrammed ILC1s to adopt phenotypical hallmarks of NK cells (178). Since the down-regulation of EOMES in NK cells mediated by TGF-β drove the NK cells to adopt an ILC1 phenotype, EOMES appears as a major signaling hub that dictate NK cell identity (181, 182). Numerous TFs including AIOLOS, PRDM1 (BLIMP1), FOXO1, IRF2, RUNX3, and KLF2 regulate the late developmental stages of NK cells with main eﬀects on terminal maturation and eﬀector functions. PRDM1 (encoding BLIMP1) was shown to be regulated by T-bet and IL-15. Further, BLIMP1- deﬁcient mice had fewer KLRG1+ mature NK cells. Although granzyme B expression was altered in PRDM1-deﬁcient NK cells, eﬀector functions, including cytotoxicity, remained normal (183). A similar phenotype was reported for mice deﬁcient in the IKAROS zinc ﬁnger TF member AIOLOS. NK cells developed in AIOLOS-deﬁcient mice but terminally diﬀerentiated CD11b+ NK cells were reduced. While NK cell eﬀector functions were largely maintained, Aiolos−/−NK cells were hyper-responsive to tumor cells, resulting in superior tumor surveillance (184). Transcriptional Regulation of NK Cell Development p NK cell development and function are regulated by a plethora of TFs expressed at diﬀerent developmental stages, and at each stage these sets of TFs constitute regulatory networks for the establishment of distinct phenotypes. While numerous TFs that regulate pivotal steps during NK cell development were identiﬁed, the regulation of NK cell development is much less understood on a molecular level. As a consequence, the NK cell-speciﬁc target genes of TFs are insuﬃciently deﬁned. Although TFs were proposed to mainly act during one stage of NK cell development, it should be considered that they might regulate NK cell development at various stages and depending on the amount of TFs being expressed (i.e. TF gradients). Additional diﬃculties in assembling the available data into a satisfying model are represented by the limitations in accurately p Mice deﬁcient for the TF ETS-1 had a strong reduction in mature NK cell numbers (164). Similar to NFIL3, ETS-1 was already expressed in CHILPs and also in NKPs. ETS-1 regulated the ﬁtness of CHILPs, but the eﬀects on NK cell development are probably emerging later with reduction at pre-NKP and rNKP stages, where ETS-1 might regulate T-bet and ID2 (165, 166). MEF is another member of the ETS TF family, which regulated essential functions during NK and NKT cell development, whereas B and T cells developed in normal proportions (167). While MEF-deﬁcient mice have reduced NK cells and impaired eﬀector function, including cytotoxicity and IFN-γ production, mechanistic insights are scarce (167). Important regulators of NK cell development include ID2 and GATA-3, known to regulate early ILC commitment (21, 125). July 2020 \| Volume 11 \| Article 813 10 NK and ILC Development Stokic-Trtica et al. However, due to the phenotype of the gene-deﬁcient mice and their expression pattern, it seems more likely that these TFs mediate their decisive eﬀects after NK cell commitment. Mice deﬁcient in ID2 or GATA-3 developed NKPs and immature NK cells but had a maturation defect of NK cells. In contrast, they lacked the other ILC lineages (12, 28, 105, 109, 126). While GATA-3 is required for ILC1 development (21, 28, 168, 169), it was dispensable for the development but not for the maturation of NK cells. GATA-3-deﬁcient NK cells had an immature phenotype, were poor producers of IFN-γ, and showed defects in BM egress because they are retained in the BM due to the high CXCR4 expression (28, 126, 169). Frontiers in Immunology \| www.frontiersin.org Transcriptional Regulation of NK Cell Development Consistent with a role later in development, RUNX and CBF-β were also crucial for NK cell memory formation following MCMV infection (198). Finally, TFs that constitute regulatory network during NK cell development represent a nice example of how these proteins act as a part of a complex context that dictates their function and how compensatory mechanisms in their absence could, in some situations, buﬀer the entire system. DEVELOPMENT OF ILC1s/TISSUE-RESIDENT NK CELLS NK cells and ILC1s share many phenotypical and functional properties that make the diﬀerentiation between these two innate lymphocyte subsets, especially in humans, very challenging (59). In addition, ILC1s comprise several subsets of lymphocytes previously referred to as “immature,” “tissue-resident,” or “unusual” NK cells before the revised nomenclature in 2013. These include TRAIL+ NK cells (142) and thymic NK cells (168) or (after the revised nomenclature) ILC1s in the BM, the lamina propria (21), the epithelium of the intestine [intraepithelial (ie)ILC1s] (20), the salivary glands (199), the adipose tissue (200), or the uterus (201). ILC1 subsets diﬀer in terms of dependency on TFs during development, e.g. EOMES and NFIL3, and cytokines, e.g. IL-7 and IL-15 (21–23, 36, 120, 168, 202). Although these subsets are often all referred to as ILC1s, it is very diﬃcult to conclude whether diﬀerent developmental requirements reﬂect the tissue adaption of one cell lineage or diﬀerent cell lineages of phenotypically similar cells. Besides the heterogeneity and the tissue adaptation of ILC1s, diﬀerences between mouse and human ILC1s add an additional layer of complexity to the topic. For example, it is well-established that murine liver TRAIL+ NK cells express and are developmentally dependent on T-bet but not EOMES. However, the human liver contains a population of CD56bright lymphocytes, which phenotypically resembled ILC1s but expressed high levels of EOMES and only low levels of T-bet (203). Nevertheless, the functional and the phenotypical characterizations of diﬀerent subsets of ILC1s are contributing to a better understanding of their biology and diversity as well as enabling their separation in a more comprehensive way. While being recognized as tissue-resident cells, ILC1s have been residing in various tissues, expressing speciﬁc markers that are represented in Figure 3B and which will be discussed below. p γ p y p ( ) Based on CD56 expression, a unique subset of ILC1s was also described in the intestinal epithelium of humans. NKp44+ CD103+ and NKp44−CD103−ieILC1s were discriminated with similar functional properties, such as strong IFN-γ production. In addition, ieILC1s showed signs of TGF-β imprinting, such as CD103 expression, and were phenotypically diﬀerent from cNK cells as illustrated by the expression of CD160, CD49a, CXCR6, CD69, and CD39, which were also found on ILC1s in other organs (141). Unlike thymic NK cells, ieILC1s lacked the expression of CD127 (IL-7Rα) but did express IL-2Rβ chain. Transcriptional Regulation of NK Cell Development Diﬀerent strategies were used to genetically interfere with RUNX to investigate the function in vivo, including the overexpression of dominant-negative RUNX3 or the conditional deletion of RUNX3 or CBF-β. While RUNX3 regulated the development of ILC1s and ILC3s by diﬀerent mechanisms, ILC2 development remained intact and RUNX proteins protected ILC2s from Although EOMES is also expressed by non-hematopoietic cells as well as in CD8+ T cells, where the TF regulates CD8 memory formation, among ILCs, EOMES represents a speciﬁc TF for NK cells (16, 176, 177). Moreover, mice harboring a conditional deletion of EOMES using NKp46Cre completely lacked NK cells but still contain other ILC lineages (178, 179). Therefore, EOMES represents an attractive candidate for the speciﬁc targeting of NK cells by using, for example, NKp46Cre Eomesﬂ/ﬂmice to exclude eﬀects on T cells. While epigenetic studies provide evidence that the EOMES and the T-bet promoters are both in an open chromatin conﬁguration July 2020 \| Volume 11 \| Article 813 Frontiers in Immunology \| www.frontiersin.org 11 NK and ILC Development Stokic-Trtica et al. elevated expression of this TF in comparison to splenic cNK cells (168). Phenotypically, thymic NK cells resembled ILC1s rather than NK cells because of the lack of CD11b and Ly49 receptors and their expression of CD69. However, whether thymic NK cells belong to the same lineage as ILC1s and TRAIL+ liver NK cells requires further clariﬁcation, especially because they express EOMES and DX5, which are usually not found on ILC1s (202). Thymic NK cells were reduced in Foxn1−/−mice, which do not develop a functional thymus, suggesting that the thymus is an organ required for the generation of this ILC1 subset. Data obtained in reporter mice for TCR-δ germ-line transcription suggest that thymic NK cells might be derived from lymphocytes with T cell potential (205). This is in line with data showing that primitive, double-negative T cell progenitors still possess the potential to diﬀerentiate into cells that phenotypically resemble NK cells (206). Further, it was proposed that thymic NK cells might be the counterpart of CD56bright NK cells, which are potent IFN-γ producers but have weak cytotoxic potential (168). an exhaustion-like phenotype (192–194). Concerning NK cell development, the deletion of either RUNX3 or CBF-β altered NK cell development via the regulation of CD122 and IL- 15 responsiveness. This was accompanied by reduced numbers of CD11b+ and CD43+ mature NK cells and enhanced cytokine production (195–197). Frontiers in Immunology \| www.frontiersin.org DEVELOPMENT OF ILC1s/TISSUE-RESIDENT NK CELLS The murine counterpart of human ieILC1s localizing within the gut epithelium co-expressed CD160, NKp46, and NK1.1 (20). Examining the developmental pathway of ieILC1s in mice, Fuchs et al. demonstrated the requirement of NFIL3 and T-bet. These ieILC1s were in part independent of IL-15Rα, indicating that intraepithelial ILC1s are developmentally distinct from cNK cells (20). Functionally, and similar to ex-ILC3s, ieILC1s were linked to the immunopathology in the αCD40 model of colitis due to their IFN-γ production (20, 52, 207). Further, ILC1s were enriched in patients with Crohn’s disease and may, therefore, contribute to the development of inﬂammatory bowel disease similar to lamina propria ILC1s (19, 20, 208). In the lamina propria of the intestine, it was challenging to identify ILC1s because of the sizeable populations of NK cells and ex-ILC3s, which all expressed the prototypic makers of ILC1s, such as NKp46 and NK1.1. Using double-reporter mice for EOMES (labeling NK cells) and fate-labeling for RORγt (labeling all ILC3s independent of their RORγt expression), a subset of lymphocytes within NKp46 and NK1.1 lymphocytes was deﬁned, which expressed T-bet. This population within NKp46+ NK1.1+ lymphocytes lacked EOMES and RORγt expression and did not have a history of RORγt expression either. Further, such ILC1s were developmentally dependent on T-bet, NFIL3, and GATA- 3, but not EOMES or RORγt. Phenotypically, intestinal ILC1s expressed markers associated with ILC1s in diﬀerent tissues such as CD127, CD160, or CD49a, lacked markers of cNK cells such as CD11b and CD62L, and showed low Ly49 receptor expression. Despite expressing both CD127 and CD122, ILC1s were strictly IL-15-dependent and did not require IL-7. Upon The characteristic feature of thymic non-conventional NK/ILC1 is that they express CD127 and developmentally depend on IL-7 signaling (168). This is in contrast to splenic and BM cNK cells, the phenotype and the function of which were not perturbed in the absence of IL-7. To a lesser extent, thymic NK cells required IL-15 for their development similar to NK cells (204). Moreover, thymic non-conventional NK cells depended on GATA-3 for their development and showed an July 2020 \| Volume 11 \| Article 813 Frontiers in Immunology \| www.frontiersin.org 12 NK and ILC Development Stokic-Trtica et al. which are produced by conventional dendritic cells upon infection. After activation with IL-12, ILC1s respond with IFN-γ secretion to limit viral load and thereby contribute to early antiviral immunity at sites of primary viral infection (210). EPIGENETIC AND microRNA-MEDIATED REGULATION OF NK CELL AND ILC1 DEVELOPMENT ILC1s, also referred to as tissue-resident NK (trNK) or TRAIL+ NK cells in the liver (142), diﬀered from conventional NK cells since they expressed only T-bet as the key TF in mice, and this expression is favored in the liver microenvironment (16, 29, 35, 36). On the contrary, the BM provides a microenvironment that promotes lower expression levels of T-bet in NK cells, enabling the subsequent expression of EOMES (35). Another remarkable diﬀerence between cNK cells and ILC1s was the expression of the “homolog of BLIMP1 in T cells” (HOBIT) in ILC1s (213). This TF is speciﬁcally up-regulated in tissue-resident cells and controlled the expression of molecules associated with tissue residency, such as CD49a and CD69. Interestingly, HOBIT was essential for liver ILC1s but not for ILC1s in other organs investigated (210, 213). In addition, the development of ILC1s in the liver was demonstrated to be dependent on PLZF expression and independent of NFIL3, contrary to NK cells (36, 98). Among epigenetic modiﬁcations, the deubiquitination of histone H2A by MYSM1 is important for NK cell generation as the deletion of this enzyme is causing maturation defects in NK cells (219). The MYSM1 histone H2A deubiquitinase also contributed to the development of ILC1s in other organs. In addition to modifying histones, MYSM1 also functions as a transcriptional regulator of ID2 expression during the maturation of NK cells by recruiting NFIL3 to the Id2 gene locus. MYSM1 was involved in maintaining an active chromatin conﬁguration at the Id2 locus (219), further promoting its expression. Another epigenetic mechanism that regulates NK cell development involves repressive histone marks such as the tri- methylation of lysine residue 27 of Histone 3 protein during early NK cell diﬀerentiation (220). In the absence of this marker through the repression of EZH2 enzymatic activity (enhancer of zeste homolog 2), ILC1 and NK cell lineage commitment was enhanced, together with increased NK cell survival and NKG2D-mediated cytotoxicity (220). TRAIL represents a prototypic marker of liver ILC1s as it is constitutively expressed on both mouse and human ILC1s, and together with CD49a and CD69, it has been used for separating liver ILC1s from NK cells. This type II transmembrane protein causes apoptosis primarily in tumor cells by binding to certain death receptors. Recent ﬁndings are suggesting that TRAIL expression is regulated by the activation of the NKp46 receptor in ILC1s since NKp46-deﬁcient mice lack this eﬀector protein (214–216). DEVELOPMENT OF ILC1s/TISSUE-RESIDENT NK CELLS The genetic ablation of liver ILC1s is leading to increased MCMV load in mice; hence, NK cell responses are not the only early antiviral response in mice. In addition to rapidly responding to IL-12, “memory-like” qualities have been reported for ILC1s in models of contact hypersensitivity and MCMV infection. This is remarkable because these cells were originally considered as “immature NK” cells due to the lack of surface markers characteristic of mature NK cells (142). ILC1s were described to mediate tissue-resident memory responses to MCMV depending on glycoprotein m12 (217). Furthermore, previous reports have already linked liver ILC1s to memory responses during contact hypersensitivity reactions (29, 218). However, the mechanism underlying recognition of haptens by ILC1s following memory responses remains elusive. transfer into alymphoid mice, ILC1s were a stable lineage without diﬀerentiation potential into cNK cells or ex-ILC3s and could also be found in the BM (35). BM ILC1s phenotypically overlap with the previously described immature NK cells based on markers such as CD69 (140). However, markers often connected to immature NK cells, such as CD69, TRAIL, or CD51, are rather found on ILC1s, and it should also be noted that they are not expressed before or after that developmental stage during NK cell development. In addition, CD69 is considered to be a marker for cell activation or tissue residency, which is associated with activated rather than with immature lymphocytes (136, 140). Therefore, additional studies have to address the potential heterogeneity within EOMES−NK1.1+ cells, previously termed “immature NK cells” in the BM. transfer into alymphoid mice, ILC1s were a stable lineage without diﬀerentiation potential into cNK cells or ex-ILC3s and could also be found in the BM (35). BM ILC1s phenotypically overlap with the previously described immature NK cells based on markers such as CD69 (140). However, markers often connected to immature NK cells, such as CD69, TRAIL, or CD51, are rather found on ILC1s, and it should also be noted that they are not expressed before or after that developmental stage during NK cell development. In addition, CD69 is considered to be a marker for cell activation or tissue residency, which is associated with activated rather than with immature lymphocytes (136, 140). Therefore, additional studies have to address the potential heterogeneity within EOMES−NK1.1+ cells, previously termed “immature NK cells” in the BM. DEVELOPMENT OF ILC1s/TISSUE-RESIDENT NK CELLS Although cytokine IL-12 was ﬁrst described as a NK cell- stimulating factor (209), IL-12 elicited stronger eﬀects on ILC1s than on NK cells, consistent with higher expression levels of the components of IL-12 receptor on ILC1s (21, 62, 210). While ILC1s were potent producers of IFN-γ and TNF, they expressed less perforin, indicating that they are less cytotoxic and rather mediate the cytotoxic eﬀect by TNF receptors such as TRAIL. Functionally, a lack of perforin-mediated cytotoxicity or a loss of NK cell identity resulted in decreased immunosurveillance of tumors (181, 182, 211). However, data from diﬀerent infection models suggest that there is a spatial and a temporal division of labor between NK cells and ILC1s. ILC1s protected the digestive tract from Toxoplasma gondii, Clostridium diﬃcile, or MCMV infections, which are controlled to a large degree by IFN-γ secreted by ILC1s (21, 210, 212). Taken together, the experimental evidence obtained from knockout mice suggests that ILC1s constitute a separate tissue- resident lineage distinct from cNK cells. Further investigation is required to answer questions of ILC1 diversity. Frontiers in Immunology \| www.frontiersin.org EPIGENETIC AND microRNA-MEDIATED REGULATION OF NK CELL AND ILC1 DEVELOPMENT The number of NK1.1+ cells in the organs of Dicer-1 mutant mice was aﬀected, along with the impaired maturation of NK cells. NK cells without miR showed a diminished function, including reduced target cell cytotoxicity and IFN-γ production. Additionally, in Dicer- 1-deﬁcient mice, the IL-15 receptor signaling in NK cells was impaired. This ﬁnding explains, at least in part, the decreased survival of NK cells and the observed perturbations in NK cell maturation. The eﬀects of single miRs, such as miR142, miR155, miR150, and miR15/16, revealed speciﬁc eﬀects and potential target genes. The conserved miR142 sequence encodes two highly expressed mature miRNAs, 142-3p and 142-5p, which have diﬀerent mRNA targets (221). The target of the miR142-3p is the 3′ UTR of Itgav gene that encodes integrin-αV. In the absence of miR142- 3p, this integrin was up-regulated in ILC1s and promoted their survival. The other product of the miR142 sequence, miR142-5p, was targeting the 3′ UTR suppressor of cytokine signaling 1 (Socs1) gene, a negative regulator of IL-15 signaling. Thus, in the absence of miR142-5p, SOCS1 un-antagonized, leading to impaired IL-15 signaling (221). While the down-regulation of RORγt and the up-regulation of T-bet occurs at steady state in CCR6−ILC3s, the plasticity of NK cells or ILC2s might require a trigger, such as chronic inﬂammation. The conversion of ILC2s to an ILC1-like phenotype is triggered by cytokines, such as IL-1, IL-12, and IL-18, and was described in the context of chronic obstructive pulmonary disease (237–240). This process is connected to the up-regulation of T-bet, and the genetic deletion of T-bet using NKp46Cre resulted in enhanced ILC2 responses, suggesting that the balance of the lineage-specifying TFs GATA-3 and T-bet determines ILC2 plasticity (241). In humans, miR155 was shown to down-regulate SH2 containing 5′ inositol phosphatase (SHIP1), which in part contributes to the regulation of IFN-γ production following stimulation (225). In mice, miR155 targeted the 3′ UTR of Noxa transcripts during homeostasis and of Socs1 transcripts during the activation of NK cells (226). The direct functional target of miR-150 and miR15/16 was the TF c-Myb, through which the maturation program was controlled (222, 224). p y Whether the conversion of NK cells to ILC1-like cells is occurring at a steady state is diﬃcult to evaluate because of the lack of fate-labeling studies for the NK lineage-specifying TF EOMES. EPIGENETIC AND microRNA-MEDIATED REGULATION OF NK CELL AND ILC1 DEVELOPMENT However, fate-labeling was carried out using Cre under the NKp46 promoter, which is expressed in NK cells, ILC1s, CCR6−ILC3s, and subsets of γδ T cells (138). While the down-regulation of NKp46 was described for ILC3s (236), the results did not provide evidence that NK cells down-regulate NKp46 at steady state (138). It should be considered though that conversion in other ILC lineages, which also express NKp46, would not be detected using this fate-labeling strategy. The ﬁrst evidence for the potential conversion of NK cells into ILC1-like cells came from studies that investigated the unusual subsets of ILC1s in the salivary gland. Unlike ILC1s in other organs, the salivary gland ILC1s co-expressed EOMES and T- bet but did not developmentally depend on either of these TFs and also not on NFIL3, suggesting that they have diﬀerent developmental requirements (199, 242). In addition, the salivary gland ILC1s depicted hallmarks of tissue-resident cells, such as TGF-β imprinting, that was also reported for ILC1s in diﬀerent organs, for instance, the intestine (20). ILC1s in the salivary gland were reduced in the absence of TGF-β signaling, and the phenotypical markers of ILC1s, such as CD49a and TRAIL were down-regulated, whereas EOMES was up-regulated (199). Furthermore, NK cells that were hyper-responsive to TGF-β, due to the genetic manipulation of TGF-βRI or deletion of SMAD4, developed an ILC1-like phenotype in the salivary gland or within tumor tissue. As a consequence, these ILC1-like NK cells failed to control tumor growth or viral infection with EPIGENETIC AND microRNA-MEDIATED REGULATION OF NK CELL AND ILC1 DEVELOPMENT Apart from the regulation of gene expression on the transcriptional level, another epigenetic mechanism is required for the proper development of ILC1s and the adequate maturation of NK cells. Available data implicate small non- coding RNA molecules (221–226), such as microRNAs (miRs), to regulate posttranscriptional gene expression by binding to the 3′ untranslated region (UTR) of mRNAs and inducing either suppression or mRNA translation or its degradation (227). Deletion of the RNase III enzyme Dicer-1, an enzyme required for the generation of single-stranded 20–25 bp long non-coding Another important functional hallmark of liver pro- inﬂammatory ILC1s is that they are activated via IL-12, July 2020 \| Volume 11 \| Article 813 Frontiers in Immunology \| www.frontiersin.org 13 NK and ILC Development Stokic-Trtica et al. (44, 52, 207, 229, 232, 233). T-bet deﬁciency was vice versa reported to promote colitis in response to Helicobacter typhlonius that was mediated by IL-17A-producing ILC3s (234, 235). In humans, diﬀerentiation of ILC3s toward CD127+ ILC1s was described in the intestine of patients with Crohn’s disease and was promoted by cytokines IL-2 and IL-12 and CD14+ DCs. Interestingly, this process was found to be reversible and stimulated by IL-1β, IL-23, retinoic acid, and CD14− DCs (19, 229, 230). Data obtained in fate-labeling studies in mice using either RORγ tCre (230) or NKp46Cre (236) also support the model that the plasticity of ILC3s is a reversible process. Signals regulating NKp46 expression on CCR6−ILC3s included the Notch-T-bet axis as a positive regulator and TGF- β signaling as a negative regulator (236). Altogether these studies provide evidence for the reversible plasticity of CCR6− ILC3s toward ILC1s, mediated by signals that regulate RORγt and T-bet. RNA molecules, in NKp46-expressing cells revealed the role of miRs in murine NK cells and ILC1s (223). The number of NK1.1+ cells in the organs of Dicer-1 mutant mice was aﬀected, along with the impaired maturation of NK cells. NK cells without miR showed a diminished function, including reduced target cell cytotoxicity and IFN-γ production. Additionally, in Dicer- 1-deﬁcient mice, the IL-15 receptor signaling in NK cells was impaired. This ﬁnding explains, at least in part, the decreased survival of NK cells and the observed perturbations in NK cell maturation. RNA molecules, in NKp46-expressing cells revealed the role of miRs in murine NK cells and ILC1s (223). Frontiers in Immunology \| www.frontiersin.org SPECIFIC TARGETING TO UNCOVER FUNCTIONAL SPECIALIZATION OF GROUP 1 ILC SUBSETS Despite progress in the generation of genetically modiﬁed mice, speciﬁc targeting of ILCs remains a major challenge in the ﬁeld because of the large overlap in gene expression between ILCs and T cells as well as other immune cells. Since a systematic review of genetic models for the investigation of ILC function was recently published (243), we aim to focus the discussion on NK cell receptor (NKR)+ ILCs that comprise conventional NK cells, ILC1s, and CCR6−ILC3s. Concerning NKR+ ILCs, speciﬁc targeting of each subset is further complicated by shared receptors such as NKG2D, NKp46, and NK1.1 and TFs such as T-bet, NFIL3, or TOX within NKR+ ILCs, making them alone not a suitable target (141). While antibody-mediated depletion strategies using αNK1.1 or αThy1 were eﬀective, more speciﬁc depletion strategies were developed using genetic models based on NKp46Cre mice (138, 153, 244). While NKp46 is fairly speciﬁc to group 1 ILCs, a second allele is required to ensure speciﬁcity among group 1 ILCs, which is often a ﬂoxed mouse for an essential TF such as EOMES or RORγt. Following the targeting strategy, the generation of NKp46Cre Eomesﬂ/ﬂresulted in the selective ablation of NK cells, thus allowing a deﬁnitive conclusion about the contribution of NK cells in an experimental autoimmune encephalomyelitis model (178, 179). NKp46Cre Rorc(γt)ﬂ/ﬂmice were likewise generated to investigate redundant and non-redundant functions of ILC3s during Citrobacter rodentium infection and in colitis models (233, 245). While these two strains provide speciﬁc targeting for NK cells and CCR6−ILC3s, respectively, the genetic mouse models for ILC1s are even more diﬃcult to develop. NKp46Cre Tbx21ﬂ/ﬂmice lacked ILC1s (179), but a contribution of NK cells or CCR6−ILC3s in these mice could not be excluded because NK cells and CCR6−ILC3s have a migration or maturation defect in T-bet-deﬁcient mice. It should be also considered that the phenotype might be dependent on which line of NKp46Cre deleter mice is used (179, 241). Furthermore, mice deﬁcient for the TF HOBIT were used to investigate ILC1 function in the liver because ILC1s, but not NK cells, are reduced in the liver of these mice. However, the use of this mouse line is limited to TRAIL+ NK cells and not ILC1s in other organs (210, 213). Therefore, the goal for NKp46Cre to delete a selective TF important for ILC1 subsets in many organs is still not achieved. REGULATION OF NK CELL DEVELOPMENT AND FUNCTION BY RECEPTOR–LIGAND INTERACTION While ILC plasticity after lineage commitment is now well-established to occur, additional investigation is required to elucidate how the plastic behavior of ILCs could be therapeutically harnessed. PLASTICITY TOWARD GROUP 1 ILCs Although ILCs comprise separate lineages of innate lymphocytes deﬁned by distinct lineage-specifying TFs, a considerable amount of plasticity after fate commitment was reported for most ILC lineages in mice and humans, often connected to a certain tissue microenvironment or in the context of inﬂammation (228). Plasticity is characterized by the down-regulation of lineage-specifying TFs, such as RORγt for ILC3s or GATA- 3 for ILC2s, and acquisition of master TFs of alternative cell fates, acquisition of phenotypic characteristics of other ILC lineages (e.g. up-regulation of NK cell receptors), and production of cytokines not associated with the original lineage. The plasticity of ILCs was ﬁrst described for ILC3s (52, 229). Fate-labeling for RORγt expression revealed that ILC3s were able to diﬀerentiate into cells phenotypically resembling ILC1s (referred to as ex-RORγt+ ILC3s or ex-ILC3s) (52, 229–231). This process was accompanied by the gradual up-regulation of ILC1 signature genes such as T-bet, NK receptors (NKp46, NK1.1, and NKG2D), and cytokine receptors (IL12Rβ2), as well as eﬀector functions (19, 21, 44, 53, 55). During this process, ex-ILC3s became IFN-γ-producing lymphocytes, which were responsive to several cytokines, including IL-12 and IL- 23, and promoted inﬂammation and immunopathology in experimental models of colitis and Salmonella enterica infection July 2020 \| Volume 11 \| Article 813 Frontiers in Immunology \| www.frontiersin.org 14 Stokic-Trtica et al. NK and ILC Development REGULATION OF NK CELL DEVELOPMENT AND FUNCTION BY RECEPTOR–LIGAND INTERACTION cytomegalovirus (181, 182). Some of the eﬀects that TGF-β has on the NK/ILC1 fate decisions are mediated via the balance of the master TFs T-bet and EOMES. Notably, it was reported that the forced expression of EOMES driven by the T-bet promoter turned ILC1s into cells with NK cell properties (178). However, it remains unclear whether this occurs in vivo, and if yes, under which conditions. cytomegalovirus (181, 182). Some of the eﬀects that TGF-β has on the NK/ILC1 fate decisions are mediated via the balance of the master TFs T-bet and EOMES. Notably, it was reported that the forced expression of EOMES driven by the T-bet promoter turned ILC1s into cells with NK cell properties (178). However, it remains unclear whether this occurs in vivo, and if yes, under which conditions. The activation of NK cells is mediated to a large degree by the integration of stimulatory and inhibitory signals as measured by the engagement of NK receptors by its cognate ligands. NK cells need to be calibrated during development to become activated if a deﬁned threshold of stimulatory to inhibitory signals is exceeded, a process coined “NK cell education” or “licensing.” Classical NK cell education is linked to self recognition and mediated by inhibitory receptors for class I MHC, such as Ly49 receptors or KIR (82, 246–248). Thus, this process requires the timely expression of the corresponding ligands for the receptors involved in the education process. Moreover, besides the type of MHC molecule expressed and the type of receptors on NK cells, the strength of class I MHC– Ly49 receptor interaction also deﬁnes the quality and the quantity of NK cell education (249). In connection with this, it was observed that the absence of MHC I on the surface of cells, by the genetic deletion of β2-microglobulin, TAP, or KbDb, resulted in the hypo-responsiveness of NK cells (250–252). In line with these ﬁndings, NK cells with mutations in ITIMs required for inhibitory signaling were functionally impaired. Further, the deletion of intracellular downstream signaling molecules, SH- 2-domain-containing protein tyrosine phosphatase 1 (SHP1) and SH-2-domain-containing inositol-5-phosphatase (SHIP), resulted in the hypo-responsiveness of NK cells (253–255). On a molecular level, the MHC I education process was linked to the reorganization of the nanostructure of immunoreceptors and conﬁnement in domains, thus generating the basis for diﬀerent activation thresholds (251, 252, 256). Frontiers in Immunology \| www.frontiersin.org REFERENCES 6. Mebius RE, Rennert P, Weissman IL. Developing lymph nodes collect CD4+CD3- LTbeta+ cells that can diﬀerentiate to APC, NK cells, and follicular cells but not T or B cells. Immunity. (1997) 7:493–504. doi: 10.1016/S1074-7613(00)80371-4 1. Herberman RB, Nunn ME, Holden HT, Lavrin DH. Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. II characterization of eﬀector cells. Int J Cancer. (1975) 16:230–9. doi: 10.1002/ijc.2910160205 1. Herberman RB, Nunn ME, Holden HT, Lavrin DH. Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. II characterization of eﬀector cells. Int J Cancer. (1975) 16:230–9. doi: 10.1002/ijc.2910160205 7. Satoh-Takayama N, Vosshenrich CA, Lesjean-Pottier S, Sawa S, Lochner M, Rattis F, et al. Microbial ﬂora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense. Immunity. (2008) 29:958–70. doi: 10.1016/j.immuni.2008.11.001 2. Herberman RB, Nunn ME, Lavrin DH. Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. I distribution of reactivity and speciﬁcity. Int J Cancer. (1975) 16:216–29. doi: 10.1002/ijc.2910160204 2. Herberman RB, Nunn ME, Lavrin DH. Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. I distribution of reactivity and speciﬁcity. Int J Cancer. (1975) 16:216–29. doi: 10.1002/ijc.2910160204 8. Cella M, Fuchs A, Vermi W, Facchetti F, Otero K, Lennerz JK, et al. A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity. Nature. (2009) 457:722–5. doi: 10.1038/nature07537 3. Kiessling R, Klein E, Pross H, Wigzell H. “Natural” killer cells in the mouse. II cytotoxic cells with speciﬁcity for mouse Moloney leukemia cells characteristic of the killer cell. Eur J Immunol. (1975) 5:117–21. doi: 10.1002/eji.1830050209 3. Kiessling R, Klein E, Pross H, Wigzell H. “Natural” killer cells in the mouse. II cytotoxic cells with speciﬁcity for mouse Moloney leukemia cells characteristic of the killer cell. Eur J Immunol. (1975) 5:117–21. doi: 10.1002/eji.1830050209 9. Cupedo T, Crellin NK, Papazian N, Rombouts EJ, Weijer K, Grogan JL, et al. Human fetal lymphoid tissue-inducer cells are interleukin 17-producing precursors to RORC+ CD127+ natural killer-like cells. Nat Immunol. (2009) 10:66–74. doi: 10.1038/ni.1668 4. Kiessling R, Klein E, Wigzell H. “Natural” killer cells in the mouse. I cytotoxic cells with speciﬁcity for mouse Moloney leukemia cells speciﬁcity and distribution according to genotype. Eur J Immunol. (1975) 5:112–7. doi: 10.1002/eji.1830050208 10. Luci C, Reynders A, Ivanov Ii, Cognet C, Chiche L, Chasson L, et al. SPECIFIC TARGETING TO UNCOVER FUNCTIONAL SPECIALIZATION OF GROUP 1 ILC SUBSETS Although it remains elusive if the adaptation of NK cell reactivity to sustained activation by stimulatory ligands due to overexpression is the underlying similar mechanism described for NK cell education, the ﬁndings become relevant in the context of anti-tumor immunity where, for instance, NKG2D ligands might be continuously expressed or shedded from the tumor cells, thus saturating their receptors. Although it is controversial if the chronic expression or shedding of NKG2D ligands should be regarded as a tumor escape mechanism or if it is promoting tumor immunosurveillance, these ﬁndings indicate the importance of the regulation of NK cell activity by receptor– ligand interaction (265–267). Apart from the chronic expression of induced-self ligands on tumor cells, blocking antibodies for inhibitory receptors targeting KIR or NKG2A are evaluated blockade of inhibitory receptors on NK cells has the potential to complement T cell immunotherapy because the eﬃciency of T cell checkpoint blockade correlated with the production of neoantigens by tumor cells present on MHC I. However, the tumor cells that did not produce neoantigens or escaped MHC I-peptide recognition by CD8+ T cells (268) could be recognized and lysed by NK cells expressing inhibitory receptors to detect the presence of MHC I. However, data available so far might indicate that there is a narrow therapeutic window deﬁned by the blocking of the inhibitory receptor and the eﬀects on NK cell education, rendering the cells hypo-responsive (267). In summary, NK cells need education mediated by the engagement of inhibitory and stimulatory receptors during development. NK cell education is required for both adequate reactivity and tolerance toward self. Blocking of inhibitory NK cell receptors during anti-tumor therapy can complement checkpoint blockade and illustrates the transfer of basic knowledge for human therapy. ACKNOWLEDGMENTS We thank Dr. Divija Deshpande, Dr. Katja Jarick, Caroline Tizian, and Akriti Kanth for critically reading the manuscript. The ﬁgures were created with BioRender.com. of induced-self ligands on tumor cells, blocking antibodies for inhibitory receptors targeting KIR or NKG2A are evaluated in clinical trials to promote anti-tumor immunity (267). The FUNDING This work was supported by grants from the German Research Foundation (DFG; KL 2963/1-1 and KL 2963/2-1 to CK) and the European Research Council (ENTRI to CK). This work was supported by grants from the German Research Foundation (DFG; KL 2963/1-1 and KL 2963/2-1 to CK) and the European Research Council (ENTRI to CK). AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. SPECIFIC TARGETING TO UNCOVER FUNCTIONAL SPECIALIZATION OF GROUP 1 ILC SUBSETS In addition, data obtained in models that overexpressed ligands for stimulatory receptors such as m157 or NKG2D ligands, in which the NK cells were persistently exposed to a receptor engagement, revealed that NK cells adapted to this stimuli, for instance by the down-regulation of the stimulatory receptor (262–264). Although it remains elusive if the adaptation of NK cell reactivity to sustained activation by stimulatory ligands due to overexpression is the underlying similar mechanism described for NK cell education, the ﬁndings become relevant in the context of anti-tumor immunity where, for instance, NKG2D ligands might be continuously expressed or shedded from the tumor cells, thus saturating their receptors. Although it is controversial if the chronic expression or shedding of NKG2D ligands should be regarded as a tumor escape mechanism or if it is promoting tumor immunosurveillance, these ﬁndings indicate the importance of the regulation of NK cell activity by receptor– ligand interaction (265–267). Apart from the chronic expression of induced-self ligands on tumor cells, blocking antibodies for NK cells from MHC I-deﬁcient mice were hypo-responsive, a similar paradoxical phenotype was uncovered for NKG2D- deﬁcient mice having hyper-responsive NK cells despite the lack of an important stimulatory NK cell receptor. Notably, mice deﬁcient for NKG2D (Klrk1−/−) were hyper-responsive, resulting in the superior control of MCMV infection and tumor growth (257, 258). However, immunosurveillance of tumors expressing NKG2D ligands was impaired in NKG2D-deﬁcient mice (259). Mechanistically, NKG2D regulated signaling via the natural cytotoxicity receptor NKp46 and the signaling molecule CD3ζ (258). While the precise timing of NK cell education is not well-deﬁned, some studies suggested that NK cell education is not limited to a time window during development but represents a continuous process. This is supported by studies that used adoptive transfer of uneducated NK cells in MHC I-suﬃcient hosts that could restore NK cell functionality (260, 261). In addition, data obtained in models that overexpressed ligands for stimulatory receptors such as m157 or NKG2D ligands, in which the NK cells were persistently exposed to a receptor engagement, revealed that NK cells adapted to this stimuli, for instance by the down-regulation of the stimulatory receptor (262–264). SPECIFIC TARGETING TO UNCOVER FUNCTIONAL SPECIALIZATION OF GROUP 1 ILC SUBSETS Interestingly, Ly49s were strongly underrepresented but not totally absent on ILC1s, suggesting diﬀerences between NK cell and ILC1 education. However, with respect to ILC1s, fundamental questions remain unanswered. These questions include whether ILC1s require an education process at all and, if so, whether the education is regulated by cell-bound immunoreceptor–ligand interaction. If this is true, how much of education is regulated by inhibitory receptors such as CD94/NKG2A expressed by ILC1s? Due to the lack of data for ILC1 education, we focus on the regulation of NK cell development and activation (21, 141). NK cell education not only is limited to self recognition of MHC I molecules but also involves self recognition of non-MHC ligands such as those provided by the receptor– ligand pairs CD155-TIGIT, CD48-2B4, and CLR-b-NKRP1-B (252). Moreover, it became apparent that stimulatory receptors mediating induced-self recognition are involved in the NK cell education process as well. NKG2D is a stimulatory receptor, which recognizes induced-self ligands and which regulates NK cell education (90). NKG2D is already expressed early on from the NKP stage. For induced-self ligands, it is however incompletely understood when these ligands are expressed under homeostatic conditions and which cell types would be involved in this process (132, 257, 258). Parallel to the ﬁnding that July 2020 \| Volume 11 \| Article 813 Frontiers in Immunology \| www.frontiersin.org 15 NK and ILC Development Stokic-Trtica et al. NK cells from MHC I-deﬁcient mice were hypo-responsive, a similar paradoxical phenotype was uncovered for NKG2D- deﬁcient mice having hyper-responsive NK cells despite the lack of an important stimulatory NK cell receptor. Notably, mice deﬁcient for NKG2D (Klrk1−/−) were hyper-responsive, resulting in the superior control of MCMV infection and tumor growth (257, 258). However, immunosurveillance of tumors expressing NKG2D ligands was impaired in NKG2D-deﬁcient mice (259). Mechanistically, NKG2D regulated signaling via the natural cytotoxicity receptor NKp46 and the signaling molecule CD3ζ (258). While the precise timing of NK cell education is not well-deﬁned, some studies suggested that NK cell education is not limited to a time window during development but represents a continuous process. This is supported by studies that used adoptive transfer of uneducated NK cells in MHC I-suﬃcient hosts that could restore NK cell functionality (260, 261). REFERENCES Diﬀerentiation of Type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages. Cell. (2014) 157:340–56. doi: 10.1016/j.cell.2014. 03.030 41. Walker JA, Oliphant CJ, Englezakis A, Yu Y, Clare S, Rodewald HR, et al. Bcl11b is essential for group 2 innate lymphoid cell development. J Exp Med. (2015) 212:875–82. doi: 10.1084/jem.20142224 22. Spits H, Artis D, Colonna M, Diefenbach A, Di Santo JP, Eberl G, et al. Innate lymphoid cells - a proposal for uniform nomenclature. Nat Rev Immunol. (2013) 13:145–9. doi: 10.1038/nri3365 42. Yu Y, Wang C, Clare S, Wang J, Lee SC, Brandt C, et al. The transcription factor Bcl11b is speciﬁcally expressed in group 2 innate lymphoid cells and is essential for their development. J Exp Med. (2015) 212:865–74. doi: 10.1084/jem.20142318 23. 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https://openalex.org/W4247410765	https://bmcbioinformatics.biomedcentral.com/counter/pdf/10.1186/1471-2105-14-S17-A1	English	null	Proceedings of the Twelfth Annual UT-ORNL-KBRIN Bioinformatics Summit 2013	BMC bioinformatics	2,013	cc-by	3,880	© 2013 Rouchka and Flight; 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. Proceedings of the Twelfth Annual UT-ORNL-KBRIN Bioinformatics Summit 2013 Eric C Rouchka1, Robert M Flight2 From 12th Annual UT-ORNL-KBRIN Bioinformatics Summit 2013 Buchanan, TN, USA. 22-24 March 2013 ten short talks were selected from 43 submitted poster abstracts. The University of Tennessee (UT), the Oak Ridge National Laboratory (ORNL), and the Kentucky Biomedical Research Infrastructure Network (KBRIN), have collabo- rated over the past twelve years to share research and educational expertise in bioinformatics. One result of this collaboration is the joint sponsorship of an annual regional summit to bring together researchers, educators and stu- dents who are interested in bioinformatics from a variety of research and educational institutions. This summit pro- vides unique opportunities for collaboration and forging links between members of the various institutions. This year, the Twelfth Annual UT-ORNL-KBRIN Bioinfor- matics Summit was held at Paris Landing State Park in Buchanan, TN from March 22-24, 2013. A total of 182 participants pre-registered for the summit, with 116 from various Tennessee institutions and 54 from various Kentucky institutions. A number of additional participants came from universities and research institutions from other states and countries, e.g. University of British Columbia, University of Arkansas Medical Sciences, Michigan State University, University of Cincinnati, Iowa State University, etc. Sixty-six registrants were faculty, with an additional 46 students, 43 staff, and 92 postdoc- toral participants. Correspondence: eric.rouchka@louisville.edu 1Department of Computer Engineering and Computer Science, University of Louisville, Duthie Center for Engineering, Louisville, KY 40292, USA Full list of author information is available at the end of the article INTRODUCTION Open Access Rouchka and Flight BMC Bioinformatics 2013, 14(Suppl 17):A1 http://www.biomedcentral.com/1471-2105/14/S17/A1 Rouchka and Flight BMC Bioinformatics 2013, 14(Suppl 17):A1 http://www.biomedcentral.com/1471-2105/14/S17/A1 Friday workshops y p Ramin Homayouni (University of Memphis) and Zhongming Zhao (Vanderbilt University) opened the Bioinformatics Summit with a workshop titled “Tools and Applications for Next Gen Sequencing.” Michael Dickens from the University of Memphis began the workshop with an overview of bioinformatics tools used in the assembly and annotation of de novo genomes. This presentation covered various tools involved in the pipeline aspects of sequencing and quality control, de novo assembly, genome annotation, and manual annota- tion. This included a discussion of WebApollo [http:// genomearchitect.org], a web-based community annota- tion integrated with JBrowse [1]. Pelin Jia from Vander- bilt University followed with a discussion of a pipeline for variant calling within NGS data. She discussed var- ious aspects of the pipeline, including quality control using FastQC [http://www.bioinformatics.babraham.ac. uk/projects/fastqc/]; sequence mapping; post-processing in terms of alignment, marking duplicates, realignment, and base recalibration; variant calling; variant filtering; and variant annotation. The conference program consisted of three days of presentations. The first afternoon consisted of two work- shops, one for Next-Generation Sequence Analysis, and a second on analysis of data resulting from the Conditions Affecting Neurocognitive Development and Learning in Early childhood (CANDLE) project. The remainder was dedicated to scientific presentations divided into three plenary sessions on Next-Generation Sequencing, Transla- tional Bioinformatics, and Systems Biology. In addition, The second half of the opening workshop consisted of workshops on two NGS tools. The first of these tools was MuTect [2] discussed by Huy Vuong from Vanderbilt University. MuTect is a tool used for detection of somatic mutations in cancer which incorporates information from the Catalog of Somatic Mutations in Cancer (COSMIC) [3] as well as tools for calling single nucleotide variants (SNVs). The second tool covered by Qingguo Wang was VirusFinder [4], a tool for detecting viruses and their inte- gration sites using next-generation sequencing data. Celeste Luketic from Life Technologies closed the first workshop with a discussion of the Ion TorrentTM PGMTM Rouchka and Flight BMC Bioinformatics 2013, 14(Suppl 17):A1 http://www.biomedcentral.com/1471-2105/14/S17/A1 Rouchka and Flight BMC Bioinformatics 2013, 14(Suppl 17):A1 http://www.biomedcentral.com/1471-2105/14/S17/A1 Page 2 of 4 genome sequencing of the chimp genome [11] shows that humans and chimps share a 99% similarity in non-coding regions, with nearly identical protein coding sequences. However, a number of regions have been found where the sequence is much more divergent in humans than between chimpanzees and other distantly related species. Session II: translational bioinformatics Session II: translational bioinformatics Saturday morning began with a presentation titled “Channotyping Epilepsy – Complexity in Ion Channel Gene Profiles and Personal Risk Prediction”, by Dr. Tara Klassen (Baylor College of Medicine). Dr. Klassen opened the talk by taking the vantage point that perhaps ion channels are the best markers for disease. Over 40 genetic disorders, or channelopathies, caused by ion channels have been characterized, including spinocere- bellar ataxia type 13 [15], long and short QT syndrome [16], cystic fibrosis [17], retinitis pigmentosa [18], and several forms of epilepsy. Epilepsy is a spectrum of disor- ders, affecting 2.2 million people in the United States and 65 Million worldwide [19]. Since the same ion channel gene can cause different excitability disorders in different tissues, a cohort was studied at the Baylor College of Medicine hospitals, including 152 patients with idiopathic epilepsy and 139 with neurologically normal controls. A total of 237 ion channel genes were sequenced and analyzed [20]. From the exploration of the SNPs in these ion channels, it was observed that rare severe ion channel SNPs do not predict epilepsy. In fact, individuals, both with and without epilepsy, can carry multiple mutations in human epilepsy (hEP) genes. Dr. Klassen discussed that it was more of a complex combination of SNPs within hEP genes, and that a systems approach combining sequencing, transcriptomics, proteomics, and modelling was the best approach to understanding the role of ion channel SNPs in epilepsy. Friday workshops These regions, called human accelerated regions (HARs) [12,13] were further studied. A total of 728 HARs are found across the human genome, with 69% in intergenic regions, 21% in introns, 6% in UTRs, and 4% in protein coding regions [14]. HARs tend to be enriched near tran- scription factors, developmental genes, and genes impli- cated in diseases. The thought is that these regions function as transcriptional enhancers. Dr. Capra and his group have been working on developing machine learning approaches for detecting possible roles as tissue-specific enhancers of the HARs, and experimentally validating the results. and ProtonTM sequencers. These sequencers are based on a semiconductor platform that detects nucleotide incor- poration by measuring the resultant change in pH [5]. In addition to discussing the technology and the associated software, Celeste discussed applications using Ion based technologies, including the Ion AmpliSeqTM panels. These ready-to-use panels contain a set of targeted regions for specific diseases, including the cancer hotspot panel, comprehensive cancer panel, inherited disease panel, and sample ID panel for SNP genotyping. The second workshop of the afternoon focused on “Availability and Uses of CANDLE Genomic Data.” Fran Tylavsky from the University of Tennessee Health Science Center (UTHSC) kicked off the second workshop by giving an overview of a project studying the conditions affecting neurocognitive development and learning in early childhood (CANDLE) [6]. This project, which involves a total of 1474 children (1404 which are active), collected various data using 54 instruments at 24 differ- ent time-points, resulting in over 14 million pieces of data, including approximately 900,000 sequence variants at 27,000 sites. Building upon the vast amount of data available through CANDLE, Beni Mozhui from UTHSC followed with a discussion of multiscalar analysis of CANDLE using GeneNetwork [7] and PLINK [8]. GeneNetwork is a platform designed to facilitate genetic studies and integrative systems genetics and models through data storage and data analysis while PLINK is a toolset allowing for whole-genome association and popu- lation-based linkage analyses. Rob Williams (UTHSC) closed the second workshop with a hands on demonstra- tion of GeneNetwork functionality by applying it to the CANDLE dataset. Session I: next generation sequencing Jinghui Zhang (St. Jude’s Children’s Research Hospital) began the formal program with a talk titled “Analysis of next-generation sequencing data for pediatric cancer gen- omes: discoveries, challenges and lessons learned.” In this presentation, Dr. Zhang presented a summary of discov- eries resulting from the Pediatric Cancer Genome Project (PCGP), a $65 Million collaboration between St. Jude’s and Washington University in St. Louis [9]. As part of this project, paired next generation sequencing is being per- formed at the whole genome for both tumor and normal cells for each of 600 pediatric cancer patients. Dr. Zhang discussed a number of interesting discoveries, as well as the development of tools for understanding structural variations in cancer, such as CREST [10]. Following Dr. Klassen’s talk was a presentation by Stephen Wong (Weill Cornell Medical College) on “Sys- tems and Chemical Biology Strategies for Drug Reposition- ing.” In this talk, Dr. Wang discussed approaches to repositioning old drugs, given that the current cost to bring a drug to market costs $1 billion and takes 15 years [21]. Tony Capra (Vanderbilt University) followed with a talk titled “Integrating genome-scale data to predict the effect of human-specific non-coding mutations.” Dr. Capra pre- sented a summary of the evolutionary analysis between humans and our closest relatives, chimpanzees. Whole Rouchka and Flight BMC Bioinformatics 2013, 14(Suppl 17):A1 http://www.biomedcentral.com/1471-2105/14/S17/A1 Rouchka and Flight BMC Bioinformatics 2013, 14(Suppl 17):A1 http://www.biomedcentral.com/1471-2105/14/S17/A1 Page 3 of 4 Due to this prohibitive cost, the pharmaceutical industry has moved to drug repurposing, resulting in over 40 repo- sitioned drugs [22]. Using computational methodologies, Dr. Wang proposed using knowledge-based, network- based, and disease similarity-based methodologies to aid in drug repositioning. The result is DrugMap Central, which provides an integrative view of multi-dimensional drug data, including basic chemical information, targets, target-related signalling pathways, clinical trial informa- tion, and FDA approval information [23]. incorrect assumption of “guilt by association” genes which assumes that interacting genes are likely to share similar functions [29,30]. Posters and short talks The poster session was held on day two. Forty-three pos- ters were on display, from a variety of different research areas. A number of posters were also selected for short talks. These included “Making data accessible to biologists: small group assignment of correlated genes” (Antony Athippozhy, University of Kentucky); “Giving raw data a chance to talk: a demonstration of de-identified Pediatric Research Database (PRD) and exploratory analysis techni- ques for possible research cohort discovery and identifi- able high risk factors for readmission” (Teeradache Viangteeravat, Children’s Foundation Research Institute); “Power and sample size of two-stage extreme phenotype sequencing design for next generation sequencing studies” (Guolian Kang; St. Jude’s Children’s Research Hospital), “Diffsplice: the genome-wide detection of differential splicing events with RNA-seq” (Yin Hu, University of Kentucky); “A client-oriented workshop on the essentials of next gen sequencing data acquisition and bioinformatics analysis” (Pat Calie, Eastern Kentucky University); “Gene networks in the Phytophthora capsisci/Solanum lycopersi- cum pathosystem” (Jordan Bird, University of Tennessee – Knoxville); “Our strategy to achieve and document reproducible computing” (Nisrine Enyinda, St. Jude’s Children’s Research Hospital); “Isoform reconstruction through molecule inference with statistical isoform selec- tion” (Yan Huang, University of Kentucky); “Using par- tially ordered sets to represent and predict true patterns of gene response to treatments” (Nam Vo, University of Memphis); and “Query based sampling and multi-layered semantic analysis to find robust network of associa- tion between drugs and disease” (Karthikka Ramani Muthukuri, University of Memphis). For full author lists and abstracts see the rest of the supplement. Session III: systems biology Joerg Gsponer (University of British Columbia) began the Sunday sessions with a talk titled “New insights into neu- rodegeneration by computational approaches.” In this pre- sentation, Dr. Gsponer discussed the role that intrinsically unstructured proteins (IUPs) play in neurodgeneration. Up to one-third of all proteins contain large IUP regions that lack a unique structure [24]. In addition to their lack of higher order structure, many IUPs are also found to form protein aggregates [25]. Cellular systems balance the detrimental and beneficial effect of protein aggregation. IUPs are typically found in low abundance and are short lived. A number of IUPs have been shown to have roles in neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Dr. Gsponer discussed computational approaches to understanding the regulation of aggregation prone proteins, based on the complexity of the 5’ untrans- lated region (UTR). The 5’ UTR of IUPs typically contains RNA binding motifs, with one example being KHD1 which binds two-thirds of all poly-Q/N proteins [26]. Dr. Gsponer discussed the NeuroGeM Knowledgebase [http://www.chibi.ubc.ca/neurogem/] which contains 1,218 modifiers from 8 disease models, including Alzheimer’s, Huntington’s, poly-q, Parkinson’s, Spinocerebellar ataxia, and amyotrophic lateral sclerosis. He discussed Meta- analysis techniques his group is employing to integrate functional enrichment, disease-specific modifiers, and highly interconnected modifiers to predict modifiers of IUPs for further experimental validation. g We would like to thank the additional Conference Program Committee members Nigel Cooper (University of Louisville), Dan Goldowitz (University of British Columbia), Mike Langston (University of Tennessee-Knoxville), Terry Mark-Major (University of Tennessee-Memphis), Cynthia Peterson (University of Tennessee-Knoxville), Claire Rinehart (Western Kentucky University) Arnold Stromberg (University of Kentucky), Rob Williams (University of Tennessee- Memphis) and Zhongming Zhao (Vanderbilt University) for organizing an outstanding scientific program. In addition, we wish to thank Terry Mark- Major, Michelle Padgett, Whitney Rogers, and Jane Thornton for all of their Future plans p Paul Pavlidis (University of British Columbia) closed out the invited speaker portion of the 2013 Summit with a presentation “From gene lists, networks and annotations to function.” The purpose of the presented work was to present the lack of available resources for linking together the genetic basis for diseases and phenotypes. Dr. Pavlidis described Neurocarta, a knowledgebase containing 7,000 genes and 2,000 phenotypes along with supporting evi- dence linking the genes and phenotypes [27]. Neurocarta was initially developed as a neuroscience resource, and therefore has detailed information about neurodevelop- mental disorders. In addition, Dr. Pavlidis discussed issues with annotation resources, pointing to biases and redun- dancy in Gene Ontology annotations [28] along with the The 2014 Bioinformatics summit will return to the state of Kentucky and is scheduled for April 11-13, 2014 at Lake Barkley State Park. Potential focus areas include current technological trends in molecular biology, applications of next-generation sequencing, and systems biology. References y 21. Tobinick EL: The value of drug repositioning in the current pharmaceutical market. Drug news & perspectives 2009, 22(2):119-125. 1. Skinner ME, Uzilov AV, Stein LD, Mungall CJ, Holmes IH: JBrowse: a next- generation genome browser. Genome research 2009, 19(9):1630-1638. 1. Skinner ME, Uzilov AV, Stein LD, Mungall CJ, Holmes IH: JBrowse: a next- generation genome browser. Genome research 2009, 19(9):1630-1638. 22. Chong CR, Sullivan DJ Jr.: New uses for old drugs. Nature 2007, 448(7154):645-646. 2. Cibulskis K, Lawrence MS, Carter SL, Sivachenko A, Jaffe D, Sougnez C, Gabriel S, Meyerson M, Lander ES, Getz G: Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nature biotechnology 2013, 31(3):213-219. 2. Cibulskis K, Lawrence MS, Carter SL, Sivachenko A, Jaffe D, Sougnez C, Gabriel S, Meyerson M, Lander ES, Getz G: Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nature biotechnology 2013, 31(3):213-219. 23. Fu C, Jin G, Gao J, Zhu R, Ballesteros-Villagrana E, Wong ST: DrugMap Central: an on-line query and visualization tool to facilitate drug repositioning studies. Bioinformatics 2013, 29(14):1834-1836. 3. Forbes SA, Bindal N, Bamford S, Cole C, Kok CY, Beare D, Jia M, Shepherd R, Leung K, Menzies A, et al: COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic acids research 2011, 39(Database issue):D945-950. 3. Forbes SA, Bindal N, Bamford S, Cole C, Kok CY, Beare D, Jia M, Shepherd R, Leung K, Menzies A, et al: COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic acids research 2011, 39(Database issue):D945-950. 24. Gsponer J, Futschik ME, Teichmann SA, Babu MM: Tight regulation of unstructured proteins: from transcript synthesis to protein degradation. Science 2008, 322(5906):1365-1368. 25. Linding R, Schymkowitz J, Rousseau F, Diella F, Serrano L: A comparative study of the relationship between protein structure and beta- aggregation in globular and intrinsically disordered proteins. Journal of molecular biology 2004, 342(1):345-353. 4. Wang Q, Jia P, Zhao Z: VirusFinder: software for efficient and accurate detection of viruses and their integration sites in host genomes through next generation sequencing data. PloS one 2013, 8(5):e64465. 5. Rothberg JM, Hinz W, Rearick TM, Schultz J, Mileski W, Davey M, Leamon JH, Johnson K, Milgrew MJ, Edwards M, et al: An integrated semiconductor device enabling non-optical genome sequencing. Nature 2011, 475(7356):348-352. 26. References Kim HJ, Kim NC, Wang YD, Scarborough EA, Moore J, Diaz Z, MacLea KS, Freibaum B, Li S, Molliex A, et al: Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. Nature 2013, 495(7442):467-473. 6. Volgyi E, Carroll KN, Hare ME, Ringwald-Smith K, Piyathilake C, Yoo W, Tylavsky FA: Dietary patterns in pregnancy and effects on nutrient intake in the Mid-South: the Conditions Affecting Neurocognitive Development and Learning in Early Childhood (CANDLE) study. Nutrients 2013, 5(5):1511-1530. 6. Volgyi E, Carroll KN, Hare ME, Ringwald-Smith K, Piyathilake C, Yoo W, Tylavsky FA: Dietary patterns in pregnancy and effects on nutrient intake in the Mid-South: the Conditions Affecting Neurocognitive Development and Learning in Early Childhood (CANDLE) study. Nutrients 2013, 5(5):1511-1530. 27. Portales-Casamar E, Ch’ng C, Lui F, St-Georges N, Zoubarev A, Lai AY, Lee M, Kwok C, Kwok W, Tseng L, et al: Neurocarta: aggregating and sharing disease-gene relations for the neurosciences. BMC genomics 2013, 14:129. 28. Gillis J, Pavlidis P: Assessing identity, redundancy and confounds in Gene Ontology annotations over time. Bioinformatics 2013, 29(4):476-482. 7. Wu CC, Huang HC, Juan HF, Chen ST: GeneNetwork: an interactive tool for reconstruction of genetic networks using microarray data. Bioinformatics 2004, 20(18):3691-3693. 29. Gillis J, Pavlidis P: The impact of multifunctional genes on “guilt by association” analysis. PloS one 2011, 6(2):e17258. 8. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, et al: PLINK: a tool set for whole-genome association and population-based linkage analyses. American journal of human genetics 2007, 81(3):559-575. 30. Gillis J, Pavlidis P: “Guilt by association” is the exception rather than the rule in gene networks. PLoS computational biology 2012, 8(3):e1002444. doi:10.1186/1471-2105-14-S17-A1 Cite this article as: Rouchka and Flight: Proceedings of the Twelfth Annual UT-ORNL-KBRIN Bioinformatics Summit 2013. BMC Bioinformatics 2013 14(Suppl 17):A1. 9. Downing JR, Wilson RK, Zhang J, Mardis ER, Pui CH, Ding L, Ley TJ, Evans WE: The Pediatric Cancer Genome Project. Nature genetics 2012, 44(6):619-622. 10. Wang J, Mullighan CG, Easton J, Roberts S, Heatley SL, Ma J, Rusch MC, Chen K, Harris CC, Ding L, et al: CREST maps somatic structural variation in cancer genomes with base-pair resolution. Nature methods 2011, 8(8):652-654. 11. Initial sequence of the chimpanzee genome and comparison with the human genome. Nature 2005, 437(7055):69-87. 12. Authors’ details 1D f C 1Department of Computer Engineering and Computer Science, University of Louisville, Duthie Center for Engineering, Louisville, KY 40292, USA. 2 19. Epilepsy Across the Spectrum: Promoting Health and Understanding. Washington (DC);England MJ, Liverman CT, Schultz AM, Strawbridge LM 2012:. 1Department of Computer Engineering and Computer Science, University of Louisville, Duthie Center for Engineering, Louisville, KY 40292, USA. 2 2Department of Chemistry, University of Louisville, Louisville, KY 40292, USA. 2Department of Chemistry, University of Louisville, Louisville, KY 40292, USA. 20. Klassen T, Davis C, Goldman A, Burgess D, Chen T, Wheeler D, McPherson J, Bourquin T, Lewis L, Villasana D, et al: Exome sequencing of ion channel genes reveals complex profiles confounding personal risk assessment in epilepsy. Cell 2011, 145(7):1036-1048. Rouchka and Flight BMC Bioinformatics 2013, 14(Suppl 17):A1 http://www.biomedcentral.com/1471-2105/14/S17/A1 QT and short QT syndromes: a mutation update. Human mutation 2009, 30(11):1486-1511. QT and short QT syndromes: a mutation update. Human mutation 2009, 30(11):1486-1511. efforts in dealing with the conference organization details. Funding for the UT-ORNL-KBRIN Summit is provided in part by the University of Memphis Office of the Provost, Memphis Research Consortium, Kentucky Biomedical Research Infrastructure Network (KBRIN), University of Tennessee Center for Integrative and Translational Genomics, University of Tennessee Molecular efforts in dealing with the conference organization details. Funding for the UT-ORNL-KBRIN Summit is provided in part by the University of Memphis Office of the Provost, Memphis Research Consortium, Kentucky Biomedical Research Infrastructure Network (KBRIN), University of Tennessee Center for Integrative and Translational Genomics, University of Tennessee Molecular Resource Center, UT-ORNL Science Alliance, and NIH grants P20RR16481 and P20GM103436. 17. Bobadilla JL, Macek M Jr., Fine JP, Farrell PM: Cystic fibrosis: a worldwide analysis of CFTR mutations–correlation with incidence data and application to screening. Human mutation 2002, 19(6):575-606. 18. Oh KT, Weleber RG, Lotery A, Oh DM, Billingslea AM, Stone EM: Description of a new mutation in rhodopsin, Pro23Ala, and comparison with electroretinographic and clinical characteristics of the Pro23His mutation. Archives of ophthalmology 2000, 118(9):1269-1276. Acknowledgements ld l k h We would like to thank the additional Conference Program Committee members Nigel Cooper (University of Louisville), Dan Goldowitz (University of British Columbia), Mike Langston (University of Tennessee-Knoxville), Terry Mark-Major (University of Tennessee-Memphis), Cynthia Peterson (University of Tennessee-Knoxville), Claire Rinehart (Western Kentucky University) Arnold Stromberg (University of Kentucky), Rob Williams (University of Tennessee- Memphis) and Zhongming Zhao (Vanderbilt University) for organizing an outstanding scientific program. In addition, we wish to thank Terry Mark- Major, Michelle Padgett, Whitney Rogers, and Jane Thornton for all of their Page 4 of 4 Page 4 of 4 Rouchka and Flight BMC Bioinformatics 2013, 14(Suppl 17):A1 http://www.biomedcentral.com/1471-2105/14/S17/A1 References Pollard KS, Salama SR, King B, Kern AD, Dreszer T, Katzman S, Siepel A, Pedersen JS, Bejerano G, Baertsch R, et al: Forces shaping the fastest evolving regions in the human genome. PLoS genetics 2006, 2(10):e168. 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 13. Pollard KS, Salama SR, Lambert N, Lambot MA, Coppens S, Pedersen JS, Katzman S, King B, Onodera C, Siepel A, et al: An RNA gene expressed during cortical development evolved rapidly in humans. Nature 2006, 443(7108):167-172. Submit your next manuscript to BioMed Central and take full advantage of: 14. Kostka D, Hubisz MJ, Siepel A, Pollard KS: The role of GC-biased gene conversion in shaping the fastest evolving regions of the human genome. Molecular biology and evolution 2012, 29(3):1047-1057. 15. Waters MF, Minassian NA, Stevanin G, Figueroa KP, Bannister JP, Nolte D, Mock AF, Evidente VG, Fee DB, Muller U, et al: Mutations in voltage-gated potassium channel KCNC3 cause degenerative and developmental central nervous system phenotypes. Nature genetics 2006, 38(4):447-451. y y 16. Hedley PL, Jorgensen P, Schlamowitz S, Wangari R, Moolman-Smook J, Brink PA, Kanters JK, Corfield VA, Christiansen M: The genetic basis of long 16. Hedley PL, Jorgensen P, Schlamowitz S, Wangari R, Moolman-Smook J, Brink PA, Kanters JK, Corfield VA, Christiansen M: The genetic basis of long
https://openalex.org/W3192471488	https://jbba.scholasticahq.com/article/27192.pdf	English	null	Principles of Natural Resource Economics for Bitcoin	The journal of the British Blockchain Association	2,021	cc-by	5,099	1. Resource Economics and Bitcoin being used in the present rather than the future, the ‘excess’ representing the opportunity cost of intertemporal substitution in consumption. The claim that Bitcoin is ‘digital gold’ rests first and foremost on the soundness of the idea that Bitcoin behaves like a traditional exhaustible resource1; that it is more akin to gold than any other depletable resource relies further on the premise that it is relatively scarcer than gold. To that end, it is significant that Bitcoin has been programmed to mimic the essential characteristics of an exhaustible resource: its extraction rate approaches 0 over time and the total yield feasible from ‘mining’ is limited to 21 million bitcoins. It is, therefore, worth examining what analytical value the economics of exhaustible resources provides for the case of Bitcoin. While the market value of the natural stock of an unextracted resource depends on the prevailing market price and the attendant costs of bringing the resource to the market, the opportunity costs depend on trading off future consumption possibilities with present use as well as considerations on the present value of the rent that is destroyed by extracting in the present rather than leaving the resource in situ for the future. The difference between marginal extraction costs and the price is often called the Hotelling rent in recognition of Hotelling’s seminal 1931 paper [2]. It further follows from the preceding observations that the rate of change in price of the depletable resource must equal the interest rate that a miner uses to discount the future, and this is known as the Hotelling r -percent growth rule. Whenever marginal extraction costs are zero, the price of the resource in stock and that of the unmined resource are equivalent and the Hotelling rule applies equally to both. If, however, extraction costs increase over time, the price of the resource rises at less than the discount interest. The economics of exhaustible resources has one rather simple and compelling analytical premise. It is that the opportunity costs incurred from current extraction and consumption of an exhaustible resource must be weighed against the fact that limited supplies ought to generate returns over time. A miner must, therefore, consider both the market value of a resource and the opportunity costs of current extraction in its investment decisions. Abstract To assess claims such as Bitcoin is ‘digital gold’ it makes sense to examine whether Bitcoin exhibits features common to other exhaustible natural resources that are the concern of natural resource economists. We therefore present some foundational ideas in the economics of exhaustible resources and examine their relevance to Bitcoin. There are several useful similarities but also some key differences, chiefly with respect to how miners manage inventories, or their ‘inventory policy’. Therefore, to highlight this aspect, we use a simple model for any physical natural resource and introduce sensitivity to a capital-to-energy ratio. The resulting policy for Bitcoin miner over a halving cycle is not unlike a traditional miner in that optimal inventories are determined by optimal capital investments over the entire duration of the cycle. Keywords: Bitcoin, miner, exhaustible resources, inventory policy JEL Classifications: Q3, D21 and G31 Keywords: Bitcoin, miner, exhaustible resources, inventory policy Keywords: Bitcoin, miner, exhaustible resources, inventory policy JEL Classifications: Q3, D21 and G31 JEL Classifications: Q3, D21 and G31 being used in the present rather than the future, the ‘excess’ representing the opportunity cost of intertemporal substitution in consumption. PEER REVIEWED RESEARCH OPEN ACCESS ISSN Online: 2516-3957 ISSN Print: 2516-3949 https://doi.org/10.31585/jbba-4-2-(2)2021 1 See [1] for a recent comparison of Bitcoin (specifically, price and hashrate behaviour) in terms of established results in energy economics regarding the oil and gas industry. Principles of Natural Resource Economics for Bitcoin Prateek Goorha Bridgewater State University, Massachusetts, USA Correspondence: goorha@sent.com Received: 16 March 2021 Accepted: 23 June 2021 Published: 4 August 2021 Correspondence: goorha@sent.com Correspondence: goorha@sent.com Correspondence: goorha@sent.com Received: 16 March 2021 Accepted: 23 June 2021 Published: 4 August 2 Correspondence: goorha@sent.com Received: 16 March 2021 Accepted: 23 June 2021 Published: 4 August 2021 p g @ Received: 16 March 2021 Accepted: 23 June 2021 Published: 4 August 2021 PEER REVIEWED RESEARCH Published Open Access under the CC-BY 4.0 Licence 1. Resource Economics and Bitcoin The price of any such depletable resource should exceed its marginal cost of extraction to capture any logic of an exhaustible resource Thus, all things being equal, an increase in the discount rate implies a higher price for the unextracted resource and would incentivise a faster rate of extraction. In Bitcoin, 1 See [1] for a recent comparison of Bitcoin (specifically, price and hashrate behaviour) in terms of established results in energy economics regarding the oil and gas industry. The JBBA \| Volume 4 \| Issue 2 \| 2021 Published Open Access under the CC-BY 4.0 Licence 1 1 while the rate of extraction is algorithmically fixed for any given halving cycle, mining effort can readily be increased. Further, miners arguably discount hyperbolically for the simple reason that the total remaining in situ stock is known in advance as well as the fact that extraction costs are likely to rise exponentially into the future as all miners increase efforts. higher the difficulty6 of the cryptographic problem and vice versa, essentially adjusting the size of the resource field for miners based directly on their efforts. This isn’t very different from any exhaustible natural resource that has increasing extraction costs over the long run with periods of falling costs that eventuate from new discoveries of resource sites or cost-saving technologies. In contrast with any other natural resource, Bitcoin’s exhaustion trajectory is far more deterministic. Many of the other features immanent to Bitcoin are, as a matter of fact, similar in nature to the assumptions made by Hotelling [2] in that Bitcoin mining is competitive, 7 the overall stock of bitcoins is known exactly and that, while technology for mining does improve, it does so in lock-step with the algorithmically adjusted difficulty for mining. Thus, the Hotelling rule ought to provide at least a useful starting point for the case of Bitcoin. A relationship between Bitcoin and the economics of natural resources has been examined in a few other papers. [3] presents a continuous-time model for the inventory policy of miners in Bitcoin that permits examining how miners optimise over the income generated from transaction fees, while also accounting for risks that emanate from demand- side shocks. 2 See [5] for a useful review of the literature inspired by Hotelling. 3 We assume a basic familiarity Bitcoin; for the uninitiated, we suggest reading [6] and the literature cited in that paper. 4 In this regard, [7] shows that the marginal cost of mining provides a strong support for the price of Bitcoin, making the analogy to natural resources stronger and to a pure speculative asset weaker. 5 Recall that this involves using the SHA-256 hashing function twice, compressing arbitrary sized inputs into a fixed-length output in the process. 6 Recall that the difficulty is adjusted by the Bitcoin code for every 2016 blocks, based upon whether the hashpower deployed over the network is trailing or leading a target of 10-minutes per block. 7 The intuition is relatively straightforward. When an exhaustible resource is mined under conditions of monopoly, it will be extracted at a more gradual pace and price will remain above the marginal rate of extraction as it grows at a relatively more stable rate. Conversely, when the same resource is mined competitively, the rate of extraction will be higher, over a shorter horizon, and the price will grow faster over the entire period. 8 This is not to say that, just like any other natural resource that is competitively exploited under rules of free access, mining in Bitcoin does not create social costs. Rather, the point is that competitive extraction creates an externality on network security in Bitcoin, which is an effect not seen with physical natural resources. 1. Resource Economics and Bitcoin By contrast, the ambition of this chapter is to present a simple model that places emphasis on highlighting the parallels in Bitcoin with natural resource mining and examines the inventory policy of Bitcoin miners over the course of a halving cycle. In this ambition, a notable contribution is the empirical analysis presented in [4]. The authors discuss the importance of the Hotelling rule to natural resource and energy economics, provide a useful review of the literature and, using Bitcoin as a case study, show strong support in the data that mining rents in Bitcoin are associated with the market rate of return. As opposed to exhaustible natural resources, where the Hotelling rent depends on whether the resource is left in situ, decisions on timing when to mine bitcoin, however, isn’t a free variable. While miners frequently do pool their resources into one of several larger mining pools to maximise their chances of finding a block, in general, mining cannot be unitised in Bitcoin in any meaningful way. Thus, bitcoins are mined competitively and with a strictly decreasing yield over time – a block reward that halves roughly every four years. Further, with Bitcoin, there is no possibility of unexpected discoveries or new technologies making currently inaccessible reservoirs of resources suddenly available for exploitation. 1.1 Some Slight Differences The Hotelling r-percent growth rule is sensitive to several factors in practice that have useful analogies in Bitcoin. 2 ,3 These include: (a) the marginal cost incurred by the miner in exploration and extraction of the resource, which in the case of Bitcoin depends on the network difficulty and the requisite hashrate4; (b) the perceived scarcity of the resource, i.e. the point on Bitcoin’s overall trajectory of bitcoins mined relative to its absolute cap or total depletion; (c) the level of competition in mining, or, for Bitcoin, the relative hashrate of a miner to the overall hashrate being deployed by other miners across the network. 2 See [5] for a useful review of the literature inspired by Hotelling. 1.2 And a Key Point of Difference For a non- perishable good like Bitcoin, this creates a situation for intertemporal arbitrage and increases the marginal propensity to save in earlier periods. Indeed, both these patterns – price growth rates far exceeding the discount rate and a high marginal propensity to save – have been characteristic of the majority of Bitcoin’s history. In addition to reservation demand by miners, demand for bitcoins is often as a hedge against inflation and systemic risks; an increasing fraction of its consumers, be they individuals or institutional entities, use it as a store of value. As such, the asset is removed from active circulation and becomes the numeraire for measuring intertemporal wealth. Growing long- term inventories have the effect of modulating the Bitcoin market into even more of a traditional exhaustible and non- renewable scarce resource. In other words, when selling from inventory becomes the predominant source of supply in the market, the optimal time-path of production of traditional exhaustible resources becomes more applicable to the time- path of Bitcoin inventory depletion. For example, as demand becomes more inelastic with high levels of supply-side market concentration, the propensity to sell reduces. An increasing rate of resource depletion over time, coupled with the prospect of decreasing elasticity, requires the rate of growth in price to keep outstripping the discount rate. For a non- perishable good like Bitcoin, this creates a situation for intertemporal arbitrage and increases the marginal propensity to save in earlier periods. Indeed, both these patterns – price growth rates far exceeding the discount rate and a high marginal propensity to save – have been characteristic of the majority of Bitcoin’s history. 𝑃(𝑏𝑡−𝑣𝑡) = 1 𝑟𝑏 ⁄ 𝐸[𝑃(𝑣𝑡+ 𝑚𝑡+1 −𝑣𝑡+1)] −𝑤. The Hotelling growth rule suggests that, within each halving period for Bitcoin, prices would have to rise at least by the rate of interest for miners to be indifferent about whether to increase mining effort or to delay it. The difference between the rate of growth of the spot price and the interest rate modulates mining effort. Note that this is a different consideration for miners than their incentives to deploy costly hashrate in response to extant difficulty levels. With high capital costs for mining, short-term supply is inelastic, adding a secondary factor to inventory levels, besides planned reservation demand; the more inelastic shorter-term supply is, the more price volatility we ought to expect from changes in demand. 1.2 And a Key Point of Difference It is well established that excessive competition in the rapid exploitation of a resource leads to social waste; a common property problem of restricting access drives the familiar tragedy of the commons. Bitcoin, however, expressly relies on and exploits the incentives that create the common property problem. While the mining of bitcoins is governed by an algorithmic mechanism that encourages ‘excessive’ competition, the value of the resulting waste that accrues from this mad rush of mining is internalised to the security of the Bitcoin network, since higher network hashrates directly result in commensurate difficulty increases.8 Being a digital resource, the size of the resource ‘field’ to be explored in Bitcoin over time can, in theory, increase or decrease in proportion with the exploratory effort of the miners. The miners deploy the hashpower of their mining rigs in order to increase the probability that their efforts to solve the cryptographic problem 5 are successful, thereby earning them the right to add their block to the Bitcoin blockchain and receive the block reward. The more hashpower that is brought to bear across the network, the p 8 This is not to say that, just like any other natural resource that is competitively exploited under rules of free access, mining in Bitcoin does not create social costs. Rather, the point is that competitive extraction creates an externality on network security in Bitcoin, which is an effect not seen with physical natural resources. The JBBA \| Volume 4 \| Issue 2 \| 2021 2 Published Open Access under the CC-BY 4.0 Licence 2 Since bitcoins are not perishable, a miner’s decision on production efforts over the extraction time path is interlinked with its policy over inventory levels. In contrast to a miner that mines a perishable natural resource competitively, a miner in Bitcoin selects a time path for the rate at which it adds to its inventory rather than the rate of extraction, which is exogenous. 9 To the extent that there are no real alternate uses for dedicated mining rigs, miners can really only engage in mining other proof of work cryptocurrencies to optimise their Hotelling rent. 1.2 And a Key Point of Difference The reservation demand of miners – or the mined bitcoins that are held in inventory by miners – is influenced, in equilibrium, by the return that their stores generate, which must be equal to the return that the miners can achieve from alternate assets.9 Figure 1: Miner Rolling Inventory and Price (Data Source: ByteTree) So the available supply of bitcoins, tb , during period t depends on the coins mined during t , t m , and the stock of bitcoins sold by miners from their inventory, 1 tv − . ‘Consumption’ of bitcoins during t must equal the available supply net of the number of bitcoins that miners hold back in their inventory. Thus, 1 = t t t b m v − + and = t t t c b v − . The inventory level is drawn down if the mining costs incurred between periods exceed the expected return from bitcoin as estimated by miners, br . Thus, the price of the bitcoins held in inventory by the miners must be greater than or equal to the costs they incur to store the coins, w , and the rate of return that they expect to receive on their inventory. In other words, for < 0 tv : Figure 1: Miner Rolling Inventory and Price (Data Source: ByteTree) In addition to reservation demand by miners, demand for bitcoins is often as a hedge against inflation and systemic risks; an increasing fraction of its consumers, be they individuals or institutional entities, use it as a store of value. As such, the asset is removed from active circulation and becomes the numeraire for measuring intertemporal wealth. Growing long- term inventories have the effect of modulating the Bitcoin market into even more of a traditional exhaustible and non- renewable scarce resource. In other words, when selling from inventory becomes the predominant source of supply in the market, the optimal time-path of production of traditional exhaustible resources becomes more applicable to the time- path of Bitcoin inventory depletion. For example, as demand becomes more inelastic with high levels of supply-side market concentration, the propensity to sell reduces. An increasing rate of resource depletion over time, coupled with the prospect of decreasing elasticity, requires the rate of growth in price to keep outstripping the discount rate. subjectto ( ) = ( ) ( ( )) ( ) k t v t f k t k t  − , Assume that at time t a miner uses capital, ( ) K t , and energy, ( ) E t , as inputs in mining Bitcoin, ( ) B t . To simplify the analysis, assume that energy costs grow at a steady rate of > 0  . where = ( )    + , ( ) > 0 kf k and ( ) < 0 kk f k . where = ( )    + , ( ) > 0 kf k and ( ) < 0 kk f k . Choice over optimal inventory levels, * [0,1] v  , over the halving cycle for the miner is a function of the dynamics of the optimal capital-to-energy ratio, ( ) k t , over the period. We can assess the trajectory of the optimal inventory level, v , by appealing to the Pontryagin maximum principle ( ) PMP . To do so, we define a function, * g , for which we assume * 0 > 0 g at = 0 t , such that * * * * * * * 0 ( ) = (1 ( )) ( ( )) ( )( ( ) ( ( ) )) k k g t g v t f k t g t v t f k t  − − − − and ( ) = 0 g H . Thus, the rate of bitcoins extracted is given by the miner’s production function ( ) = ( ( ), ( )) B t F K t E t , where F provides constant returns to scale. The miner reserves some of the output as inventory ( ) V t for investment and sells the rest to the market, ( ) C t , at prevailing prices to cover expenses. Thus, ( ) = ( ) ( ) B t V t C t + . If the share of mined coins reserved for inventory is ( ); [0,1] t   , we can write ( ) = (1 ( )) ( ) C t t B t  − . If the share of mined coins reserved for inventory is ( ); [0,1] t   , we can write ( ) = (1 ( )) ( ) C t t B t  − . subjectto ( ) = ( ) ( ( )) ( ) k t v t f k t k t  − , Now assume that the deployed mining rigs become out of date at a rate > 0  , governed by difficulty adjustments as well as exogenous improvements in technology. Therefore, the growth rate of capital for the miner is given by: The PMP then says that the Hamiltonian, H : * * * * * H( , ( ), ( ), ) = (1 ) ( ( )) ( )( ( ( )) ( )) t k t g t v v f k t g t vf k t k t  − + − is maximised by the optimal inventory trajectory. is maximised by the optimal inventory trajectory. ( ) = ( ) ( ) ( ) ( ) K t t B t t K t   − . • As the cycle approaches its completion – i.e. for t nearing H − ( ) <1 g t . With ( ) <1 < [0, ] g t t t H  , we would have * * ( ) = ( ) k t k t  − and optimal inventory over the entire halving cycle would just be zero and the optimal capital-to-energy ratio would simply be given by (0) e t k  − . In terms of units of energy expended, we can redefine these variables so that we have 𝐵 𝐸= 𝑏, 𝑉 𝐸= 𝑣, 𝐶 𝐸= 𝑐 and 𝐾 𝐸= 𝑘. Permitting ( ) = ( , ) f k F k e allows us to state Bitcoin’s average energy requirement in market consumption, ( ) = (1 ( )) ( ( )). c t v t f k t − We assume that ( ) f k is concave, so that the marginal product of capital increases at a decreasing rate. • However, it is also possible for there to exist some time, 𝑡̂, within the cycle where 𝑔∗(𝑡̂) = 1, while ( ) <1 g t for < t H . For the cases where the optimal inventory is positive, the situation is governed by whether k is above or below the steady-state rate of k , or the level of k that satisfies ( ) = kf k . 1.2 And a Key Point of Difference On the other hand, there are two countervailing effects that arise on market prices from a natural resource being stored in inventory, especially in the presence of speculative capital. While increasing inventories during periods of declining prices results in price depressions becoming less severe, disposing stocks from inventories curtails price spikes during periods of relative shortages in market supply. Figure 1 illustrates these effects for Bitcoin over the course of a year beginning in March 2020, using the metric of miner rolling inventory (MRI), which exceeds 100 when miners sell from inventories at a faster pace than they mine. However, as the stock of a durable good increases, demand must grow at a faster rate than the discount rate for price to increase, regardless of the costs and rate of resource extraction. This can be seen as the minimum threshold for the rate of growth in demand for a stock-to-flow ratio to have a secular effect on price. The price profile has a U-shape for partially durable resources with growth in demand when mining is competitive. [5] Whether the logic applies to Bitcoin depends on whether it can be seen as partially durable. To the extent that the prospect of adverse regulation, elastic rehypothecation, lost wallets and hacked accounts decreases the fully durable characteristic of Bitcoin, this becomes more relevant. 9 To the extent that there are no real alternate uses for dedicated mining rigs, miners can really only engage in mining other proof of work cryptocurrencies to optimise their Hotelling rent. The JBBA \| Volume 4 \| Issue 2 \| 2021 Published Open Access under the CC-BY 4.0 Licence Published Open Access under the CC-BY 4.0 Licence 3 2. Modeling the Bitcoin Miner’s Inventory Policy benefit from a given halving cycle [0, ] H as 0 ( ) H c t dt  , which then also determines the miner’s strategy over inventory, ( ) v t , where 0 t H  . Given the particular importance of inventories, it is worth considering a stylised model for inventory policy in Bitcoin that underscores the relationship of the policy to optimising capital investments. As a digital resource, mining in Bitcoin depends most critically on the hashing power of the mining ‘rigs’ and their energy efficiency. Thus, the model gives scenarios for which inventory levels can vary between 0, 1 and some fraction between for a given cycle, depending directly on the optimal capital-to-energy ratio. In other words, the miner’s optimisation problem can be stated as, 0 Max (1 ( )) ( ( )) H v t f k t dt −  , subjectto ( ) = ( ) ( ( )) ( ) k t v t f k t k t  − , References: The model could be usefully extended to allow for a determinate time path of extraction in Bitcoin, since miners are often faced with additional considerations for their inventory policy. First, miners can readily adjust their optimal capital stock upwards in reaction to a bull market. They cannot, however, reduce capital stock swiftly in a bear market. While inventories can certainly help offset the costs of overcapitilisation in a bull market, they can also build during the bear market in anticipation of the next upward swing in prices and demand. Thus, rather than permitting obsolescence from higher difficulty adjustments in the network, miners are forced to be forward-looking in their inventory policy. Second, to the extent that a determinate extraction path forces the hands of miners in Bitcoin, the option value of investment can come from timing over selecting optimal capital levels (as in [9]) or from simply building up inventories. [1] Fantazzini, Dean and Nikita Kolodin (2020) “Does the Hashrate Affect the Bitcoin Price?”, Journal of Risk and Financial Management, Vol. 13, No. 11, pp. 263. [2] Hotelling, Harold (1931) “The Economics of Exhaustible Resources”, Journal of Political Economy, Vol. 39, pp. 137–75. [3] Dai, Min; Wei Jiang; Steven Kou and Cong Qin (2021) “From Hotelling to Nakamoto: The Economics of Bitcoin Mining”, NUS RMI Working Paper Series, No. 2021-01. [4] Landry, Craig E; Dylan Turner and Jeffrey H. Dorfman (2019) “Hotelling Meets Crypto-Currency: Do Bitcoin Rents Follow Hotelling’s Rule?”, SSRN Working Paper. [5] Devarajan, Shantayanan and Anthony C. Fisher (1981) “Hotelling’s ‘Economics of Exhaustible Resources’: Fifty Years Later”, Journal of Economic Literature, Vol. 19, No. 1, pp. 65–73. subjectto ( ) = ( ) ( ( )) ( ) k t v t f k t k t  − , So, if at 𝑡< 𝑡̂, ( ) >1 g t , it is the case that 𝑘∗(𝑡̂) is less than k and then (0) k was less than k , and we should expect ( ) =1 v t for all 𝑡< 𝑡̂. • However, it is also possible for there to exist some time, 𝑡̂, within the cycle where 𝑔∗(𝑡̂) = 1, while ( ) <1 g t for < t H . For the cases where the optimal inventory is positive, the situation is governed by whether k is above or below the steady-state rate of k , or the level of k that satisfies ( ) = kf k . So, if at 𝑡< 𝑡̂, ( ) >1 g t , it is the case that 𝑘∗(𝑡̂) is less than k and then (0) k was less than k , and we should expect ( ) =1 v t for all 𝑡< 𝑡̂. Logically, the capital deployment path for the miner depends on both the amount that is invested, in terms of capital’s energy requirement, and through considerations over its obsolescence and associated considerations on the availability of energy. Therefore, ( ) = ( ) ( ( )) ( ) ( ) k t v t f k t k t   − + . • Between these two extrema for the optimal inventory strategy, over the halving cycle there may be some positive spans of time for which ( ) =1 g t or, in other words, Crucially, the halving cycles for the block reward plays a key role in Bitcoin. It is, therefore, useful to consider a miner’s The JBBA \| Volume 4 \| Issue 2 \| 2021 Published Open Access under the CC-BY 4.0 Licence 4 ( ) = 0 g t . This suggests that * ( ( )) = kf k t  and *( ) = k t k for that span of time. In turn, during that time ( ) = 0 k t , the optimal inventory policy is thus given by 0 < 𝛽(𝑘̅ 𝑓(𝑘̅) ⁄ ) < 1. 3. Concluding Remarks Examining Bitcoin as a digital resource not unlike a traditional natural resource permits us to seek some useful insights from natural resource economics. Indeed, as just another resource, the model presented above ought to seem intuitive, and miners of Bitcoin ought to behave largely like miners of any other physical resource. [6] Goorha, Prateek (2020) “Bitcoinomics 101: principles of the Bitcoin market”, Economics Bulletin, Vol. 40, Issue 1, 163–176. [7] Hayes, Adam S. (2019) “Bitcoin price and its marginal cost of production: support for a fundamental value”, Applied Economics Letters, Vol. 26, Issue 7, Pages 554–560. The relevance of scarcity of a non-renewable resource to economic growth has largely only been muted on account of positive elasticities of substitution in production or by technological breakthroughs [10]. A contrary logic is applicable to scarce resources that are used as stores of wealth to the extent that they become effective additions to a diversified portfolio. It is particularly worth considering the effects of substitutability between Bitcoin and other physical stores of wealth, such as gold, rare collectibles and real estate.10 [8] Levhari, David, and Robert S. Pindyck (1981) “The Pricing of Durable Exhaustible Resources”, The Quarterly Journal of Economics, Vol. 96, Issue. 3, pp. 366–377. [9] Arrow, Kenneth and Anthony C. Fisher (1974) “Environmental Preservation, Uncertainty and Irreversibility”, Quarterly Journal of Economics, Vol. 88, pp. 312–320. [10] Solow, Robert M. (2016) “Resources and Economic Growth”, The American Economist, Vol. 61, Issue. 1, pp. 52–60. Competing Interests: None declared. [11] Cebrián-Hernández, Ángeles and Enrique Jiménez- Rodríguez (2021) “Modeling of the Bitcoin Volatility through Key Financial Environment Variables: An Application of Conditional Correlation MGARCH Models”, Mathematics, Vol. 9, Issue 3, pp. 267. Author’s contribution: Prateek Goorha is the main author responsible for writing the manuscript, collecting data, proofreading, etc. 10 Most recently, [11] shows uncorrelation between Bitcoin and both gold and oil and correlation with stocks of companies associated with cryptocurrencies and electronic payment systems. The JBBA \| Volume 4 \| Issue 2 \| 2021 The JBBA \| Volume 4 \| Issue 2 \| 2021 5 5 Published Open Access under the CC-BY 4.0 Licence Published Open Access under the CC-BY 4.0 Licence
W4387363077.txt	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0292470&type=printable	en		Fear, stress, anxiety, depression and insomnia related to COVID-19 among undergraduate nursing students: An international survey	PloS one	2,023	cc-by	6,179	PLOS ONE RESEARCH ARTICLE Fear, stress, anxiety, depression and insomnia related to COVID-19 among undergraduate nursing students: An international survey Mohammed Al Maqbali ID1☯, Norah Madkhali2☯, Alexander M. Gleason ID3☯, Geoffrey L. Dickens ID4,5☯* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Fatima College of Health Sciences, Al Ain, United Arab Emirates, 2 Jazan University, Jizan, Saudi Arabia, 3 Fatima College of Health Sciences, Abu Dhabi, UAE, 4 Mental Health Nursing Department of Nursing, Midwifery and Health Faculty of Health and Life Sciences, Northumbria University, Newcastle-Upon-Tyne, United Kingdom, 5 Adjunct Professor Western Sydney University, Penrith, Australia ☯ These authors contributed equally to this work. * geoffrey.dickens@northumbria.ac.uk Abstract OPEN ACCESS Citation: Al Maqbali M, Madkhali N, Gleason AM, Dickens GL (2023) Fear, stress, anxiety, depression and insomnia related to COVID-19 among undergraduate nursing students: An international survey. PLoS ONE 18(10): e0292470. https://doi.org/10.1371/journal.pone.0292470 Editor: Ramona Bongelli, University of Macerata: Universita degli Studi di Macerata, ITALY Received: February 19, 2023 Accepted: September 21, 2023 Published: October 5, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0292470 Copyright: © 2023 Al Maqbali 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. The emergence of COVID-19 has produced unprecedented change in daily life activities leading to major impacts on psychological wellbeing and sleep among individuals worldwide. The study aimed to assess levels of fear, stress, anxiety, depression, and insomnia among undergraduate nursing students in four countries two years after the start of the pandemic. An international, multi-centre cross-sectional electronic survey was conducted between December 2021 and April 2022. An on-line questionnaire was distributed via Qualtrics® and JISC® software. Instruments included the Fear of COVID-19 Scale, the Perceived Stress Scale, the Hospital Anxiety and Depression Scale and the Insomnia Severity Index, and a demographics and academic background questionnaire. The independent variables included demographic and academic backgrounds, while fear level, stress, anxiety, depression, and insomnia were the dependent variables. A total of 918 undergraduate nursing students from KSA, Oman, UK, and UAE were participants in the study. Students presented with stress (91.6%), anxiety (69.1%), depression (59.8%), and insomnia (73.2%). The participants’ mean Fear of COVID-19 Scale score was 12.97 (SD = 6.14). There were significant positive relationships between fear of COVID-19, stress, anxiety, depression, and insomnia. Undergraduate nursing students experienced moderate to severe levels of Fear of COVID-19, stress, anxiety, depression, and insomnia two years after the onset of the COVID-19 pandemic. Psychological intervention and peer support are needed to reduce the long-term adverse outcomes of mental health problems and insomnia. It is important to introduce education about crisis management of infectious disease during pandemics into the nursing curriculum to increase student knowledge and improve their preparedness for such emergencies. Data Availability Statement: As the data are collected from four countries, they are not publicly available due to the Rules Governing the Ethics of Scientific Research. Other researchers may obtain PLOS ONE \| https://doi.org/10.1371/journal.pone.0292470 October 5, 2023 1 / 12 PLOS ONE access to the data directly from the Ethics Review Committee at Fatima College of Health Sciences (fchs.ethics@fchs.ac.ae). Funding: The authors received no specific funding for this work. Competing interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Psychological distress among nursing students 1 Introduction The world has changed considerably since December 2019 when the novel coronavirus disease (COVID-19) emerged in Wuhan City, Hubei province, China, and rapidly spread worldwide [1]. The World Health Organization (WHO) declared COVID-19 to be a pandemic on January 30, 2020 [2]. COVID-19 has posed a serious threat to human health, notably there have been more than 750 million fatalities associated with the disease [3]. Additionally, the uncertain nature of consequences of the disease has caused unexpected changes in social life, work, and travel activities. Since the moment WHO declared COVID-19 as a pandemic, governments throughout the world implemented strict measures including lockdowns to reduce the transmission rate of the virus [4]. Consequently, higher education institutions were closed and most shifted to remote modes of educational delivery. Nursing students experienced a decrease in traditional classroom learning due to the pandemic, and at the same time, they were required to engage in essential clinical practice places as part of their education. This double impact affected their learning experience and demanded additional responsibilities during their training. Several researchers found that nursing students might experience of anxiety and stress during clinical placement [5, 6]. It is known that, even outside the context of a global pandemic, nursing students indicate that difficult learning materials, stringent examinations, long hours of study, the challenges of clinical placements, and the physical and emotional demands of programs can lead to mental health problems such as, stress, anxiety, and depression [7–9]. Previous studies during Severe Acute Respiratory Syndrome and Middle East Respiratory Syndrome outbreaks revealed that nursing students experienced high stress levels due to their increased risk of infection from direct patient contact [10–12]. Similarly, the mental health of pre-registration nursing students has been affected during the COVID-19 pandemic, as evidenced by reports of negative emotions, fear, confusion, pessimism, sleep disturbance, and an increasing number of psychological problems [13, 14]. This can lead to increased attrition rates from nursing education [15]. However, the mental health status of undergraduate nursing students after two years of the COVID-19 pandemic has yet to be explored. The contribution of this study, therefore, is to ascertain the mental health status of nursing students two years after the WHO first identified COVID-19 as a pandemic. This is crucial in enabling better planning for interventions to prevent and manage the mental health problems of nursing students in the event of the emergence of similar or other diseases in the future. The specific objectives of this study were to describe the prevalence of fear, stress, anxiety, depression, and insomnia, and their relationship with academic demographic variables, among undergraduate nursing students in Kingdom of Saudi Arabia (KSA), Oman, United Kingdom (UK), and United Arab Emirates (UAE) after more than two years of the COVID-19 pandemic. 2 Methods 2.1 Study design This international collaborative study used a web-based cross-sectional design and was conducted in four countries (KSA, Oman, UK, and UAE). The sample consisted of undergraduate nursing students who were attending higher education institutions in participating countries (UK—Northumbria University, Oman—Oman College of Health Sciences, Saudi Arabia— Jazan University, UAE—Fatima College of Health Sciences). The study questionnaire was created using Qualtrics1 and JISC1 software for electronic distribution. The participants were recruited via email invitations from their respective institutions. All data collection was PLOS ONE \| https://doi.org/10.1371/journal.pone.0292470 October 5, 2023 2 / 12 PLOS ONE Psychological distress among nursing students conducted between December 2021 and April 2022. The inclusion criteria for participating in the study were an ability to speak and write in English, being above 18 years of age, and being a pre-registration nursing student. Ethical approval was obtained from committees at each institution by research and ethical review and approval Committee, Oman College of Health Sciences (OCHS/REC/PROPSAL-APPROVED/1/2021), Standing Committee for Scientific Research, Jazan University (REC43/03/047), research and ethical review and approval Committee, Fatima College of Health Sciences (78220), and Northumbria University research ethics committee (35636). A written informed consent statement was presented on the first screen of the survey tool and participants were required to click a button to confirm their consent before they could complete the survey. If the participant chose not to consent, then they were directed to the end of the survey. 2.2 Measures Data on demographic and academic backgrounds were gathered, encompassing information such as age, sex, marital status, year of study, and type of learning (online only, online and attendance, attendance only). Additionally, students were asked about their COVID-19 infection, vaccination status, and participation in clinical placements throughout the pandemic. Fear level was determined using Fear of COVID-19 Scale (FCV-19S) [16]. This is a selfrated 7-item scale scored on 5-point Likert scales ranging from 1 (strongly disagree) to 5 (strongly agree). Total scores range from 7 to 35, with higher scores indicating higher levels of COVID-19-related fear. A cut-off of 17.5 indicates high fear level [17] and was adopted for this study. In previous research, Cronbach’s α values were found to be 0.82, which demonstrated good internal reliability [16]. The FCV-19S has been thoroughly validated as a tool to measure fear of COVID-19 among nursing students from various countries and demonstrated excellent internal consistency [18]. In this study the Cronbach’s α was 0.88. Stress was measured using the Perceived Stress Scale (PSS), a 10 item scale with response on a 4-point scale ranging from 0 (never) to 4 (very often), with overall scores ranging from 0 to 40 [19]. Scores �14 indicate the presence of moderate stress [19]. The PSS-10 version is reported to have acceptable internal consistency (α = 0.70) [20]. The PSS has undergone extensive validation in nursing students and consistently demonstrated good internal consistency, with a reported Cronbach’s alpha coefficient of 0.93 [21]. In the current study Cronbach’s α was 0.81. The Hospital Anxiety and Depression Scale (HADS). comprises 14 items assessing anxiety (7-items; HADS-A) and depression (7-items; HADS-D) [22]. Each is rated on a 5-point response scale (from 0 to 4). The scores in each subscale are computed by summing the corresponding items (possible scores of 0–21 for each. subscale). A score of 0–7 is considered normal, 8–10 a borderline case, and 11–21 a case exhibiting anxiety or depression [22]. The cutoff value of 8 and above for both HADS-A and HADS-D [23] was adopted for this study. The HADS has demonstrated very good internal consistency among nursing student (Cronbach’s α = 0.82) [24]. In the current study, Cronbach’s α was 0.63 for the HADS-A and 0.63 for the HADS-D. The Insomnia Severity Index (ISI) is a seven item self-report questionnaire assessing the nature, severity, and impact of insomnia [25]. A 5-point scale is used to rate each item (0 = no problem; 4 = very severe problem), yielding a total score ranging from 0 to 28. The total score is interpreted as follows: absence of insomnia (0–7); sub-threshold insomnia (8–14); moderate insomnia (15–21); and severe insomnia (22–28). Cut-off scores of 10 have been used to identify possibility of insomnia [25]. Previous research has reported adequate psychometric PLOS ONE \| https://doi.org/10.1371/journal.pone.0292470 October 5, 2023 3 / 12 PLOS ONE Psychological distress among nursing students properties of the ISI among nursing student (Cronbach’s α = .86) [26]. The Cronbach’s α of the ISI in this study was 0.71. 2.3 Data analysis The questionnaires were thoroughly reviewed for accuracy and completeness after the data collection process. Participants who decided not to respond to certain questions or left one or more questions unanswered were not included in the valid data set due to missing or incomplete data. Data were entered into and analysed using IBM SPSS Statistics (Version 25.0). Frequencies and percentages were used as descriptive statistics. Caseness for each of the psychometric constructs was assigned dichotomously using the cut-off scores indicated above. The Chi-square test was utilised for comparisons to determine group differences. Pearson’s correlation coefficient was used to determine the relationships among variables. Logistic regression was performed, to understand the relationships between high level of fear, stress, anxiety, depression, insomnia, and other study variables. For all analyses performed, a P < .05 was considered statistically significant. 3 Results A total of 1053 questionnaires were collected. After excluding incomplete questionnaires, the study was left with 918 valid questionnaires from nursing students. Most of the participants were female (85.7%, n = 787), and single (86.9%, n = 798). The largest age group was those aged 21 to 22 years (47.2%, n = 443). The participants came from a range of academic year of study: 33.7% fourth years, 33.1% third years, 23.9% second years, and 9.4% first years. About 70 percent had engaged in a mixed type of learning. The majority had received two or more doses of the COVID-19 vaccination (96.1%, n = 882). One thirds had been infected by COVID-19 (32.6%, n = 299). The mean (SD) of FCV-19S was 16.86 (SD = 6.14), PSS was 21.87 (SD = 5.64), HADS-A was 10.25 (SD = 3.53), HADS-D 9.11 (SD = 3.53), and ISI 12.97 (SD = 5.17). Table 1 shows the prevalence rates of each scale, as follows: fear 46.2%, stress 91.6%, anxiety 69.1%, depression 59.8%, and insomnia 73.2%, based on the specified cut-off scores for each scale. Respondents from KSA (52.5%) were more likely to score at or above the cut-off level on the FCV-19S compared with those from other countries, indicating greater proportion of students with fear of COVID-19. The prevalence of anxiety was significantly higher in UAE 80%, UK 79%, and KSA a 71% than in Oman 59%, χ2 (4) = 20.314, P< 0.001. A similar difference was observed for depression prevalence in KSA 70%, UAE 64%, and UK 63%, compared to in Oman 41%, χ2 (4) = 23.92, P< 0.001. Participants from UAE and KSA had significantly higher prevalence of insomnia compared to participants from the UK and Oman. Only for stress were there no significant differences between countries. Pearson correlation coefficients (Table 2) indicated significant relationships between fear, stress, anxiety, depression, and insomnia scores. 3.1 Factors predicting fear, stress, anxiety, depression, and insomnia All variables were selected for entry into the logistic regression models. The models for fear (F [17, 918] = 59.92, P< 0.001), stress (F [17, 918] = 41.23, P = 0.001), anxiety (F [17, 918] = 40.90, P< 0.001), depression (F [17, 918] = 86.14, P< 0.001) and insomnia (F [17, 918] = 75.10, P< 0.001) were statistically significant. Table 3 shows that married status was the strongest predictor of high fear level with married individuals almost three times as likely to score above the cut-off than unmarried ones (OR 2.91 CI,0.99–8.51, P = 0.05). Second- and third-year students, those assigned on placement PLOS ONE \| https://doi.org/10.1371/journal.pone.0292470 October 5, 2023 4 / 12 PLOS ONE Psychological distress among nursing students Table 1. Demographic characteristic of participants (N = 918). Total n KSA (n = 442) % n % OMAN (n = 289) n % UK (n = 97) UAE (n = 90) n n % % Gender Male 131 14.3 65 14.7 61 21.1 4 4.1 1 1.1 Female 787 85.7 377 85.3 228 78.9 93 95.9 89 98.9 18–20 280 30.5 78 17.6 125 43.3 54 55.7 23 25.6 21–22 433 47.2 239 54.1 135 46.7 22 22.7 37 41.1 More than 23 205 22.3 125 28.3 29 10 21 21.6 30 33.3 Married 97 10.6 60 13.6 12 4.2 12 12.4 13 14.4 Single 798 86.9 375 84.8 276 95.5 70 72.2 77 85.6 Others 23 2.5 7 1.6 1 0.3 15 15.5 0 0 86 9.4 16 3.6 50 17.3 19 19.6 1 1.1 13.3 Age .00 Marital Status .00 Academic Year Level First .00 Second 219 23.9 83 18.8 89 30.8 35 36.1 12 Third 304 33.1 161 36.4 73 25.3 43 44.3 27 30 Fourth 309 33.7 182 41.2 77 26.6 0 0 50 55.6 266 29 98 22.2 153 52.9 3 3.1 12 13.3 Type of Learning at present Time Face-To-Face p .00 .00 Online 15 1.6 5 1.1 2 0.7 5 5.2 3 3.3 Mixed 637 69.4 339 76.7 134 46.4 89 91.8 75 83.3 Have you had a Confirmed case of COVID-19 .00 Yes 299 32.6 127 28.7 61 21.1 59 60.8 52 57.8 No 619 67.4 315 71.3 228 78.9 38 39.2 38 42.2 Have you taken any of COVID-19 vaccine? .00 Yes, two doses or more 882 96.1 437 98.9 266 92 92 94.8 87 96.7 Yes, one dose 29 3.2 2 0.5 23 8 2 2.1 2 2.2 No 7 0.8 3 0.7 0 0 3 3.1 1 1.1 Do you assigned to be on placement during the pandemic .00 Yes 449 48.9 146 33 161 55.7 73 75.3 69 76.7 No 469 51.1 296 67 128 44.3 24 24.7 21 23.3 Low Fear (<17.5) 494 53.8 210 47.5 166 57.4 69 71.1 49 54.4 High Fear (�17.5) 424 46.2 232 52.5 123 42.6 28 28.9 41 45.6 FCV-19S .00 PSS .35 Non-Stress (<14) 77 8.4 31 7 26 9 12 12.4 8 8.9 Stress (�14) 841 91.6 411 93 263 91 85 87.6 82 91.1 No anxiety (HADS(A) <8) 284 30.9 128 29 118 40.8 20 20.6 18 20 Anxiety (HADS(A) �8) 634 69.1 314 71 171 59.2 77 79.4 72 80 No depression (HADS(D) <8) 369 40.2 132 29.9 169 58.5 36 37.1 32 35.6 Depression (HADS(D) �8) 549 59.8 310 70.1 120 41.5 61 62.9 58 64.4 No insomnia (ISI <10) 246 26.8 94 21.3 84 29.1 50 51.5 18 20 Insomnia (ISI �10) 672 73.2 348 78.7 205 70.9 47 48.5 72 80 HADS anxiety .00 HADS depression .00 ISI .00 https://doi.org/10.1371/journal.pone.0292470.t001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0292470 October 5, 2023 5 / 12 PLOS ONE Psychological distress among nursing students Table 2. Relationships between fear, stress, anxiety, depression, and insomnia. Fear Fear Stress Anxiety Depression Total ISI 1 .135 .092 .132 .199 1 .350 .294 .327 1 .102 .153 1 .289 Stress Anxiety Depression Total ISI Score 1 ** p <0.01 https://doi.org/10.1371/journal.pone.0292470.t002 during the pandemic, and those from the UK had significantly higher levels of Fear of COVID. Being female was the only significant predictor of stress (OR 2.89[CI, 1.55–5.39], P< 0.001). Only being a student from Oman appeared to be a predictor of anxiety (OR 0.34[CI, 0.18– 0.64], P< 0.001). Four variables predicted depression: a student being in their second academic year (OR 2.37[CI, 1.70–3.83], P< 0.001), being female, those between 21 and 22 years of age, and participants from Oman. The last logistic regression model showed that the significant predictors for insomnia in pre-registration nursing students were being in the first academic year (OR 3.03 [CI, 1.41–6.49], P< 0.001), second academic year (OR 1.82[CI, 1.08–3.05], P = 0.02) and location in Oman or UK. Table 3. Logistic regression analyses of factors associated with higher fear, depression, anxiety, stress, sleep disturbance odds ratio (95% CI). Higher Fear OR (95% CI) Stress P OR (95% CI) Anxiety P OR (95% CI) Depression P OR (95% CI) Insomnia P OR (95% CI) P Gender Male Ref Female Ref Ref 2.89(1.55–5.39) .00 Ref Ref 1.49(0.99–2.25) .05 0.61(0.37–1.00) .04 Age More than 23 Ref Ref Ref Ref Ref 18–20 21–22 0.64(0.44–0.95) .02 Marital Status Others Married Ref Ref Ref Ref Ref Ref Ref Ref Ref 2.91(0.99–8.51) .05 Single Academic Year Level Fourth Ref First 2.07(1.06–4.03) .03 3.03(1.41–6.49) .00 Second 2.07(1.31–3.27) .00 2.48(1.07–5.77) .03 Third 1.68(1.17–2.40) .00 2.37(1.7–3.83) .00 1.82(1.08–3.05) .02 Do you assign a placement during the pandemic No Yes Ref Ref Ref Ref 1.91(1.40–2.60) .00 1.99(1.15–3.44) .01 Ref 1.51(1.07–2.14) .02 Country UAE Ref Ref Ref Ref Ref KSA Oman UK 0.34(0.18–0.64) .00 0.42(0.24–0.74) .00 0.48(0.25–0.93) .04 0.37(0.19–0.72) .00 0.13(0.06–0.27) .00 https://doi.org/10.1371/journal.pone.0292470.t003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0292470 October 5, 2023 6 / 12 PLOS ONE Psychological distress among nursing students 4 Discussion This study was conducted to determine the psychological disorders associated with the COVID-19 pandemic among pre-registration nursing students in four countries. It is one of the few large-scale studies that has assessed the effects of the pandemic on various aspects of psychological health in this group. The study found that the pre-registration nursing students experienced symptoms of stress from mild to severe (91.6%), anxiety (69%), depression (59.8%), and insomnia (73.2%). Rates in this study were higher compared to those in a systematic review and meta-analyses of 17 studies conducted by Mulyadi et al. [27] involving 13,247 nursing students where prevalence rates of probable stress were (30%), anxiety (32%), depression (52%), and insomnia (27%) during the COVID-19 pandemic. In addition, prevalence rates in this study were higher in comparison with those in the general population in a meta-analyses of 999 studies where rates were: stress (32%) anxiety (28%), depression (27%), and insomnia (32%) during the COVID19 pandemic [28]. The differences between the findings might be explained by the various isolation measures that were used by the countries to limit the spread of COVID-19. This is important as the timing of the implementation of these measures can influence the severity of the adverse psychological sequelae [29]. Besides the different methods used in these studies, the definitions and sampling methods used also made the comparisons between the findings difficult to interpret. The mean level of fear of COVID-19 was 16.86 (SD = 6.14) out of a possible score of 35. This finding is in line with the rather limited previous literature [30–32]. Yet, scores in this study were lower than those reported from studies conducted in Mexico (25.71) [33] and Thailand (25.6) [34]. This may be explained by those studies having been conducted at the start of the pandemic when there was greater uncertainty of the effects of COVID-19 on humans. Another possible reason might also be attributed to differences in vaccine availability and distribution, impacting individuals’ perception of safety and well-being between the studies. In this study, there were significant differences between countries in terms of prevalence of fear, anxiety, depression, and insomnia. The reasons for this might be due to variation in individual countries at the time of conducting the study in terms of the level and nature of COVID-19 restrictions including social isolation, lockdown, and the associated economic challenges. Additionally, the variations in mental health outcomes among countries can also be influenced by factors like the availability and accessibility of healthcare services, public health infrastructure, cultural attitudes towards mental health, and the level of social support systems in place during the pandemic [35]. These country-specific elements contribute to the diverse impacts on individuals’ mental well-being during the global health crisis. The results of this study indicate a significant association between stress, anxiety, depression, and insomnia and fear of COVID-19. Previous studies reported similar findings that indicated a direct influence between the stress, anxiety, depression, insomnia and higher level of fear among undergraduate nursing students [36–38]. This suggests that the effects of the pandemic on psychological wellbeing were broad and that a range of strategies might be required to reduce the variety of symptoms in future. The results suggested that being married is the strongest predictor of a higher level of fear. A possible reason for this is the added contribution of fear of transmitting the infection to their families; this seems especially likely given that more than half of these participants were assigned to clinical placement during the pandemic. In the present study, being female was the strongest predictor of both stress and anxiety. These results are consistent with other studies [39, 40]. Academic year of study was the strongest predictor of depression, namely second year and insomnia, namely first year. This might be due to repercussions on education in terms of PLOS ONE \| https://doi.org/10.1371/journal.pone.0292470 October 5, 2023 7 / 12 PLOS ONE Psychological distress among nursing students barriers and difficulties of lack of technology skills [41]. This finding suggests that the pandemic may have had a greater impact on those in the early part of the nursing course who would have greater uncertainty about clinical placement and may have had insufficient time to forge close support networks with fellow students. Thus, in future pandemics there may be a need to prioritise supportive interventions for students during pandemics can potentially apply to other situations besides pandemics. The underlying principle is that during any crisis or challenging circumstances, individuals may face increased stress, anxiety, depression, insomnia and disruptions in their lives. This can be particularly true for students who might be dealing with additional pressures related to their education and personal lives, such as financial burdens, family responsibilities, health concerns, or social challenges. The current study revealed a high prevalence of stress, anxiety, and depression even two years after the onset of the pandemic. A systematic review involving 89 studies found that implementing online mental health consultation was beneficial to reduce depression and anxiety and improve psychological well-being of college students [42], suggesting this might be a way to support nursing students in the future. Higher education institutions should provide online training courses and counselling services to help students overcome their psychological problems. Regretfully, research has shown that nursing programs generally do not offer sufficient training on crisis-coping strategies [43, 44] and it is recommended that the nursing education curriculum is strengthened regarding all aspects of infectious diseases. A possible limitation of this study is that self-reported questionnaires may not accurately reflect mental health problems and the actual level of fear. Recruitment of participants through online platforms and emails might have led to a lower response rate as some individuals may not regularly check and respond to their emails. Another possible limitation is the large differences in spread of the virus, death rates, and restrictions in different countries. 5 Conclusion This is the first study to examine the psychological impact of COVID-19 pandemic among undergraduate nursing students in KSA, Oman, UK, and UAE. This study revealed that the rate of fear of COVID-19, stress, anxiety, depression, and insomnia among undergraduate nursing students is between moderate to severe level. The main implication of the current findings is that higher education institutions need to support and monitor students’ psychological needs and explore the benefits of interventions to reduce the level of psychological symptoms. Likewise, it is important to embed and include disaster and pandemic management in the nursing curriculum to increase the student knowledge and preparedness for such emergencies. Further, researchers should continue to examine the mental health symptoms in this population as the impact of the pandemic may persist over time. Nursing students of today will in the future be working as nurses, and it is important to nurture them at the time of outbreaks of infectious diseases. 6 Relevance for clinical practice Higher education Institutions should consider the various factors that affect the development and maintenance of stress, anxiety, depression, and sleep among undergraduate nursing students. These include their backgrounds, perceptions, and feelings, to minimize their risk of experiencing these conditions in the future. Professional and academic tutors should additionally provide support and help students develop resilience during their internship. In addition to training students how to take care of a patient with an infectious disease, disaster management education should also be included in the nursing curricula to help students develop effective disaster management response skills. PLOS ONE \| https://doi.org/10.1371/journal.pone.0292470 October 5, 2023 8 / 12 PLOS ONE Psychological distress among nursing students Acknowledgments Gratitude to all individuals who took part in the research for their willingness to give up their time to complete the questionnaires. Author Contributions Conceptualization: Mohammed Al Maqbali, Norah Madkhali, Geoffrey L. Dickens. Data curation: Mohammed Al Maqbali, Norah Madkhali, Alexander M. Gleason, Geoffrey L. Dickens. Formal analysis: Mohammed Al Maqbali, Norah Madkhali, Alexander M. Gleason, Geoffrey L. Dickens. Funding acquisition: Mohammed Al Maqbali, Norah Madkhali, Alexander M. Gleason, Geoffrey L. Dickens. Investigation: Mohammed Al Maqbali, Norah Madkhali, Alexander M. Gleason, Geoffrey L. Dickens. Methodology: Mohammed Al Maqbali, Norah Madkhali, Alexander M. Gleason, Geoffrey L. Dickens. Project administration: Mohammed Al Maqbali, Norah Madkhali, Geoffrey L. Dickens. Resources: Mohammed Al Maqbali. Software: Mohammed Al Maqbali, Geoffrey L. Dickens. Supervision: Mohammed Al Maqbali, Geoffrey L. Dickens. Validation: Mohammed Al Maqbali. Visualization: Mohammed Al Maqbali. Writing – original draft: Mohammed Al Maqbali, Norah Madkhali, Alexander M. Gleason, Geoffrey L. Dickens. Writing – review & editing: Mohammed Al Maqbali, Norah Madkhali, Alexander M. Gleason, Geoffrey L. Dickens. References 1. 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. New England Journal of Medicine. 2020; 382: 1199–1207. https://doi.org/10.1056/NEJMoa2001316 PMID: 31995857 2. World Health Organization. Statement on the second meeting of the International Health Regulations (2005) Emergency Committee regarding the outbreak of novel coronavirus (2019-nCoV). 2020 [cited 14 Jun 2020]. 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https://openalex.org/W4362425990	https://figshare.com/articles/journal_contribution/Supplementary_Data_from_Vandetanib_Designed_to_Inhibit_VEGFR2_and_EGFR_Signaling_Had_No_Clinical_Activity_as_Monotherapy_for_Recurrent_Ovarian_Cancer_and_No_Detectable_Modulation_of_VEGFR2/22440550/1/files/39891412.pdf	English	null	Supplementary Data from Vandetanib, Designed to Inhibit VEGFR2 and EGFR Signaling, Had No Clinical Activity as Monotherapy for Recurrent Ovarian Cancer and No Detectable Modulation of VEGFR2	null	2,023	cc-by	86	Supplemental Table 1. Antibodies used for RPTA Antibody Company Dilution ERK Cell Signaling, Danvers, MA 1:1000 pERK Cell Signaling, Danvers, MA 1:1000 EGFR Cell Signaling, Danvers, MA 1:500 pEGFR Cell Signaling, Danvers, MA 1:500 VEGFR2 Cell Signaling, Danvers, MA 1:1000 pVEGFR2 Cell Signaling, Danvers, MA 1:1000 AKT Cell Signaling, Danvers, MA 1:1000 pAKT Cell Signaling, Danvers, MA 1:1000 P27 kip 1 Cell Signaling, Danvers, MA 1:1000 P38 Cell Signaling, Danvers, MA 1:1000 Cleaved PARP Cell Signaling, Danvers, MA 1:1000 Supplemental Table 1. Antibodies used for RPTA
https://openalex.org/W4285264852	https://www.e3s-conferences.org/articles/e3sconf/pdf/2022/17/e3sconf_eregce2022_01024.pdf	English	null	A Critical Review on Regional Ecological Environment Assessment	E3S web of conferences	2,022	cc-by	3,757	A Critical Review on Regional Ecological Environment Assessment The purpose of EEA is to analyze the ecological environmental impact of a region due to human activities and the impact on humans, mainly involving ecological risk assessment, ecological environmental vulnerability assessment, ecological health assessment, ecological footprint, etc, this attributes to carry out ecological management and ecological protection policies, etc[2-3].There are many types of EEA objects based on different perspectives, from the assessment area, they can be divided into administrative EEA and thematic EEA. Administrative ecological environmental assessment studies the impact of various human social activities on a certain administrative area[4-5]. Thematic EEA refers to the assessment of the ecological environment of a certain type of geographic area, such as plateaus[6], lakes[7], highways[8], cities[9],mine [10], etc. According to the element attributes of the assessment object, it can be divided into comprehensive element assessment and partial single element assessment. Comprehensive element assessment takes the study area as an ecosystem and uses landscape ecology[11], ecosystem services[12] and other methods to evaluate the ecological environment from a macro perspective. Partial single element assessment is the evaluation of key elements in the ecosystem, such as diatoms [13], heavy metal toxic elements[14-15], etc. 1 Introduction In order to meet the development needs of industrialization and urbanization, the scope and intensity of human activities continue to expand, ecosystem areas such as urban and rural natural environments and nature reserves have been shrinking, resulting in a substantial shrinkage of ecological resources. At the same time, environmental problems of different scales such as land and water pollution, heat island effect, and global warming caused by human activities have become more and more serious, the degradation of the ecological environment has become a bottleneck restricting the development of human economy. The ecological environment assessment(EEA) could contribute to provide a scientific basis for formulating ecological environmental protection policies and measures, as well as ecological coordination and sustainable development[1].Therefore, it is of great significance to carry out regional ecological environment monitoring and evaluation timely. The article summarizes the content and development of EEA, discusses the main methods of current EEA and the application of remote sensing in EEA, and finally summarizes the shortcomings of current EEA. 2.2 The development of EEA 2.1 Contents of EEA * Corresponding author: yangmx@iwhr.com E3S Web of Conferences 350, 01024 (2022) E3S Web of Conferences 350, 01024 (2022) E3S Web of Conferences 350, 01024 (2022) E3S Web of Conferences 350, 01024 (2022) E3S Web of Conferences 350, 01024 (2022) EREGCE 2022 E3S Web of Conferences 350, 01024 (2022) EREGCE 2022 https://doi.org/10.1051/e3sconf/202235001024 https://doi.org/10.1051/e3sconf/202235001024 A Critical Review on Regional Ecological Environmen Assessment Xinghan Wang1, Pengfei Jia2 and Mingxiang Yang1,* 1China Institute of Water Resources and Hydropower Research, Beijing; 10038, China 2CITIC CONSTRUCTION, CO., LTD © 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/). © 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/). A Critical Review on Regional Ecological Environment Assessment Abstract. With the continuous advancement of industrialization and urbanization, the relationship between mankind and the ecological environment has become increasingly tense, and the ecological environment assessment has become a research hotspot in recent years. The article summarizes the research content and development process of ecological environment assessment, lists various mainstream assessment methods and introduces their application characteristics, and then divides the weight determination methods into subjective weighting, objective weighting and subjective and objective combination, and analyses their advantages and disadvantages; Meanwhile, the application of remote sensing technology in ecological environment assessment research is analyzed. Finally, the main problems of ecological environment assessment work are summarized and its future development direction is pointed out. Ecological environment refers to the entirety of various ecosystems composed of biological communities and non-biological natural factors. The purpose of EEA is to analyze the ecological environmental impact of a region due to human activities and the impact on humans, mainly involving ecological risk assessment, ecological environmental vulnerability assessment, ecological health assessment, ecological footprint, etc, this attributes to carry out ecological management and ecological protection policies, etc[2-3].There are many types of EEA objects based on different perspectives, from the assessment area, they can be divided into administrative EEA and thematic EEA. Administrative ecological environmental assessment studies the impact of various human social activities on a certain administrative area[4-5]. Thematic EEA refers to the assessment of the ecological environment of a certain type of geographic area, such as plateaus[6], lakes[7], highways[8], cities[9],mine [10], etc. According to the element attributes of the assessment object, it can be divided into comprehensive element assessment and partial single element assessment. Comprehensive element assessment takes the study area as an ecosystem and uses landscape ecology[11], ecosystem services[12] and other methods to evaluate the ecological environment from a macro perspective. Partial single element assessment is the evaluation of key elements in the ecosystem, such as diatoms [13], heavy metal toxic elements[14-15], etc. Ecological environment refers to the entirety of various ecosystems composed of biological communities and non-biological natural factors. 2.1 Contents of EEA In 1964, the International Conference on In 1964, the International Conference on https://doi.org/10.1051/e3sconf/202235001024 https://doi.org/10.1051/e3sconf/202235001024 E3S Web of Conferences 350, 01024 (2022) EREGCE 2022 E3S Web of Conferences 350, 01024 (2022) EREGCE 2022 E3S Web of Conferences 350, 01024 (2022) E3S Web of Conferences 350, 01024 (2022) Environmental Quality first proposed the concept of environmental impact assessment. In 1969, the United States promulgated the National Policy Act (NEPA) and established the world ’ s first environmental assessment system, it is also the beginning of the world’s EEA work. Subsequently, the U.S. Environmental Commission further improved it and added relevant content of environmental impact assessment (EIA). EIA is a process that proposes the potential environmental impacts of the development project and proposes appropriate measures to avoid, reduce or compensate for these impacts (called mitigation measures) [16-17]. on-site ecosystem, use Shannon-Wiener index to represent biodiversity, this method can better reflect the relationship between the biological community and the ecological environment; Wang estimated the economic value of China's forest species diversity based on the biodiversity evaluation method[25].The ecological footprint method evaluates the sustainable development of the ecosystem by calculating the profit and loss difference between the ecological carrying capacity and the size of the ecological footprint; this method is suitable for urban EEA with good basic data; Świąder use the ecological footprint method to evaluate the ecological environment carrying capacity in Wroclaw, Poland[26].Index evaluation method is to evaluate each index participating in the evaluation separately, and finally use the weighted sum method to achieve the effect of comprehensive evaluation of the ecological environment. Index evaluation method has the characteristics of difficulty in weighting and quantitative evaluation, because it needs to establish an evaluation system, but it can be more comprehensively evaluated,so it is widely used in ecological environment evaluation, for example, the Ecological Environment Index (EI) proposed in the "Technical Specifications for Evaluation of Ecological Environment Condition (Trial)" promulgated and implemented by the State Environmental Protection Administration of China,the Environmental Quality Index (NWF) proposed by the United States and Canada's Total Environmental Quality Index (EQI)[3].There are many eco-environmental assessment methods and each has its own focus; therefore, when dealing with different eco-environmental assessment issues, it should be determined according to the specific assessment objectives. 3.2 Index weight determination method The indicator weighting methods in EEA are mainly divided into subjective weighting method (SW) and objective weighting method (OW). SW determines the index weight based on the researcher's prior knowledge and subjective judgment. It is highly subjective. Commonly used methods are as follows: Analytic Hierarchy Process (AHP)[27], Delphi Method[28], Fuzzy Mathematics (FM)[29], etc. OW determines the weight of each indicator based on the correlation between various indicator data. This type of weighting method is less artificially affected and can objectively reflect the relationship between indicators, common OW methods are: Entropy Method (EWM)[29], Principal Component Analysis (PCA)[30], Random Forest (RF)[31],Convolutional Neural Network (CNN) [32], etc. At present, many studies combine the advantages of subjective and objective weighting methods to determine the index weights, which not only avoids the excessive subjectiveness of objective weighting methods, but also combines prior knowledge to judge the importance of the evaluation indicators in the study area, effectively reducing the evaluation Error of result, for example, Li used the PCA-AHP-TOPSIS method to estimate the ecological environment index of the area along the 2.1 Contents of EEA In the mid-1970s, some developed countries in Europe and the United States improved the shortcomings of EIA and extended its application to the planning level and policy level, this is the strategic environmental assessment (SEA). SEA is used to solve the environmental impact of strategic decisions and designed to incorporate environmental sustainability into strategic decision-making[18]; In 1989, the World Bank stipulated that all major projects under its supervision required ecological environmental assessment[19]. So far, SEA has been widely used worldwide, mainly in the countries of the Organization for Economic Co-operation and Development (OECD) and the client countries of the World Bank, these countries have established the SEA system in the form of legislation, and have formulated the SEA framework to a certain extent[20]. In 2001, the United Nations Foundation and other organizations launched the Millennium Ecosystem Assessment (MA) project, which was implemented from 2001 to 2005.MA uses a new conceptual framework to record, analyze and understand the impact of environmental changes on ecosystems and human activities, it is a summary of the comprehensive evaluation of global ecosystems[21-22]; For the first time, MA has systematically and comprehensively revealed the status and change trends of various ecosystems on a global scale. Since 2005, MA has conducted hundreds of ecosystem assessments of different scales and different themes[23], which has also brought EEA research into a new stage of development. 3.1 Assessment method After years of eco-environmental assessment and research by experts all over the world, there are currently some more mature eco-environmental assessment methods, such as fuzzy discrimination, biodiversity evaluation, ecological footprint, Index evaluation,etc. Fuzzy discrimination is an assessment method that transforms qualitative evaluation into quantitative evaluation based on the principles of fuzzy mathematics, finally, a certain value between 0 and 1 is used to indicate the relationship between each assessment index and the system, this method is applicable to both large-scale and small-scale areas, Sami comprehensive environmental assessment of the farm system with the help of fuzzy discriminant method[24].The Biodiversity assessment method is based on the investigation of the 2 https://doi.org/10.1051/e3sconf/202235001024 https://doi.org/10.1051/e3sconf/202235001024 E3S Web of Conferences 350, 01024 (2022) EREGCE 2022 E3S Web of Conferences 350, 01024 (2022) EREGCE 2022 E3S Web of Conferences 350, 01024 (2022) E3S Web of Conferences 350, 01024 (2022) Beijing-Hangzhou Grand Canal [33], avoiding the subjectivity and extensiveness of conventional multi-factor decision analysis. Beijing-Hangzhou Grand Canal [33], avoiding the subjectivity and extensiveness of conventional multi-factor decision analysis. computational efficiency. It also has advantages such as free and parallel, it is now widely used in remote sensing image classification, land use change monitoring, etc.Chen[40] used Landsat data from the GEE platform to extract four indicators including vegetation index, humidity component, heat and dryness to evaluate and monitor the quality of the ecological environment in China's Three-River Source Region. Based on Landsat images from the GEE platform, Mahdianpari[41]used random forests to assess the spatial dynamics of wetlands in Newfoundland, Canada from 1985 to 2015.In the future, remote sensing technology based on cloud computing and big data analysis will be widely used in EEA, on this basis, with the help of artificial intelligence, knowledge base, etc. to mine the ecological environment big data, enhance the use value of big data, and provide more accurate and efficient services for the decision-making management of the ecological environment. 4 Application of Remote Sensing in EEA EEA focuses more on the integration of data and information[34], so the accuracy of data acquisition is very important, in the early days, the evaluation index data was easily restricted by time and space scales, With the development of space science and computer technology, 3S technology has been widely used in ecological environment evaluation. The spatial resolution, time resolution, and spectral resolution of remote sensing data continue to increase, which improves the real-time and operability of remote sensing technology; combines remote sensing image data with long-term statistics and observation data to strengthen the research on dynamic evaluation of the ecological environment. The spatial resolution, time resolution, and spectral resolution of remote sensing data continue to increase, which improves the real-time and operability of remote sensing technology; combines remote sensing image data with long-term statistics and observation data to strengthen the research on the dynamic evaluation of ecological environment. Yao based on Landsat8 data, selected vegetation coverage, bare soil index and slope as evaluation indicators, using Index evaluation method to evaluate the highway ecological environment quality[35].Ying uses ASTER GDEM and Landsat data to extract the three indicators of vegetation coverage, soil index and soil moisture, and determines the weight of each indicator through the Delphi method, and then uses the index evaluation method to analyze the ecological environment quality of the Wujiang River Basin in Guizhou Province, China[36]. 5 Conclusion In the era of global climate change and information exchange, monitoring regional ecological environmental changes is of great significance for humans to solve ecological problems. The maturity of 3S technology, the development of big data, and the optimization of machine learning algorithms have further improved the methods and theories of EEA research. Eco-environmental assessment objects are also more targeted, from early large-scale macro-evaluation to partial thematic regional eco-environmental assessment, which provides a theoretical basis for solving typical regional eco-environmental problems. However, due to the regional differences in different research areas, although many EEA work combines local geography, ecology, and social conditions to construct an assessment system, there is still a lack of complete EEA systems suitable for various objects and scales. In addition, the weights of evaluation indicators and the classification of evaluation results tend to be subjective or objective, the use of a single subjective or objective weighting method will lead to deviations in the evaluation results, and inevitable errors in the grading process will cause the evaluation results to be subjective. The future ecological environment evaluation work should fully consider the actual situation of the study area, construct an appropriate evaluation system and determine the index weights according to local conditions, and use big data, emerging technology platforms, etc. to conduct ecological environment evaluation from a multidisciplinary perspective. In 2013, Xu[30] improved China’s EI index and constructed a new remote sensing ecological index RSEI based on PCA using greenness, humidity, heat, and dryness indicators,and verified its effective application in EEA. Based on high-resolution remote sensing images, Hao[37] selected the fractional vegetation cover (FVC), water density (WD), impervious surface coverage (ISC), net primary production (NPP) and land surface temperature (LST) evaluated and analysis of the ecological environmental impact caused by urban expansion in Beijing, China. Based on Landsat data, Zhang[38] used the RSEI index to evaluate the ecological environmental quality of Nanjing, China from 1990 to 2013, and predicted the development trend of ecological environmental quality. Wu[39] selected fractional vegetation cover (FVC), leaf area index (LAI), total primary productivity (GPP), land surface temperature (LST), and wet (Wet) retrieved from MODIS data as evaluation indicators. assessment the ecological environment of China's Tibet from 2006 to 2016. References 1. Sun D, Zhang J, Zhu C, et al. 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https://openalex.org/W2797796088	https://europepmc.org/articles/pmc5895029?pdf=render	English	null	The physiological cost of diazotrophy for Trichodesmium erythraeum IMS101	PloS one	2,018	cc-by	15,404	The physiological cost of diazotrophy for Trichodesmium erythraeum IMS101 * tboatman@chelsea.co.uk * tboatman@chelsea.co.uk * tboatman@chelsea.co.uk Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Tobias Boatman was supported by a UK Natural Environment Research Council PhD studentship (NE/J500379/1 DTB). Funding obtained by RJG and TL. Abstract Trichodesmium plays a significant role in the oligotrophic oceans, fixing nitrogen in an area corresponding to half of the Earth’s surface, representing up to 50% of new production in some oligotrophic tropical and subtropical oceans. Whilst Trichodesmium blooms at the sur- face exhibit a strong dependence on diazotrophy, colonies at depth or at the surface after a mixing event could be utilising additional N-sources. We conducted experiments to establish how acclimation to varying N-sources affects the growth, elemental composition, light absorption coefficient, N2 fixation, PSII electron transport rate and the relationship between net and gross photosynthetic O2 exchange in T. erythraeum IMS101. To do this, cultures were acclimated to growth medium containing NH4 + and NO3 - (replete concentrations) or N2 only (diazotrophic control). The light dependencies of O2 evolution and O2 uptake were measured using membrane inlet mass spectrometry (MIMS), while PSII electron transport rates were measured from fluorescence light curves (FLCs). We found that at a saturating light intensity, Trichodesmium growth was ~ 10% and 13% lower when grown on N2 than with NH4 + and NO3 -, respectively. Oxygen uptake increased linearly with net photosynthesis across all light intensities ranging from darkness to 1100 μmol photons m-2 s-1. The maxi- mum rates and initial slopes of light response curves for C-specific gross and net photosyn- thesis and the slope of the relationship between gross and net photosynthesis increased significantly under non-diazotrophic conditions. We attribute these observations to a reduced expenditure of reductant and ATP for nitrogenase activity under non-diazotrophic conditions which allows NADPH and ATP to be re-directed to CO2 fixation and/or biosynthe- sis. The energy and reductant conserved through utilising additional N-sources could enhance Trichodesmium’s productivity and growth and have major implications for its role in ocean C and N cycles. RESEARCH ARTICLE Editor: Douglas A. Campbell, Mount Allison University, CANADA Editor: Douglas A. Campbell, Mount Allison University, CANADA Received: December 21, 2017 Accepted: March 26, 2018 Published: April 11, 2018 Copyright: © 2018 Boatman 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: Douglas A. Campbell, Mount Allison University, CANADA Received: December 21, 2017 Accepted: March 26, 2018 Published: April 11, 2018 Copyright: © 2018 Boatman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Boatman TG, Davey PA, Lawson T, Geider RJ (2018) The physiological cost of diazotrophy for Trichodesmium erythraeum IMS101. PLoS ONE 13 (4): e0195638. https://doi.org/10.1371/journal. pone.0195638 Diazotrophy Diazotrophic cyanobacteria are able to meet their daily nitrogen quota by fixing dinitrogen (N2). N2 þ 16ATP þ 8Hþ þ 8e ! 2NH3 þ H2 þ 16ADP þ 16Pi ð1Þ ð1Þ N2 þ 16ATP þ 8Hþ þ 8e ! 2NH3 þ H2 þ 16ADP þ 16Pi While N2 fixation is an extremely energy demanding process, Trichodesmium incurs addi- tional costs related to the protection of nitrogenase from the irreversible inhibition of photo- synthetically evolved O2 [9, 19, 20]. The separation of O2 evolution and N2 fixation is regulated over a diurnal cycle of N2 fixation and photosynthesis [21], involving daily synthesis and deg- radation of nitrogenase [22, 23] and alternation of photosynthetic activity states [24]. Tempo- ral separation occurs over short timescales, where peak rates of photosynthesis (~ 10 am) and N2 (~ 12 pm) fixation vary over a diel period. Spatial separation occurs via diazocytes, which are reversibly specialised cells for nitrogen fixation [25, 26]. Diazocytes contain the necessary proteins to perform photosynthetic CO2 fixation and N2 fixation. However, it has been sug- gested that when fixing N2, cells increase cyclic electron transport around PSI to enhance ATP synthesis [21, 24], thus allowing the cells to meet the energetic demands of N2 fixation (Eq 1). Introduction In marine ecosystems, phytoplankton primary production is often limited by the bioavailabil- ity of fixed N [1–3], where N-sources (e.g. NO3 -, NO2 -, NH4 +, urea etc) are quickly depleted by fast growing phytoplankton [4]. A significant fraction (~ 25 Tg N yr-1) of N in the euphotic zone is lost via sedimentation to the deep ocean as particulate organic nitrogen (PON), making Competing interests: The authors have declared that no competing interests exist. 1 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Trichodesmium utilising additional N-sources NO3 - concentrations higher at greater depth [5–7]. Whilst areas of upwelling transport NO3 - into the euphotic zone, there are vast regions of the oligotrophic open oceans that are depen- dent on the input of new N from N2-fixing cyanobacteria. Among the most important marine diazotrophs are Trichodesmium sp., which can form extensive surface blooms in the tropical and subtropical oceans [8–12]. NO3 - concentrations higher at greater depth [5–7]. Whilst areas of upwelling transport NO3 - into the euphotic zone, there are vast regions of the oligotrophic open oceans that are depen- dent on the input of new N from N2-fixing cyanobacteria. Among the most important marine diazotrophs are Trichodesmium sp., which can form extensive surface blooms in the tropical and subtropical oceans [8–12]. Previous studies have highlighted Trichodesmium’s capacity to assimilate various forms of combined N-sources [13–17]. It is commonly assumed that Trichodesmium obtains most of its nitrogen quota from N2 fixation, however field-based measurements of N2 fixation show wide temporal and spatial variability [18]. The causes of this variability remain unclear, but environ- mental factors such as the availability of combined nitrogen may be a contributing factor. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 where glutamine is subsequently transformed to 2-oxoglutarate and reduced using NADPH, forming two moles of glutamate. where glutamine is subsequently transformed to 2-oxoglutarate and reduced using NADPH, forming two moles of glutamate. 2 Oxoglutarate þ Glutamine ! 2½Glutamate ð6Þ ð6Þ Thus, for every mole of glutamate produced, one mole each of NH3, NADPH, ATP and 2-oxoglutarate are required. Additionally, ATP is required for the active transport of inorganic NH4 + or NO3 - into the cell [28]. Different N-sources require different amounts of energy and reductant and as such can be ordered into a hierarchy of energy requirements; where diazotro- phy requires the highest investment of electrons and ATP, followed by NO3 -, NO2 - and then NH3. Utilising additional N-sources erythraeum IMS101 growth, light dependency of gross and net O2 photosynthesis, PSII electron transport rates and elemental composition to different N- sources; investigating the physiological cost of performing diazotrophy. Materials and methods T. erythraeum IMS101 was semi-continuously cultured to achieve fully acclimated balanced growth at three N-source treatments (N2, NH4 + and NO3 -), at a targeted 380 μatm CO2 con- centration, saturating light intensity (400 μmol photons m-2 s-1), 12:12 light:dark (L:D) cycle and optimal temperature (26 ˚C ± 0.2) (3 treatments in total) for ~ 2 months (~ 30 generations). Utilising additional N-sources Global warming is increasing sea surface temperatures (SSTs) which is enhancing water strati- fication and decreasing vertical mixing [29], potentially increasing the area of N-limited oceans. Whilst detrimental to many phytoplankton, a reduced flux of NO3 - into the upper mixed layer will increase the competitive advantage of diazotrophs for other limiting nutrients (i.e. Fe or P). Trichodesmium colonies have been observed migrating to the nutricline [30, 31] to facilitate the luxury uptake of polyphosphates before returning to the surface. Whilst at these depths, cells are exposed to NO3 - concentrations greater than those at the surface. As such, Trichodesmium colonies may be assimilating and storing (i.e. cyanophycin granules) more combined N than the blooms frequently measured on the surface [32]. This could have major implications for growth rates, primary productivity and biogeochemical cycles [33]. Our approach comprises a systematic experiment where T. erythraeum IMS101 was grown over long durations, at three N-source treatments, with controlled and well-defined growth conditions, ensuring fully acclimated, balanced growth had been achieved. Our aims were to assess the response of T. erythraeum IMS101 growth, light dependency of gross and net O2 photosynthesis, PSII electron transport rates and elemental composition to different N- sources; investigating the physiological cost of performing diazotrophy. Global warming is increasing sea surface temperatures (SSTs) which is enhancing water strati- fication and decreasing vertical mixing [29], potentially increasing the area of N-limited oceans. Whilst detrimental to many phytoplankton, a reduced flux of NO3 - into the upper mixed layer will increase the competitive advantage of diazotrophs for other limiting nutrients (i.e. Fe or P). Trichodesmium colonies have been observed migrating to the nutricline [30, 31] to facilitate the luxury uptake of polyphosphates before returning to the surface. Whilst at these depths, cells are exposed to NO3 - concentrations greater than those at the surface. As such, Trichodesmium colonies may be assimilating and storing (i.e. cyanophycin granules) more combined N than the blooms frequently measured on the surface [32]. This could have major implications for growth rates, primary productivity and biogeochemical cycles [33]. O h h T h S Our approach comprises a systematic experiment where T. erythraeum IMS101 was grown over long durations, at three N-source treatments, with controlled and well-defined growth conditions, ensuring fully acclimated, balanced growth had been achieved. Our aims were to assess the response of T. Uptake of additional N-sources Like other facultative diazotrophic cyanobacteria spp., Trichodesmium can exploit other forms of nitrogen including NH4 +, NO3 -, urea and amino acids [16, 27]. These N compounds are transported into the cell via permeases, metabolised to NH4 + and then incorporated into car- bon skeletons through the glutamine synthetase (GS) and glutamine 2-oxoglutarate amino- transferase (GOGAT) pathways. This process is mediated by nitrate reductase (Eq 2) and nitrite reductase (Eq 3). NO3 þ 2e þ 2Hþ ! NO2 þ H2O ð2Þ NO2 þ 6e þ 8Hþ ! NHþ 4 þ 2H2O ð3Þ ð2Þ ð3Þ For cyanobacteria, nitrate reductase is located in the cytosol and uses NADPH to catalyse the transfer of two electrons. The NO2 - formed by nitrate reductase is further reduced to NH4 + via the transfer of six electrons. Thus, the reduction of NO3 - to NH4 + can be expressed as; NO3 þ 8e þ 10Hþ ! NHþ 4 þ 3H2O ð4Þ ð4Þ Amino acids are synthesised from ammonia (NH3) via the GS-GOGAT pathway. The initial GS pathway requires ATP and glutamate as a substrate; Amino acids are synthesised from ammonia (NH3) via the GS-GOGAT pathway. The initial GS pathway requires ATP and glutamate as a substrate; Glutamate þ NH3 þ ATP ! Glutamine þ ADP þ Pi ð5Þ Glutamate þ NH3 þ ATP ! Glutamine þ ADP þ Pi ð5Þ PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 2 / 24 Trichodesmium utilising additional N-sources PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Experimental setup Cultures were acclimated to the CO2 and light intensity for ~ 4 months (~ 60 generations) under diazotrophic conditions before the addition of NH4 + or NO3 -. Cultures were gradually enriched over a 2/3-week period by increasing the dilution ratio of YBCII media containing NH4 + or NO3 - (100 μM). T. erythraeum IMS101 was grown using YBCII medium [34] at 1.5 L volumes in 2 L pyrex bottles that were acid-washed and autoclaved prior to culturing. Daily growth rates were quantified from changes in baseline fluorescence (Fo) measured between 09:00 to 10:30 on dark-adapted cultures (20 minutes) using a FRRfII FastAct Fluorometer System (Chelsea Technologies Group Ltd, UK). Cultures were regarded as fully acclimated and in balanced growth when both the slope of the linear regression of ln Fo versus time and the ratio of live cell to acetone extracted (method detailed below) baseline fluorescence (Fo) were constant PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 3 / 24 Trichodesmium utilising additional N-sources following every dilution with fresh YBCII medium. Cultures were kept at the upper section of the exponential growth phase through periodic dilution with new growth media at 3–5 day intervals. Illumination was provided side-on by fluorescent tubes (Sylvania Luxline Plus FHQ49/T5/840). Cultures were constantly mixed using magnetic PTFE stirrer bars and aer- ated with a filtered (0.2μm pore) air mixture at a rate of ~ 200 mL s-1. The CO2 concentration was regulated (± 2 μatm) by mass flow controllers (Bronkhorst, Newmarket, UK). CO2-free air was supplied by an oil free compressor (Bambi Air, UK) via a soda lime gas-tight column which was mixed with a 10% CO2 in-air mixture from a gas cylinder (BOC Industrial Gases, UK). The CO2 concentration was continuously monitored and recorded by an infra-red gas analyser (Li-Cor Li-820, Nebraska USA), calibrated weekly by a standard gas (BOC Industrial Gases). Throughout all culturing, the inorganic carbon chemistry (S1 File) and dissolved inor- ganic NH4 + and NO3 - concentrations (S2 File) were determined prior to diluting with fresh media. Samples for elemental composition, photosynthesis-light response curves, fluores- cence light curves (FLC), in vivo light absorption and acetylene reduction assays were col- lected at the same time of day, approximately 4 and 6 hours into the photo-phase of the L:D cycle. Measuring O2 exchange by membrane inlet mass spectrometry (MIMS) Light dependent rates of O2 production and consumption were measured with a membrane inlet mass spectrometer (MIMS), using an 18O2 technique modified from McKew et al. [35] (S3 File). MIMS measurements consisted of three biological replicates per treatment (S4 File). Chlorophyll a concentrations at the point of sampling ranged from 80 to 245 μg Chla L-1. p y p p g g μg Changes in 16O2 and 18O2 and thus O2 consumption (Uo) and O2 evolution (Eo) were calcu- lated using the following equations [36]; U0 ¼ 1 þ 16O 2 18O 2 D18O2 Dt ð7Þ E0 ¼ D16O2 Dt 16O 2 18O 2 D18O 2 Dt ð8Þ U0 ¼ 1 þ 16O 2 18O 2 D18O2 Dt ð7Þ ð7Þ E0 ¼ D16O2 Dt 16O 2 18O 2 D18O 2 Dt ð8Þ ð8Þ where Uo is the rate of O2 consumption calculated from the decrease of 18O2 over time (i.e. Δ 18O2/Δt), which takes into account the relative concentration of 18O2 compared to 16O2 (i.e. 1 + 16O2/18O2) and Eo is the rate of gross O2 evolution calculated from the increase in 16O2 over time (Δ16O2/Δt), where the decline of 18O2 (i.e. Δ18O2/Δt) and 18O2 is corrected for rela- tive to the concentration of 16O2. Chlorophyll a- and C-specific rates were obtained by divid- ing U0 and E0 by the concentration of Chla and particulate organic carbon, respectively. Rates were multiplied by 1.073 to spectrally correct to the culturing LEDs (S1 Fig). where Uo is the rate of O2 consumption calculated from the decrease of 18O2 over time (i.e. Δ 18O2/Δt), which takes into account the relative concentration of 18O2 compared to 16O2 (i.e. 1 + 16O2/18O2) and Eo is the rate of gross O2 evolution calculated from the increase in 16O2 over time (Δ16O2/Δt), where the decline of 18O2 (i.e. Δ18O2/Δt) and 18O2 is corrected for rela- tive to the concentration of 16O2. Chlorophyll a- and C-specific rates were obtained by divid- ing U0 and E0 by the concentration of Chla and particulate organic carbon, respectively. Rates were multiplied by 1.073 to spectrally correct to the culturing LEDs (S1 Fig). PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Measuring O2 exchange by membrane inlet mass spectrometry (MIMS) p y p y g ( g) Photosynthesis-light (P-E) curves for gross (E0 Chl(C)) and net photosynthesis (Pnet Chl(C) = E0 Chl(C)-U0 Chl(C)) were fitted to the equations from Platt and Jassby [37]; Photosynthesis-light (P-E) curves for gross (E0 Chl(C)) and net photosynthe E0 Chl(C)-U0 Chl(C)) were fitted to the equations from Platt and Jassby [37]; E ChlðCÞ 0 ¼ E ChlðCÞ 0m 1 e aChlðCÞ g E E ChlðCÞ 0m ! " # ð9Þ ð9Þ Pnet ChlðCÞ ¼ Pnet ChlðCÞ m 1 e aChlðCÞ n E Pnet ChlðCÞ m þ R ChlðCÞ d ð10Þ ð10Þ where E0m Chl(C) and Pnetm Chl(C) are the maximum gross and net O2 evolution rates; αg Chl(C) where E0m Chl(C) and Pnetm Chl(C) are the maximum gross and net O2 evolution rates; αg Chl(C) PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 4 / 24 Trichodesmium utilising additional N-sources and αn Chl(C) are the initial light-limited slopes for gross and net photosynthesis; Rd is the dark respiration rate; and E is the light intensity (μmol photons m-2 s-1). Curve fitting was per- formed on each replicate separately to calculate mean (± S.E.) curve fit parameterisations (Sig- maplot 11.0). and αn Chl(C) are the initial light-limited slopes for gross and net photosynthesis; Rd is the dark respiration rate; and E is the light intensity (μmol photons m-2 s-1). Curve fitting was per- formed on each replicate separately to calculate mean (± S.E.) curve fit parameterisations (Sig- maplot 11.0). The maximum quantum efficiency of gross (ɸmgross) and net (ɸmnet) O2 evolution was cal- culated as follows; ɸm ¼ aC gðnÞ aC eff ð11Þ ð11Þ where the C-specific initial slope for gross (αg C) or net (αn C) O2 evolution was divided by the C-specific, effective light absorption coefficient (aeff C). where the C-specific initial slope for gross (αg C) or net (αn C) O2 evolution was divided by the C-specific, effective light absorption coefficient (aeff C). Measuring nitrogenase activity by acetylene reduction Acetylene reduction rates were measured using gas chromatography (ATI Unicam 610 series). Gaseous samples were injected into the GC column head (60 ˚C), carried via N2 gas through a Porapak N column (100 ˚C) to a flame ionising detector (100 ˚C). Peak areas of acetylene and ethylene were quantified by an integrated chromatograph data acquisition unit (Shimadzu C-R8A Integrator) and were converted into concentrations via an acetylene and ethylene stan- dard curve performed with standard gases (Scientific and Technical Gases Ltd., UK). Triplicate 6 mL samples of each biological replicate culture were placed into 12 mL exetainer, screw capped glass vials (Labco Ltd, UK). Exactly 1.2 mL of the headspace was removed and replaced with a 1.2 mL sample of acetylene (BOC Industrial Gases, UK) (headspace = 20% acetylene). The vials were gently inverted for 1 minute before 250 μL of headspace was injected into the GC column for an initial measurement of acetylene and ethylene concentrations (T0). Vials were incubated at 26 ˚C and 400 μmol photons m-2 s-1 in an aluminium temperature block and were gently inverted every 10 minutes to prevent trichomes from settling on the bottom or aggregating at the meniscus. After 1 hour, a second 250 μL gaseous headspace was injected into the GC column for the post-incubation measurement (T1). Temperature and pressure was measured during each set of measurements and accounted for in the calculations. The rate of ethylene production was calculated with the assumption that the concentrations of acetylene and ethylene within the media were always in equilibrium to those in the headspace; DC2H2 ¼ C2H2ðT1Þ C2H2ðT0Þ t VðIÞ ð12Þ ð12Þ where (ΔC2H2) is the ethylene production rate (μmol C2H4 h-1), C2H2(T0) and C2H2 (T1) are the ethylene concentrations in the headspace at the start (T0) and end (T1) of the incubation, V(I) is the volume of gaseous sample injected into the GC column (L-1) and t is the incubation time (min). N2 fixation rates were calculated to a Chla (μmol N2 (mg Chla)-1 h-1) and total carbon (μmol N2 (mg C)-1 h-1) basis; N2 fixation ¼ DC2H2 ½Chl aðCÞ 103 0:25 ð13Þ ð13Þ where ΔC2H2 (μmol h-1) is divided by the Chla or total carbon concentration (mg) and multi- plied by 0.25 under the assumption that reduction of four moles of acetylene is equivalent to reduction of one mole of dinitrogen. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Fluorescence light curves (FLCs) A 2 mL sample of each replicate culture was used to measure a fluorescence light curve (FLC) [38]. The FLCs were measured with a FRRfII FastAct Fluorometer System, using a white LED actinic light source (Chelsea Technologies Group Ltd, UK). Each FLC lasted 1 hour; compris- ing 12 light steps which ranged from 10 to 1600 μmol photon m-2 s-1, each lasting 5 minutes in duration. The FLCs provided measurements of the light absorption cross-section of PSII pho- tochemistry (σPII´), the average time constant for the re-opening of a closed PSII reaction cen- tre (τf´) and the operating efficiency of PSII photochemistry (Fq´/Fm´); Fqʹ Fmʹ ¼ Fmʹ Fʹ Fmʹ ð14Þ ð14Þ where Fm´ is the maximum fluorescence in the light-adapted state and F´ is the steady-state fluorescence at any point. Photosystem II (PSII) electron transport rates were normalised to a Chla (mol e- (g Chla)-1 h-1) and total carbon (mol e- (g C)-1 h-1) basis; ETRChlðCÞ ¼ Fqʹ Fmʹ E ðaChlðCÞ FAQPIIÞ 3600 SCF ð15Þ ð15Þ where Fq´/Fm´ is the operating efficiency of PSII photochemistry; E is the light intensity (mol photons m-2 s-1), aChl(C) is the Chla-specific (C-specific) effective light absorption (m2 g-1 Chla and m2 g-1 C, respectively), FAQPII is the fraction of absorbed photons directed to PSII, which was set to 0.5 [39], with the assumption that the quantum yield of electron transport of one trapped photon within a reaction centre is equal to 1 [40]; 3600 converts seconds to hours and SCF is a spectral correction factor of 1.194, which converts electron transport rates to the cul- turing LED spectrum (S1 Fig). where Fq´/Fm´ is the operating efficiency of PSII photochemistry; E is the light intensity (mol photons m-2 s-1), aChl(C) is the Chla-specific (C-specific) effective light absorption (m2 g-1 Chla and m2 g-1 C, respectively), FAQPII is the fraction of absorbed photons directed to PSII, which was set to 0.5 [39], with the assumption that the quantum yield of electron transport of one trapped photon within a reaction centre is equal to 1 [40]; 3600 converts seconds to hours and SCF is a spectral correction factor of 1.194, which converts electron transport rates to the cul- turing LED spectrum (S1 Fig). Measuring nitrogenase activity by acetylene reduction PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 5 / 24 Trichodesmium utilising additional N-sources Fluorescence light curves (FLCs) ETR curves were modelled using a P-E equation [37], performed on each individual repli- cate using a Marquardt–Levenberg least squares algorithm to generate the best fit (R2 > 0.993); ETR ¼ ETRmʹ 1 e aETR E ETRmʹ e bETR E ETRmʹ ð16Þ ð16Þ where ETRm´ is the hypothetical Chla(C)-specific maximum electron transport rate that would be achieved if there was no photoinhibition (mol e- (g Chla(C))-1 h-1); αETR is the initial slope of the Chla(C)-specific ETR-light curve (mol e- (g Chla(C))-1 h-1 (μmol photons m-2 s-1)-1); βETR is the parameter that accounts for downregulation and/or photoinhibition at supra-opti- mal light intensities (mol e- (g Chla(C))-1 h-1 (μmol photons m-2 s-1)-1); and E is the light inten- sity (μmol photons m-2 s-1). The realised maximum PSII electron transport rate in the presence of photoinhibition (ETRm), light intensity at which ETR is maximal (Eopt), the light-saturation parameter (Ek) PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 6 / 24 Trichodesmium utilising additional N-sources Trichodesmium utilising additional N-sources and the light inhibition parameter (Ep) were calculated from the fitted parameters as follows: and the light inhibition parameter (Ep) were calculated from the fitted parameters as follows: ETRm ¼ ETRm 0 aETR aETR þ bETR bETR aETR þ bETR bETR aETR ð17Þ ð17Þ Eopt ¼ ETRm 0 aETR ln aETR þ bETR bETR ð18Þ ð18Þ Ek ¼ ETRm aETR ð19Þ ð19Þ Ep ¼ ETRm bETR ð20Þ ð20Þ The ratio of PSII electron transport to gross O2 evolution (E0) under light-limitation (Feα) and light-saturation (Fem) were calculated as follow; Fea ¼ aETR ag ð21Þ Fem ¼ ETRm E0m ð22Þ Fea ¼ aETR ag ð21Þ ð21Þ Fem ¼ ETRm E0m ð22Þ ð22Þ Cellular elemental composition and light absorption Samples for determining particulate organic carbon (POC), nitrogen (PN) and phosphorus (PP) (S5 File), chlorophyll a (S6 File) and in vivo light absorption (S7 File) were collected with each MIMS measurement, with each sample being a biological replicate. Modelling the in vivo light absorption from pigment absorption spectra In vivo light absorption was reconstructed using the light absorption spectra of Chla and photoprotective carotenoids (PPC) taken from Woźniak et al. [41] and the light absorption spectra of phycourobilin (PUB1, PUB2, PUBx, PUB4, PUB5a, PUBb, PUB5d, PUB5g and PUB5j), phycoerythrin (PE1, PE2a, PE2b and PE3b), alloplastocyanin (APC) and plastocyanin (PC1 and PC2) taken from Ku¨pper et al. [42] (S2 Fig). The Chla-specific light absorption coefficient was modelled as the sum of the contribution of all pigments; aChl modðlÞ ¼ X i bi aiðlÞ ð23Þ ð23Þ where aChl mod is the modelled in vivo light absorption at a specific wavelength (λ = 400–700 nm); βi is the contribution of each pigment to aChl mod and ai is the pigment-specific spectral absorption coefficient of pigment i, in m2 (g pigment i)-1. hl The modelled in vivo light absorption spectra (aChl mod (λ)) was optimised to the measured spectra between 400 and 700 nm using a reduced sum of squares method (Sigmaplot 11.0). If a zero value was returned for a βi parameter, that pigment was removed from the model and the curve fit reapplied. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 7 / 24 Trichodesmium utilising additional N-sources Inorganic C-chemistry, growth rate and cell composition Balanced growth of T. erythraeum IMS101 was 0.34 d-1 when grown on N2, increasing by 10% and 13% when grown in the presence of NH4 + and NO3 -, respectively (Table 1). Particulate C: N, C:P and N:P ratios were all influenced by the presence of additional N-sources. When compared to the N2 treatment, C:N decreased by 36% and 43% for the NH4 + and NO3 - treat- ments, respectively. Ratios of C:P and N:P were comparable between NH4 + and NO3 - treat- ments, but were significantly lower (~ 60% and 35%, respectively) compared to the N2 treatment (Table 1). Ratios of Chla:C were 80% and 67% higher for the NH4 + and NO3 - treat- ments than for the N2 treatment, while Chla:N was not significantly different between treat- ments (Table 1). Carbon and Chla-specific N2 fixation rates were highest for the N2 treatment, decreasing significantly by 84% and 80% (Chla-specific) and 73% and 68% (C-specific) for the NH4 + and NO3 - treatments, respectively (Table 1). The inorganic carbon concentration, pH and alkalinity (AT) did not vary significantly amongst N-source treatments. Overall, CO2 drawdown ranged between 78 to 92 μatm from the target concentration (i.e. 380 μatm) for all N-source treatments (Table 2) and exhibited lit- tle variability over a diurnal cycle (S3 Fig). Inorganic N concentrations were > 1 μM for the N2 treatment and were ~ 8 μM for the NH4 + and NO3 - treatments at the point of dilution (Table 2). Abbreviations; C:N, C:P and N:P ratios are mol:mol, Chla:C and Chla:N ratios are mg:mol (n = 3). Letters in parenthesis indicate significant differences between N- source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A]. Light absorption The effective light absorption coefficients were not significantly different between N-source treatments, nor were the modelled absorption coefficients significantly different to the mea- sured coefficients; with modelled coefficients being only 1 to 3% higher across all N-source treatments (Table 3). In vivo light absorption spectra (Fig 1) exhibited peaks at ~ 440 nm (Chla), ~ 490–500 nm (phycourobilin; PUB), ~ 540 and 568 nm (phycoerythrin; PE), ~ 620 nm (phycocyanin; PC), ~ 640 nm (allophycocyanin; APC) and ~ 675 nm (Chla) (Table 3). Chlorophyll a and photo- protective carotenoids (PPC) dominated light absorption, together accounting for ~ 65% of Table 1. The median (± S.E.) balanced growth rates and mean elemental stoichiometry and N2 fixation rates for T. erythraeumIMS101 when acclimated to three N- source conditions (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). Variables Units N2 NH4 + NO3 - Growth rate d-1 0.340 (0.038)[A] 0.375 (0.011)[B] 0.384 (0.005)[B] Elemental Stoichiometry C:N mol:mol 6.9 (0.7) 4.4 (0.9) 3.9 (0.7) C:P mol:mol 122.6 (7.0)[B] 47.9 (2.4)[A] 36.9 (2.9)[A] N:P mol:mol 18.1 (1.3)[B] 11.8 (2.2) 9.9 (1.0)[A] Chla:C mg:mol 134 (8)[A] 239 (4)[B] 222 (2)[B] Chla:N mg:mol 906 (43) 1041 (209) 855 (154) N2 Fixation Chla-specific μmol N (mg Chla)-1 h-1 14.75 (1.66)[B] 2.35 (0.49)[A] 2.84 (0.44)[A] C-specific μmol N (mg C)-1 h-1 0.16 (0.01)[B] 0.04 (0.01)[A] 0.05 (0.01)[A] Table 1. The median (± S.E.) balanced growth rates and mean elemental stoichiometry and N2 fixation rates for T. erythraeumIMS101 when acclimated to three N- source conditions (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C) Table 1. The median (± S.E.) balanced growth rates and mean elemental stoichiometry and N2 fixation rates for T. erythraeumIMS101 when acclimated to three N- source conditions (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C) ates and mean elemental stoichiometry and N2 fixation rates for T. erythraeumIMS101 when acclimated to three N- get CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature Abbreviations; C:N, C:P and N:P ratios are mol:mol, Chla:C and Chla:N ratios are mg:mol (n = 3). Light absorption Letters in parenthesis indicate significant differences between N- source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 8 / 24 Trichodesmium utilising additional N-sources Table 2. The growth conditions (± S.E.) for T. erythraeumIMS101 when cultured under three N-source conditions (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). Variables Units N2 NH4 + NO3 - pH Total 8.18 8.18 8.19 H+ nM 6.6 (0.1) 6.6 (0.1) 6.4 (0.2) AT μM 2483 (47) 2427 (59) 2482 (56) TCO2 μM 2066 (41) 2019 (51) 2056 (44) HCO3 - μM 1762 (35) 1723 (44) 1746 (33) CO3 2- μM 296 (7) 288 (9) 302 (12) CO2 μM 8.3 (0.2) 8.2 (0.3) 8.0 (0.4) pCO2 μatm 300 (8) 296 (9) 289 (7) NH4 + μM 0.76 (0.13) 8.33 (0.45) 0.59 (0.07) NO3 - μM 0.07 (0.07) 0.46 (0.07) 8.24 (1.31) n 35 34 10 Individual pH values were converted to a H+ concentration, allowing a mean pH value to be calculated. https://doi.org/10.1371/journal.pone.0195638.t002 Table 2. The growth conditions (± S.E.) for T. erythraeumIMS101 when cultured under three N-source conditions (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). Individual pH values were converted to a H+ concentration, allowing a mean pH value to be calculated. the total. PUB1 and PUB2 were the only pigments to exhibit significant differences, where rela- tive to the N2 treatment, the contribution of PUB1 to the total light absorption increased by 7.4% whereas PUB2 decreased by 1.3% in the presence of NO3 - (Table 3). PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Trichodesmium utilising additional N-sources Table 3. The mean (± S.E.) measured and modelled effective light absorption coefficients and the relative contribution of each photosynthetic pigment to the total light absorption under the culturing LEDs within T. erythraeumIMS101, when acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). Table 3. The mean (± S.E.) measured and modelled effective light absorption coefficients and the relative contribution of each photosynthetic pigment to the total light absorption under the culturing LEDs within T. erythraeumIMS101, when acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). Table 3. The mean (± S.E.) measured and modelled effective light absorption coefficients and the relative contribution of each photosynthetic pigment to the total light absorption under the culturing LEDs within T. erythraeumIMS101, when acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). Variables Units N2 NH4 + NO3 - aeff Chl m2 (g Chla)-1 9.9 (0.6) 7.7 (0.9) 8.1 (0.3) aeff C m2 (g C)-1 0.111 (0.013) 0.154 (0.020) 0.149 (0.006) amod Chl m2 (g Chla)-1 10.0 (0.6) 7.8 (0.9) 8.3 (0.3) amod C m2 (g C)-1 0.112 (0.013) 0.156 (0.019) 0.154 (0.005) Chla % 35.66 (0.41) 36.74 (0.40) 39.18 (2.14) PPC % 30.64 (3.19) 27.48 (3.05) 27.44 (2.87) PUB1 % 2.67 (1.36)[A] 5.04 (4.67) 10.11 (2.13)[B] PUB2 % 1.34 (0.28)[B] 1.63 (1.04) 0.06 (0.06)[A] PUBx % 0 0 0 PUB4 % 0.02 (0.02) 0 0 PUB5a % 0.42 (0.21) 0 0 PUB5b % 0.24 (0.23) 0 0 PUB5d % 0.05 (0.03) 0 0 PUBg % 0.18 (0.18) 0 0 PUBj % 0.19 (0.19) 0 0 PE1 % 8.10 (3.73) 7.51 (3.70) 3.19 (2.01) PE2a % 1.17 (0.75) 0 0 PE2b % 1.02 (0.72) 0 0 PE3b % 10.93 (2.28) 13.14 (3.60) 13.03 (0.92) APC % 5.45 (1.30) 5.17 (1.34) 5.97 (1.17) PC1 % 0.94 (0.57) 0.19 (0.19) 0 PC2 % 2.22 (1.15) 3.08 (1.61) 1.02 (0.52) Light absorption coefficients were spectrally corrected to the culture LEDs and were normalised to a chlorophyll a (m2 g Chla-1) and carbon (m2 g C-1) basis. Light-dependence of O2 exchange The C-specific maximum rate (E0m C) and initial slope (αg C) of light-dependent gross photo- synthesis increased with additional N-sources (i.e. NH4 + and NO3 -) and was highest for the NH4 + treatment relative to the N2 treatment (Table 4). There were also significant effects of additional N-sources on the Chla-specific maximum rate (E0m Chl) and initial slope of light- dependent gross photosynthesis (αg Chl) (S1 Table), however the effects were more pronounced when expressed as a C-specific rate, where E0m C increased by 143% from the N2 to the NH4 + treatment, while E0m Chl increased by only 36%. The light saturation parameter (Ek = E0m C/αg C) of gross O2 evolution did not vary signifi- cantly between N-source treatments (Table 4) and was due to covariation of αg C and E0m C. The maximum quantum efficiency of gross O2 evolution (ɸmgross = αg C/aeff C) increased signifi- cantly by 76% from the N2 to NH4 + treatment (Table 4) and was due to the relatively constant aeff C and the significant increase in αg C. C Carbon-specific dark respiration rates (Rd C) varied by ~ 24% and were slightly higher for the N2 and NH4 + treatments than the NO3 - treatment (Table 4). Light-saturated net O2 evolu- tion rates (Pnetm C) approximately trebled and more than doubled from the N2 treatment to the NH4 + and NO3 - treatments respectively (Table 4); with the initial slope (αn C) showing a similar pattern to Pnetm C. This increase in αn C for the NH4 + and NO3 - treatments resulted in the maxi- mum quantum efficiency of net O2 evolution (ɸmnet = αn C/aeff C) increasing significantly by 86% and 100% respectively, relative to the N2 treatment (Table 4). The light saturation parame- ter (Ek = Pnetm C/αg C) for net O2 evolution did not vary significantly between N-source treat- ments (Table 4). The relationship between net and gross O2 evolution was linear (Fig 2D–2F), with the slope increasing by approximately 40% when cultured in the presence of an additional N-source (Table 4). This linear relationship suggests that light-dependent O2 consumption (U0 C) was a PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 9 / 24 Abbreviations; a ff Chl and a ff C are the measured Chla- and C-specific light absorption coefficients while a d Chl and a d C are the modelled Chla- and C-specific light Light absorption coefficients were spectrally corrected to the culture LEDs and were normalised to a chlorophyll a (m g Chla ) and carbon (m g C ) basis. Abbreviations; aeff Chl and aeff C are the measured Chla- and C-specific light absorption coefficients, while amod Chl and amod C are the modelled Chla- and C-specific light absorption coefficients. amod Chl and amod C were constructed from a range of pigment light absorption spectrums (λ = 400–700); comprising chlorophyll a (Chla), photoprotective carotenoids (PPC), phycourobilins (PUB1, PUB2, PUBx, PUB4, PUB5a, PUBb, PUB5d, PUB5g and PUB5j), phycoerythrin (PE1, PE2a, PE2b and PE3b), alloplastocyanin (APC) and plastocyanin (PC1 and PC2). Letters in parenthesis indicate significant differences between N-source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A]. constant proportion of gross O2 evolution (E0 C) and was independent of light intensity for all N-source treatments. Subtracting the slope from unity gave the ratio of light-driven U0 C to E0 C, which was significantly lower for the N2 treatment. The ratio of gross photosynthesis (E0) to N2 fixation increased 9-fold and 6-fold for the NH4 + and NO3 - treatments relative to the N2 treatment. In addition, the ratio of net photosyn- thesis (Pnet) to N2 fixation was 12-fold and 7-fold higher for the NH4 + and NO3 - treatments rel- ative to the N2 treatment (Table 4). Light absorption coefficients were spectrally corrected to the culture LEDs and were normalised to a chlorophyll a (m2 g Chla-1) and carbon (m2 g C-1) basis. Abbreviations; aeff Chl and aeff C are the measured Chla- and C-specific light absorption coefficients, while amod Chl and amod C are the modelled Chla- and C-specific light absorption coefficients. amod Chl and amod C were constructed from a range of pigment light absorption spectrums (λ = 400–700); comprising chlorophyll a (Chla), photoprotective carotenoids (PPC), phycourobilins (PUB1, PUB2, PUBx, PUB4, PUB5a, PUBb, PUB5d, PUB5g and PUB5j), phycoerythrin (PE1, PE2a, PE2b and PE3b), alloplastocyanin (APC) and plastocyanin (PC1 and PC2). Letters in parenthesis indicate significant differences between N-source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A]. ally corrected to the culture LEDs and were normalised to a chlorophyll a (m2 g Chla-1) and carbon (m2 g C-1) basis. Trichodesmium utilising additional N-sources Fig 1. The mean (± S.E.) Chla (a-c) and C-specific (d-f) in vivo light absorption spectra for T. erythraeumIMS101 (n = 3). Cultures were acclimated to three N-source treatments (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). The solid black line is the measured light absorption spectra (grey area represents the S.E.) while the dashed line is the modelled light absorption spectra. Fig 1. The mean (± S.E.) Chla (a-c) and C-specific (d-f) in vivo light absorption spectra for T. erythraeumIMS101 (n = 3). Cultures were acclimated to three N-source treatments (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). The solid black line is the measured light absorption spectra (grey area represents the S.E.) while the dashed line is the modelled light absorption spectra. Fig 1. The mean (± S.E.) Chla (a-c) and C-specific (d-f) in vivo light absorption spectra for T. erythraeumIMS101 (n = 3). Cultures were acclimated to three N-source treatments (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). The solid black line is the measured light absorption spectra (grey area represents the S.E.) while the dashed line is the modelled light absorption spectra. https://doi.org/10.1371/journal.pone.0195638.g001 https://doi.org/10.1371/journal.pone.0195638.g001 photochemistry (σPII) and the time constant for the re-opening of a closed PSII reaction centre (τf) in the dark-adapted state were not significantly different between N-source treatments. Furthermore, both σPII´ and τf´ exhibited no light-dependency, remaining relatively constant across the entire range of actinic light intensities (Fig 3, Table 5). The light intensity at which ETR was maximal (Eopt) was significantly lower (by ~ 120 μmol photons m-2 s-1) for the NH4 + treatment relative to the N2 treatment (Fig 4). The Chla and C-specific maximum electron transport rate and initial slope (αETR) of the ETR-light curves were not significantly different between N-source treatments (Table 5, S2 Table). In contrast, the light-saturated photoinhibition slopes (βETR) were significantly different, with β increasing by 5% and 10% for the NO3 - and NH4 + treatments, relative to the N2 treatment (Table 5). PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Light-dependence of PSII electron transport The operating efficiency of PSII photochemistry (Fq'/Fm´) increased at low light intensities, reaching a maximum at ~ 110 to 130 μmol photons m-2 s-1, before decreasing significantly with increasing light intensity (Fig 3). The light saturation parameter (Ek) and the light at which ETR was maximal (Eopt) were significantly higher for the N2 treatment than the NH4 + treatment. Conversely, the light inhibition parameter (Ep), absorption cross-section of PSII 10 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 https://doi.org/10.1371/journal.pone.0195638.g001 The ratio of PSII electron transport to gross O2 evolution under light-limitation (Feα) was ~ 4 and did not vary significantly between N-source treatments. Light saturated ratios (Fem) increased relative to Feα for all N-source treatments, with the N2 treatment being 46% and 35% higher than the NH4 + and NO3 - treatments, respectively (Table 5). 11 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Trichodesmium utilising additional N-sources Table 4. The parameters (± S.E.) of the C-specific light-response curves for gross and net photosynthetic O2 evolution of T. erythraeumIMS101 (n = 3). Parameters Units N2 NH4 + NO3 - Gross O2 evolution E0m C mmol O2 (g C)-1 h-1 6.05 (0.37)[A] 14.71 (1.20)[C] 10.98 (0.33)[B] Ek μmol photons m-2 s-1 238 (55) 227 (44) 255 (55) αg C μmol O2 (g C)-1 h-1 (μmol photons m-2 s-1)-1 27.9 (5.3)[A] 67.7 (8.2)[B] 49.8 (15.1) ɸmgross mol O2 (mol photons)-1 0.07 (0.01)[A] 0.12 (0.01)[B] 0.09 (0.03) E0:Nfix mol O2 (mol N2)-1 31 (4)[A] 289 (32)[B] 185 (57)[B] Net Photosynthesis Pnetm C mmol O2 (g C)-1 h-1 3.75 (0.27)[A] 11.48 (1.56)[B] 9.59 (0.37)[B] Ek μmol photons m-2 s-1 250 (69) 277 (8) 220 (37) αn C μmol O2 (g C)-1 h-1 (μmol photons m-2 s-1)-1 16.8 (3.4)[A] 41.5 (5.8)[B] 46.1 (8.0)[B] Rd C mmol O2 (g C)-1 h-1 -1.63 (0.19) -1.53 (0.28) -1.16 (0.76) ɸmnet mol O2 (mol photons)-1 0.04 (0.01)[A] 0.08 (0.02)[B] 0.09 (0.01)[B] Pnet:Nfix mol O2 (mol N2)-1 18 (1)[A] 207 (29)[B] 163 (36)[B] Gross (x) vs. Net (y) slope Dimensionless 0.60 (0.02)[A] 0.82 (0.03)[B] 0.83 (0.01)[B] Abbreviations; E0m C, the C-specific maximum gross O2 evolution rate; Pnetm C, the C-specific maximum net O2 evolution rate; Ek, the light saturation parameter; αg C and αn C are the C-specific initial slopes the light response curve for net and gross photosynthesis; ɸmgross and ɸmnet are the maximum quantum efficiencies of gross and net O2 evolution; Rd C, the C-specific dark respiration rate; slope, the gradient of the regression between Pnet C and E0 C; E0:Nfix and Pnet:Nfix, the ratio of gross and net photosynthesis to N2 fixation, where rates of E0 and Pnet were calculated at 400 μmol photons m-2 s-1, matching to light intensity of the N2 fixation incubations; slope, the gradient of the regression between Pnet C and E0 C. The r2 values of all curve fits were > 0.982. Letters in parenthesis indicate significant differences between CO2 treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A]. Abbreviations; E0m C, the C-specific maximum gross O2 evolution rate; Pnetm C, the C-specific maximum net O2 evolution rate; Ek, the light saturation parameter; αg C and αn C are the C-specific initial slopes the light response curve for net and gross photosynthesis; ɸmgross and ɸmnet are the maximum quantum efficiencies of gross and net O2 evolution; Rd C, the C-specific dark respiration rate; slope, the gradient of the regression between Pnet C and E0 C; E0:Nfix and Pnet:Nfix, the ratio of gross and net photosynthesis to N2 fixation, where rates of E0 and Pnet were calculated at 400 μmol photons m-2 s-1, matching to light intensity of the N2 fixation incubations; slope, the gradient of the regression between Pnet C and E0 C. The r2 values of all curve fits were > 0.982. Letters in parenthesis indicate significant differences between CO2 treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A]. Abbreviations; E0m C, the C-specific maximum gross O2 evolution rate; Pnetm C, the C-specific maximum net O2 evolution rate; Ek, the light saturation parameter; αg C and αn C are the C-specific initial slopes the light response curve for net and gross photosynthesis; ɸmgross and ɸmnet are the maximum quantum efficiencies of gross and net O2 evolution; Rd C, the C-specific dark respiration rate; slope, the gradient of the regression between Pnet C and E0 C; E0:Nfix and Pnet:Nfix, the ratio of gross and net photosynthesis to N2 fixation, where rates of E0 and Pnet were calculated at 400 μmol photons m-2 s-1, matching to light intensity of the N2 fixation incubations; slope, the gradient of the regression between Pnet C and E0 C. The r2 values of all curve fits were > 0.982. Letters in parenthesis indicate significant differences between CO2 treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A]. https://doi.org/10.1371/journal.pone.0195638.t004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Trichodesmium utilising additional N-sources Fig 2. The C-specific light response curves for gross O2 evolution, O2 consumption, net photosynthesis (n = 3) (a-c) and the relationship between gross and net O2 evolution (d-f) for T. erythraeumIMS101. Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). Chla-specific light response curves are shown in S4 Fig, while the light response curves for individual replicates are shown in S6–S8 Fig. https://doi.org/10.1371/journal.pone.0195638.g002 Fig 2. The C-specific light response curves for gross O2 evolution, O2 consumption, net photosynthesis (n = 3) (a-c) and the relationship between gross and net O2 evolution (d-f) for T. erythraeumIMS101. Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). Chla-specific light response curves are shown in S4 Fig, while the light response curves for individual replicates are shown in S6–S8 Fig. https://doi.org/10.1371/journal.pone.0195638.g002 https://doi.org/10.1371/journal.pone.0195638.g002 Growing evidence points towards nitrogenase being expressed in subsets of cells within fila- ments, called diazocytes [21, 25]. To date, no translocation transport mechanisms for N com- pounds have been observed in Trichodesmium, leading to suggestions that diazocytes release N into the external medium for use by neighbouring cells [25, 46]. This is partially supported by observations of Trichodesmium exhibiting a high capacity for NH4 + uptake during active N2 fixation [17, 18]. While such mechanisms may exist, we did not observe significant concentra- tions of dissolved inorganic NO3 - or NH4 + in the medium of our control treatment. Effect of acclimation to variation of N-sources on growth rates and elemental stoichiometry Growth rates achieved under diazotrophic conditions were similar to most previous studies [23, 43–45], as was the increase in growth rate observed under non-diazotrophic conditions [23, 43], which we attribute to the lowered demand of NADPH and ATP for nitrogenase activ- ity, where NADPH and ATP could be re-directed to CO2 fixation and/or biosynthesis. Our data shows that at saturating light intensity, the energetic cost of diazotrophy constrains Tri- chodesmium growth by ~ 13%. However, in a natural system, potential changes to inorganic carbon chemistry (influencing the activity of the carbon concentrating mechanism (CCM)), temperature (influencing enzyme activity), or other key nutrients (i.e. Fe, P), all of which were controlled in our experiments, will almost certainly influence this estimate. The decrease in C:N, C:P and N:P under non-diazotrophic conditions is consistent with previous findings [43]. The high C:N under diazotrophic conditions may be due to accumula- tion of stored glycogen, whereas the decrease in C:N under non-diazotrophic conditions is likely due to high cellular N concentrations, likely due to the luxury uptake of NH4 + and NO3 -, where surplus N is stored within cyanophycin granules [26]. Given the concurrent decrease in C:N, C:P and N:P under non-diazotrophic conditions, it is likely that utilising NH4 + or NO3 - as a N-source enables Trichodesmium cells of low carbon biomass to maintain a Chla concen- tration comparable to diazotrophic conditions. This is supported by previous observation made by Eichner et al. [43] and is also reflected by the higher Chla:C yet comparable Chla:N ratios for NH4 + or NO3 - treatments. 12 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Effect of acclimation to different N-sources on gross photosynthesis We show an effect of N-source on C-specific light saturated gross O2 evolution rates. The more than two-fold increase in the maximum O2 evolution rate and initial slope when T. ery- thraeum IMS101 was grown on NH4 + or NO3 - than when growing diazotrophically was largely due to differences in the ratio of Chla:C as chlorophyll a-specific photosynthetic parameters varied by only 36% between N2 and NH4 + treatments. 13 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Trichodesmium utilising additional N-sources Fig 3. The operating efficiency of PSII photochemistry (Fq´/Fm´) (a-c), light absorption cross-section of PSII photochemistry (σPII´) (d-f) and average time constant for the re-opening of a closed PSII reaction centres (τf´) (g-i) across a range of actinic light intensities for T. erythraeum IMS101 (n = 3). Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). https://doi.org/10.1371/journal.pone.0195638.g003 Fig 3. The operating efficiency of PSII photochemistry (Fq´/Fm´) (a-c), light absorption cross-section of PSII photochemistry (σPII´) (d-f) and average time constant for the re-opening of a closed PSII reaction centres (τf´) (g-i) across a range of actinic light intensities for T. erythraeum IMS101 (n = 3). Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). https://doi.org/10.1371/journal.pone.0195638.g003 https://doi.org/10.1371/journal.pone.0195638.g003 The increase of C-specific gross O2 evolution rates when Trichodesmium is supplied with NH4 + or NO3 - may be due to an increase in the maximum rate of CO2 fixation and/or to an increase in PSII concentration. Previous studies report high PSI:PSII ratios under diazotrophic conditions (ranging between 1.3 to 4) [47–51], which would allow cyclic photophosphoryla- tion in diazocytes to provide most of the ATP required for N2 fixation, with glycolysis and the Kreb’s cycle providing the required reducing equivalent. It may be that under non- PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 14 / 24 Abbreviations; ETRm C, the C-specific maximum electron transport rate; αETR C, the C-specific initial slope of the electron transport rate light response curve; βETR C, the C-specific light saturated slope of the electron transport rate light response curve; Ek, the light saturation parameter; Eopt, the light at which ETR is maximal; Ep, the light inhibition parameter; Fv/Fm, the maximum photochemical efficiency of PSII in the dark-adapted state; σPII, the absorption cross-section of PSII photochemistry in the dark-adapted state; τf, the average time constant for the re-opening of a closed PSII reaction centre in the dark-adapted state; Fem, the light saturated ratio of PSII electron transport to gross O2 evolution; Feα, the light limited ratio of PSII electron transport to gross O2 evolution. The r2 values of all curve fits were > 0.977. Letters in parenthesis indicate significant differences between N-source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A]. Trichodesmium utilising additional N-sources Table 5. The parameters (± S.E.) of the fluorescence light-response curves (FLCs) of T. erythraeumIMS101 (n = 3). Parameters Units N2 NH4 + NO3 - ETRm C mmol e- (g C)-1 h-1 62.5 (16.7) 70.3 (14.4) 91.0 (4.9) Ek μmol photons m-2 s-1 465 (8)[B] 421 (3)[A] 447 (20) αETR C mmol e- (g C)-1 h-1 (μmol photons m-2 s-1)-1 0.133 (0.033) 0.167 (0.033) 0.200 (0.003) βETR C mmol e- (g C)-1 h-1 (μmol photons m-2 s-1)-1 5081 (55)[A] 5577 (55)[C] 5332 (14)[B] Eopt μmol photons m-2 s-1 1263 (22)[B] 1144 (3)[A] 1216 (54) Ep μmol photons m-2 s-1 0.99 (0.11) 0.67 (0.08) 0.83 (0.09) Fv/Fm Dimensionless 0.44 (0.01) 0.35 (0.05) 0.32 (0.01) σPII nm2 PSII-1 0.353 (0.009) 0.367 (0.003) 0.380 (0.032) τf s-1 489 (5) 494 (15) 631 (105) Fem mol e- (mol O2)-1 10.5 (1.1)[B] 5.7 (1.1)[A] 7.9 (0.5) Feα mol e- (mol O2)-1 5.2 (0.8) 2.8 (0.2) 4.5 (1.0) Abbreviations; ETRm C, the C-specific maximum electron transport rate; αETR C, the C-specific initial slope of the electron transport rate light response curve; βETR C, the C-specific light saturated slope of the electron transport rate light response curve; Ek, the light saturation parameter; Eopt, the light at which ETR is maximal; Ep, the light inhibition parameter; Fv/Fm, the maximum photochemical efficiency of PSII in the dark-adapted state; σPII, the absorption cross-section of PSII photochemistry in the dark-adapted state; τf, the average time constant for the re-opening of a closed PSII reaction centre in the dark-adapted state; Fem, the light saturated ratio of PSII electron transport to gross O2 evolution; Feα, the light limited ratio of PSII electron transport to gross O2 evolution. The r2 values of all curve fits were > 0.977. Letters in parenthesis indicate significant differences between N-source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A]. h //d i /10 1371/j l 0195638 005 Table 5. The parameters (± S.E.) of the fluorescence light-response curves (FLCs) of T. erythraeumIMS101 (n = 3). Parameters Units N2 NH4 + NO3 - ETRm C mmol e- (g C)-1 h-1 62.5 (16.7) 70.3 (14.4) 91.0 (4.9) Ek μmol photons m-2 s-1 465 (8)[B] 421 (3)[A] 447 (20) αETR C mmol e- (g C)-1 h-1 (μmol photons m-2 s-1)-1 0.133 (0.033) 0.167 (0.033) 0.200 (0.003) βETR C mmol e- (g C)-1 h-1 (μmol photons m-2 s-1)-1 5081 (55)[A] 5577 (55)[C] 5332 (14)[B] Eopt μmol photons m-2 s-1 1263 (22)[B] 1144 (3)[A] 1216 (54) Ep μmol photons m-2 s-1 0.99 (0.11) 0.67 (0.08) 0.83 (0.09) Fv/Fm Dimensionless 0.44 (0.01) 0.35 (0.05) 0.32 (0.01) σPII nm2 PSII-1 0.353 (0.009) 0.367 (0.003) 0.380 (0.032) τf s-1 489 (5) 494 (15) 631 (105) Fem mol e- (mol O2)-1 10.5 (1.1)[B] 5.7 (1.1)[A] 7.9 (0.5) Feα mol e- (mol O2)-1 5.2 (0.8) 2.8 (0.2) 4.5 (1.0) Table 5. The parameters (± S.E.) of the fluorescence light-response curves (FLCs) of T. e Abbreviations; ETRm C, the C-specific maximum electron transport rate; αETR C, the C-specific initial slope of the electron transport rate light response curve; βETR C, the C-specific light saturated slope of the electron transport rate light response curve; Ek, the light saturation parameter; Eopt, the light at which ETR is maximal; Ep, the light inhibition parameter; Fv/Fm, the maximum photochemical efficiency of PSII in the dark-adapted state; σPII, the absorption cross-section of PSII photochemistry in the dark-adapted state; τf, the average time constant for the re-opening of a closed PSII reaction centre in the dark-adapted state; Fem, the light saturated ratio of PSII electron transport to gross O2 evolution; Feα, the light limited ratio of PSII electron transport to gross O2 evolution. The r2 values of all curve fits were > 0.977. Letters in parenthesis indicate significant differences between N-source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A]. Fig 4. Concurrent Chla (a-c) and C-specific (d-f) gross O2 evolution rates and PSII electron transport rates (ETR) for T. erythraeumIMS101 (n = 3). Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). https://doi.org/10.1371/journal.pone.0195638.g004 Fig 4. Concurrent Chla (a-c) and C-specific (d-f) gross O2 evolution rates and PSII electron transport rates (ETR) for T. erythraeumIMS101 (n = 3). Parameters Units N2 NH4 + NO3 - ETRm C mmol e- (g C)-1 h-1 62.5 (16.7) 70.3 (14.4) 91.0 (4.9) Ek μmol photons m-2 s-1 465 (8)[B] 421 (3)[A] 447 (20) αETR C mmol e- (g C)-1 h-1 (μmol photons m-2 s-1)-1 0.133 (0.033) 0.167 (0.033) 0.200 (0.003) βETR C mmol e- (g C)-1 h-1 (μmol photons m-2 s-1)-1 5081 (55)[A] 5577 (55)[C] 5332 (14)[B] Eopt μmol photons m-2 s-1 1263 (22)[B] 1144 (3)[A] 1216 (54) Ep μmol photons m-2 s-1 0.99 (0.11) 0.67 (0.08) 0.83 (0.09) Fv/Fm Dimensionless 0.44 (0.01) 0.35 (0.05) 0.32 (0.01) σPII nm2 PSII-1 0.353 (0.009) 0.367 (0.003) 0.380 (0.032) τf s-1 489 (5) 494 (15) 631 (105) Fem mol e- (mol O2)-1 10.5 (1.1)[B] 5.7 (1.1)[A] 7.9 (0.5) Feα mol e- (mol O2)-1 5.2 (0.8) 2.8 (0.2) 4.5 (1.0) Abbreviations; ETRm C, the C-specific maximum electron transport rate; αETR C, the C-specific initial slope of the electron transport rate light response curve; βETR C, the C-specific light saturated slope of the electron transport rate light response curve; Ek, the light saturation parameter; Eopt, the light at which ETR is maximal; Ep, the light inhibition parameter; Fv/Fm, the maximum photochemical efficiency of PSII in the dark-adapted state; σPII, the absorption cross-section of PSII photochemistry in the dark-adapted state; τf, the average time constant for the re-opening of a closed PSII reaction centre in the dark-adapted state; Fem, the light saturated ratio of PSII electron transport to gross O2 evolution; Feα, the light limited ratio of PSII electron transport to gross O2 evolution. The r2 values of all curve fits were > 0.977. Letters in parenthesis indicate significant differences between N-source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A]. https://doi org/10 1371/journal pone 0195638 t005 Table 5. The parameters (± S.E.) of the fluorescence light-response curves (FLCs) of T. erythraeumIMS101 (n = 3). Effect of acclimation to different N-sources on N2 fixation Nitrogenase activity declined significantly by 81–84% when Trichodesmium was cultured in the presence of an additional N-source. Despite being cultured under N-replete concentra- tions, Trichodesmium cells in the NH4 + and NO3 - treatments exhibited a baseline rate of N2 fix- ation. Similarly, Milligan et al. [52] reported a ~ 85% decrease when Trichodesmium was cultured in 100 μM of NO3 - for 2 weeks and Holl and Montoya [44] reported a 66% decrease when cultured in 20 μM of NO3 -, accrediting 8% of total N assimilation to diazotrophy despite the presence of additional N-sources. Maintaining the capability to perform N2 fixation under non-diazotrophic conditions, albeit at a reduced rate, could reflect Trichodesmium’s natural environment and act a potential safeguard mechanism to variable light and nutrient regimes. Noting that 16 moles of ATP are consumed per mole of N2 fixed (Eq 1) and that 2.56 moles of ATP can be produced per mole of O2 evolved by photophosphorylation linked LPET [53], we calculated that T. erythraeum IMS101 may use 20% of the ATP that could be generated from gross O2 evolution to support the observed N2 fixation rate during diazotrophic growth: Nfix E0 ¼ 1molN2 31molO2 1molO2 2:54molATP 16molATP 1molN2 ¼ 0:2 ð24Þ ð24Þ This proportion decreases to 2% and 4% for the NO3 - and NH4 + treatments, respectively, where the ratio of E0:Nfix increases to 289 for the NO3 - treatment and 185 in the NH4 + treat- ment, versus 31 in the N2 treatment (Table 4). Studies on natural populations of Trichodesmium spp. have shown that the addition of NO3 - (100 μM) in the morning can cause a gradual decrease of N2 fixation over the photic period [22]. Further studies have also shown that addition of glutamine (10 μM) immediately decreases N2 fixation rates, indicating a direct effect on enzyme activity as opposed to enzyme synthesis [54]. These observations have been accredited to accumulation of N-containing metabolites acting as potential inhibitors to the specific activity rather than abundance of nitrogenase [22, 54]. It is well known that intracellular nitrogen pools have a role in regulating nitrogenase activ- ity in diazotrophs [55, 56]. Dinitrogenase reductase catalyses the reduction of N2 to NH4 +, which is assimilated into glutamine (gln) and then into glutamate (glu) via the glutamine syn- thetase (GS, EC 6.3.1.2)/glutamate synthase (GOGAT) pathway [54]. Effect of acclimation to different N-sources on N2 fixation The intracellular pools of NH4 +, glu and gln have been identified as important feedback regulators of N uptake and metabolism, with GS activity in Trichodesmium being sensitive to both intra- and extracellular N concentrations [55]. It could be hypothesised that the activity of nitrogenase is influenced by internally recycled N (e.g. NH4 + and gln), while the synthesis of nitrogenase is influenced by newly assimilated N (e.g. NO3 -). Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). Fig 4. Concurrent Chla (a-c) and C-specific (d-f) gross O2 evolution rates and PSII electron transport rates (ETR) for T. erythraeumIMS101 (n = 3). Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). https://doi.org/10.1371/journal.pone.0195638.g004 Fig 4. Concurrent Chla (a-c) and C-specific (d-f) gross O2 evolution rates and PSII electron transport rates (ETR) for T. erythraeumIMS101 (n = 3). Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). https://doi.org/10.1371/journal.pone.0195638.g004 https://doi.org/10.1371/journal.pone.0195638.g004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 15 / 24 Trichodesmium utilising additional N-sources diazotrophic conditions and with lower nitrogenase activity, Trichodesmium enhances linear electron transport to increase NADPH production; a pathway that generates more evolved O2. Effect of acclimation to different N-sources on light-stimulated O2 consumption and the relationship between net and gross O2 evolution Net photosynthesis was significantly lower for the N2 treatment than for the NH4 + and NO3 - treatments. Despite slight variations in E0 C, the difference in net photosynthesis was princi- pally driven by O2 consumption. Approximately 68%, 32% and 29% (N2, NH4 + and NO3 -, respectively) of E0 C was consumed by O2 consuming processes, which is comparable to previ- ous observations [43, 52]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 16 / 24 Trichodesmium utilising additional N-sources Several processes demand ATP in excess of the ATP:NADPH produced through linear photophosphorylation; two most notably being N2 fixation and the operation of the CCM [57]. In this study, the carbon chemistry of all cultures was closely regulated to ensure that var- iation in O2 consumption and net photosynthesis was due to the N-source treatments only. Linearity between gross O2 evolution (E0) and O2 consumption was observed across all N- source treatments, suggesting that light-dependent O2 consumption is linked to balancing ATP to NADPH production, as opposed to serving as a mechanism to dissipate excitation energy. Diazotrophic cells consume more O2 per evolved O2 across the entire range of actinic light intensities than the NH4 + and NO3 - treatments. This suggests a higher rate of water-water cycling due to either Mehler activity or operation of plastoquinone terminal oxidase when N2 is being fixed. To maintain a sufficient supply of ATP relative to NADPH, Trichodesmium may utilise pseudocyclic photophosphorylation linked to the Mehler reaction to augment the ATP generated by linear electron transfer from water to NADP+ in addition to ATP produced by cyclic electron flow around PSI. Measurements of O2 evolution, ETR and N2 fixation were all made at one time of day (4 to 6 hours into the photo-phase of a 12:12 L:D cycle) and as such cannot be extrapolated to a diel response given the reports of temporal separation of photosynthesis and N2 fixation in Tricho- desmium [21]. Effect of acclimation to different N-sources on electron transport rates and photophysiology Like Eichner et al. [43], we observed a negligible effect of N-source on many photo-physiologi- cal parameters, including Fq´/Fm´, σPII and τf. Trichodesmium exhibited a light response typical for most cyanobacteria, where the dark-adapted photochemical yield is significantly affected by respiratory electron flow [58]. This results from a proportion of PSII reaction centres remaining in a closed state despite being in the dark and is imposed by a reduction in the plas- toquinone (PQ) pool, which prevents the oxidation of QA -. Moving from darkness to a low light intensity increases the electron flux through PSI, alleviates the bottleneck of electron transport through the Cyt b6f complex, thereby increasing Fq´/Fm´ and decreasing the re-oxi- dation time of QA -. Addition factors such as higher downregulation under dark-adapted con- ditions may also contribute to the increase in Fq´/Fm´ under low light intensities. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Implications for future oligotrophic oceans In N-limited regions of the oligotrophic open ocean, diazotrophy provides a competitive advantage by allowing cells to access N2 as an N-source against faster growing phytoplankton that rely on fixed N. Current ocean models predict a poleward shift in the 20 ˚C isotherm which could extend Trichodesmium’s niche into higher latitudes. On a global scale, this niche expansion is driven by increased SSTs; however, on regional scales persistence in an area may be dictated by Trichodesmium’s response to fluctuating nutrient regimes. At the surface in oligotrophic waters, Trichodesmium is unlikely to encounter NO2 -, NO3 - or NH4 + concentrations in excess of 0.1 μM [68], except during mixing events. While Tricho- desmium is commonly observed in the upper meters of the water column [69], observations have been recorded down to 200 m depth [70]. Thus, Trichodesmium colonies and free tri- chomes are able to migrate to the nutricline [30, 31]. Such vertical migration has been sug- gested to allow luxury uptake of phosphates before colonies return to the surface. In addition to encountering phosphates, Trichodesmium will also encounter high concentrations of NO3 - in the nutricline. As such, NO3 - uptake is likely at these greater depths or at the surface after a mixing event. Mulholland et al. [17] reported significant NO3 - uptake rates with the addition of 1 μM NO3 - to the growth media. Furthermore, Karl et al. [30] showed that concentrations of dis- solved NH4 + reached 1.5 μM L-1 and dissolved organic N (DON) reaching 13 μM L-1 during a natural bloom of Trichodesmium spp. in the North Pacific gyre. These concentrations are far greater than typical oceanic N pools and could therefore be high enough to inhibit N2 fixation rates [44]. It’s therefore possible that Trichodesmium colonies at depth may be utilising more combined N-sources than the blooms frequently measured on the surface. The energy and reductant conserved through utilising additional N-sources could significantly enhance Tri- chodesmium’s productivity and growth which could have major implications for biogeochemi- cal cycles. Our results indicate the need to seek more information on the potential for natural popula- tions of Trichodesmium to uptake fixed N-sources (e.g. NO3 -, NH4 +, labile dissolved organic nitrogen (DON)) at concentrations that migrating colonies or trichomes experience in the nutricline or that are encountered transiently after deep mixing events. Ratio of electron transport to gross O2 evolution Electrons are transferred from PSII (where O2 is evolved) to an intermediate plastoquinone pool and eventually to ferredoxin to produce NADPH [59]. A minimum of four moles of elec- trons are transported through PSII for each mole of O2 evolved at PSII. Most higher plants exhibit a linear correlation between gross O2 evolution and electron transport rate [60]. In microalgae, this relationship is often ambiguous, especially at high light intensities where the relationship can become non-linear [61, 62]. Here we show that at low light intensities, the ratio of PSII electron transport to gross O2 evolution (Feα) is close to a 4:1 ratio for all N-sources treatments. However, when light intensi- ties exceed 150 μmol photons m-2 s-1, Fe declines as ETR saturates at a higher light intensity (~ 900 μmol photons m-2 s-1) than E0 (~ 400 μmol photons m-2 s-1). Similar responses have been reported for diatoms [63], microalgae [64] and the Baltic cyanobacteria, Nostoc [65]. Few studies have measured O2 production rates in Trichodesmium [47, 66] and to our knowledge none have reported concurrent PSII electron transport rates. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 17 / 24 Trichodesmium utilising additional N-sources Interestingly, we calculated a higher Fe for the N2 cultures than for the NH4 + and NO3 - cul- tures, irrespective of using the light-limited or -saturated rates. This may be due to overesti- mating the proportion of light absorbed by PSII in the non-diazotrophic growth conditions (i.e. NH4 + and NO3 -) relative to the diazotrophic condition. Here we assumed that 50% of absorbed light was directed to PSII reaction centres and 50% to PSI reaction centres (i.e. FAQ- PII of 0.5). It’s likely that FAQPII was overestimated for diazotrophic treatment (i.e. N2) which may have had a higher ratio of PSI:PSII to support significant rates of cyclic photophosphory- lation. In addition, non-diazotrophic cells may undergo more pronounced state transitions with phycobilin proteins being redistributed between PSII and PSI. Finally, a Fe > 4 could be accredited to cyclic electron flow around PSII, which may act a mechanism to dissipate excess excitation energy under high light [67]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Implications for future oligotrophic oceans The potential signifi- cance of Trichodesmium assimilating fixed N is indicated by a modelling study by McGilli- cuddy [33] which concluded that to obtain realistic simulations of biomass and export production Trichodesmium populations in the North Atlantic must utilise fixed N. Specifically, this study indicated that 15–20% of the N quota of Trichodesmium could be due to uptake of NO3 - and NH4 +. Furthermore, although uptake of NO3 -, NH4 + or DON will decrease N2 fixa- tion rates in the short-term, as these N-sources are depleted over longer time periods, the PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 18 / 24 Trichodesmium utilising additional N-sources increase in Trichodesmium biomass may lead to increased N2 fixation and greater competition for other nutrients including Fe and P. Supporting information S1 Fig. The relative fluorescence excitation spectra of T. erythraeumIMS101 (Bold solid line) and the relative emission spectra of the Iso Light 400 LED (white) block used for O2 evolution incubations (Solid line), FRRf LED (blue) used for the saturating flashlets (Long-Dashed line), FastAct LED (white) used for the actinic light source (Short-dashed line) and the culturing LED (white) (Dotted line). (A) The fluorescence excitation was mea- sured on a 2 mL concentrated sample treated with 20 μM DCMU (final concentration) [71]. Trichodesmium cells were acclimated to 150 μmol photons m-2 s-1 on a 14:10 light:dark cycle, 26 ˚C and ambient CO2. The sample was measured using a FluorWin fluorometer scanning between 400 to 715 nm at a 1 nm resolution, with the monochromator on the detector set to 730 nm emission [72]. Spectral correction factors were calculated using the FastPro8. (B) An example of an in vivo light absorption spectra of T. erythraeum IMS101 when spectrally cor- rected to the Culture, MIMS or FRRf LED spectra. ( ) S2 Fig. Reconstructed light absorption spectra of eighteen key photosynthetic pigments present within T. erythraeum IMS101. (A) The light absorption spectra of chlorophyll a (Chla), photoprotectant carotenoid (PPC), phycoerythrin (PE), plastocyanin (PC) and alloplastocyanin (APC) pigments. (B) The light absorption spectra of phycourobilin (PUB) pigments. Each pigment spectra was normalised to the maximum peak (λ = 400–700 nm). (TIF) S2 Fig. Reconstructed light absorption spectra of eighteen key photosynthetic pigments present within T. erythraeum IMS101. (A) The light absorption spectra of chlorophyll a (Chla), photoprotectant carotenoid (PPC), phycoerythrin (PE), plastocyanin (PC) and alloplastocyanin (APC) pigments. (B) The light absorption spectra of phycourobilin (PUB) pigments. Each pigment spectra was normalised to the maximum peak (λ = 400–700 nm). (TIF) S3 Fig. Inorganic carbon chemistry (Ci) of T. erythraeum IMS101 cultures, measured at 2-hour intervals over the light period. The pH and TCO2 was measured directly, while the pCO2 concentrations were calculated via CO2SYS using the same constants as described in Boatman et al. [45] and S1 File. (TIF) p 2 g Boatman et al. [45] and S1 File. (TIF) S4 Fig. Chla-specific light response curves for gross O2 evolution, O2 consumption, net photosynthesis (n = 3) (A-C) and the relationship between gross and net O2 evolution (D-F) for T. erythraeumIMS101. Supporting information Chla and C-specific light response curves for gross O2 evolution, O2 consumption, net photosynthesis (n = 3) (A-C) and the relationship between gross and net O2 evolution (D-F) for T. erythraeumIMS101. Cultures were acclimated to a replete NO3 - concentration, at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). (TIF) S1 Table. Physiological parameters (± S.E.) of the Chla-specific light-response curves for gross and net photosynthetic O2 evolution of T. erythraeum IMS101 (n = 3). Abbrevi- ations; E0m Chl, the Chla -specific maximum gross O2 evolution rate; Pm Chl, the Chla -spe- cific maximum net O2 evolution rate; αg Chl and αn Chl are the Chla -specific initial slopes the light response curve for net and gross photosynthesis; Rd Chl, the Chla-specific dark res- piration rate. The r2 values of all curve fits were > 0.982. Letters in parenthesis indicate sig- nificant differences between CO2 treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A]. (PDF) S2 Table. Physiological parameters (± S.E.) of the fluorescence light-response curves (FLCs) of T. erythraeumIMS101 (n = 3). Abbreviations; ETRm Chl, the Chla-specific maxi- mum electron transport rate; αETR Chl, the Chla-specific initial slope of the electron transport rate light response curve; βETR Chl, the Chla-specific light saturated slope of the electron trans- port rate light response curve. The r2 values of all curve fits were > 0.977. Letters in parenthesis indicate significant differences between N-source treatments (One Way ANOVA, Tukey post hoc test; P < .05); where [B] is significantly greater than [A] and [C] is significantly greater than [B] and [A]. (PDF) Supporting information Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). (TIF) S5 Fig. Percentage of the modelled in vivo light absorption (amod) associated to each photo- synthetic pigment (λ = 400–700) for T. erythraeum IMS101. Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). Pigments include chlorophyll a (Chla), photoprotectant carotenoid (PPC), phycourobilins (PUB1, PUB2, PUBx, PUB4, PUB5a, PUBb, PUB5d, PUB5g and PUB5j), phycoerythrin (PE1, PE2a, PE2b and PE3b), alloplastocyanin (APC) and plastocyanin (PC1 and PC2). (TIF) S6 Fig. Chla and C-specific light response curves for gross O2 evolution, O2 consumption, net photosynthesis (n = 3) (A-C) and the relationship between gross and net O2 evolution (D-F) for T. erythraeumIMS101. Cultures were acclimated to N2-only, at a target CO2 S4 Fig. Chla-specific light response curves for gross O2 evolution, O2 consumption, net photosynthesis (n = 3) (A-C) and the relationship between gross and net O2 evolution (D-F) for T. erythraeumIMS101. Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). (TIF) S4 Fig. Chla-specific light response curves for gross O2 evolution, O2 consumption, net photosynthesis (n = 3) (A-C) and the relationship between gross and net O2 evolution (D-F) for T. erythraeumIMS101. Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). (TIF) S5 Fig. Percentage of the modelled in vivo light absorption (amod) associated to each photo- synthetic pigment (λ = 400–700) for T. erythraeum IMS101. Cultures were acclimated to three N-sources (N2, NH4 + and NO3 -), at a target CO2 concentration (380 μatm), saturating light intensity (400 μmol photons m-2 s-1) and optimal temperature (26 ˚C). Pigments include chlorophyll a (Chla), photoprotectant carotenoid (PPC), phycourobilins (PUB1, PUB2, PUBx, PUB4, PUB5a, PUBb, PUB5d, PUB5g and PUB5j), phycoerythrin (PE1, PE2a, PE2b and PE3b), alloplastocyanin (APC) and plastocyanin (PC1 and PC2). (TIF) 19 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Trichodesmium utilising additional N-sources S8 Fig. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Author Contributions Conceptualization: Tobias G. Boatman, Richard J. Geider. Conceptualization: Tobias G. Boatman, Richard J. Geider. Data curation: Tobias G. Boatman, Phillip A. Davey. Funding acquisition: Tracy Lawson, Richard J. Geider. Investigation: Tobias G. Boatman. Methodology: Tobias G. Boatman, Phillip A. Davey, Richard J. Geider. Project administration: Tobias G. Boatman, Richard J. Geider. Resources: Tobias G. Boatman. Resources: Tobias G. Boatman. Software: Tobias G. Boatman. Software: Tobias G. Boatman. Supervision: Tracy Lawson, Richard J. Geider. Supervision: Tracy Lawson, Richard J. Geider. Validation: Tobias G. Boatman, Phillip A. Davey, Tracy Lawson, Richard J. Geider. Visualization: Tobias G. Boatman. Writing – original draft: Tobias G. Boatman. Writing – review & editing: Tobias G. Boatman, Phillip A. Davey, Tracy Lawson, Richard J. Geider. Acknowledgments Tobias Boatman was supported by a UK Natural Environment Research Council PhD student- ship (NE/J500379/1 DTB). Tobias Boatman was supported by a UK Natural Environment Research Council PhD student- ship (NE/J500379/1 DTB). S1 File. Calculation of inorganic carbon speciation. (PDF) S2 File. Calculation of dissolved inorganic N concentration. (PDF) S3 File. Measuring O2 production and consumption. (PDF) S3 File. Measuring O2 production and consumption. (PDF) S4 File. MIMS sample preparation. (PDF) S5 File. Elemental stoichiometry. (PDF) 20 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195638 April 11, 2018 Trichodesmium utilising additional N-sources References 1. Moore CM, Mills MM, Langlois R, Milne A, Achterberg EP, La Roche J, et al. Relative influence of nitro- gen and phosphorus availability on phytoplankton physiology and productivity in the oligotrophic sub- tropical North Atlantic Ocean. Limnology and Oceanography. 2008; 53(1):291–305. 2. Moore JK, Doney SC, Lindsay K, Mahowald N, Michaels AF. Nitrogen fixation amplifies the ocean biogeochemical response to decadal timescale variations in mineral dust deposition. Tellus B. 2006; 58(5):560–72. 3. Vitousek PM, Howarth RW. Nitrogen limitation on land and in the sea: how can it occur? 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https://openalex.org/W3176126227	https://jgeb.springeropen.com/counter/pdf/10.1186/s43141-021-00182-7	English	null	Implications of targeted next-generation sequencing for bladder cancer: report of four cases	Journal of Genetic Engineering and Biotechnology /Journal of Genetic Engineering and Biotechnology	2,021	cc-by	5,275	RESEARCH Open Access Implications of targeted next-generation sequencing for bladder cancer: report of four cases Mohamed K. Khalifa1, Noha M. Bakr2,3, Amal Ramadan2,3, Khaled M. Abd Elwahab4, Esam Desoky4, Amira M. Nageeb2,3 and Menha Swellam2,3* * Correspondence: menhamswellam@gmail.com; 2Biochemistry Department, Genetic Engineering and Biotechnology Research Division, National Research Centre, El-Bohouth Street, Dokki, Giza 12622, Egypt gyp 3High Throughput Molecular and Genetic Laboratory, Center for Excellences for Advanced Sciences, National Research Centre, El-Bohouth Street, Dokki, Giza 12622, Egypt Full list of author information is available at the end of the article © 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/. (2021) 19:91 (2021) 19:91 Journal of Genetic Engineering and Biotechnology Journal of Genetic Engineering and Biotechnology Khalifa et al. Journal of Genetic Engineering and Biotechnology https://doi.org/10.1186/s43141-021-00182-7 Khalifa et al. Journal of Genetic Engineering and Biotechnology https://doi.org/10.1186/s43141-021-00182-7 Abstract Background: Bladder cancer is considered heterogeneous diseases with two major subgroups: non-muscle- invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). It is a major healthcare problem, and it is one of the leading causes of mortality worldwide. Genetic mutations are not only a cause for carcinogenesis but are also a way for treatment strategy. The present study aimed to investigate breast cancer (BRCA genes) tumor suppressor gene mutations in bladder cancer tissue and combined blood samples for patients who developed secondary tumor after or during trimodal therapy. Fresh tissue samples and their matched blood samples were collected from four patients with bladder cancer. The objective regions for the examined genes (BRCA1 and BRCA2) were sequenced using next-generation sequencing (NGS); generated BAM files were uploaded to the cloud-based Ionreporter server, and the Oncomine BRCA-specific plugin was used to analyze the paired normal and tumor sample for each patient using the default plugin parameters. Results: Intronic BRCA1 mutation c.5050-104 C >T was reported among the four investigated bladder cancer patients, and three somatic mutations were reported as follows: two of them were found to be benign rs1064793056 and rs28897679 on the Clinivar database and one nonsense pathogenic variant rs80357006. BRCA 2 gene mutation reported an exonic synonymous mutation rs397507876 in the tissue and germline DNA. Patients were treated with trimodal; however, three bladder cancer patients who reported BRCA mutations developed secondary tumors. Conclusion: Identification of mutational BRCA changes in bladder cancer is a promising marker for better treatment strategy. Further studies are encouraged on a large cohort of bladder cancer patients to confirm our findings. Keywords: Bladder cancer, Somatic mutation, Germline mutation, BRCA genes, Next-generation sequencing, Case report, Treatment, Tumor suppressor genes, Sequencing Methods After obtaining approval from the medical ethical com- mittee, the current study was carried out on four pa- tients with bladder cancer. The inclusion criteria for selected patients were based on patients with bladder cancer, and no other type of malignancies was reported; those did not fulfill these criteria were excluded. After obtaining informed consent, all patients underwent diag- nostic cystoscopy (to evaluate tumor size site number and associated pathology), and transurethral resection of bladder tumor (TUBT) was done. Samples of fresh tissue of urinary bladder tumors and blood samples were ob- tained from each patient; their clinicopathological char- acteristics are reported in Table 1. Gene mutations play a significant role in the incidence and progress of bladder cancer. It is well known that genes such as fibroblast growth factor 3 (FGFR3), retino- blastoma (RB1), gene belong to RAS genes (HRAS), total p-53 (TP53), and hamartin (TSC1) can regulate the nor- mal cell cleavage and avoid carcinogenesis. Hence, muta- tions in these genes can cause cancer [6]. In a previous study carried out in the USA, fifty-four cases with blad- der cancer were sequenced and reported mutation in breast cancer-associated protein (BAP1) in about 15% of the enrolled cases which was an earliest attempt [7], which further directs to the alteration in breast cancer gene (BRCA) gene pathway and facilitates the induction for the features of papillary histological modification in bladder cancer [7]. Also, the relation between BRCA1 mRNA and cisplatin-based neoadjuvant chemotherapy was studied [8] and emphasized that the detection of BRCA1 mRNA expression could estimate both the sensi- tivity to chemotherapy and prognosis among patients with bladder cancer [8]. Fresh tissue samples were collected and divided into two sections: one section was sent for pathological examination and stained with hematoxylin and eosin (H&E); the second tissue section was transferred to the lab in tubes with RNAse latter (storage reagent which al- lows tissues to stabilize and keep cellular RNA and re- duces the need to immediately treat tissue samples or to freeze them in liquid nitrogen for further processing) for NGS processing. Paired blood samples were collected in EDTA containing tubes for further DNA extraction and NGS processing. Structurally, the BRCA gene is complicated with slight information reported around it. Methods Generally, it is the tumor suppressor gene and consists of two genes, i.e., BRCA1 and BRCA2; regarding its function, it has been reported that it has a role in DNA repairing mechanism, controlling the growth of the cell and blocking gene mu- tations, however, each with altered role on the targets. BRCA1 gene ended with an amino acid containing his- tone (H2AX) that is able to phosphorylate through infra- red induction in a brief time, repairing DNA double stranded and assisting chromatin remodeling; hence, his- tone (H2AX) continues to be the main molecule for allowing BRCA1 gene to exert its function [9]. For BRCA2 gene, its repairing mechanism is done through RAD51 rather than H2AX. The regular expression of RAD51 certifies the repairing process through catalyzing the core reactions of homologous recombination (HR), (2021) 19:91 Khalifa et al. Journal of Genetic Engineering and Biotechnology (2021) 19:91 (2021) 19:91 Page 2 of 8 Page 2 of 8 Page 2 of 8 Khalifa et al. Journal of Genetic Engineering and Biotechnology Background with strand incursion into duplex DNA and the pairing of homologous DNA strands, thus allowing strand switch; on the other hand, overexpression or silencing of BRCA2 gene mutation leads to the collapse of the repairing process [10], causing gene error which results in the development of various tumors such as breast cancer [11], ovarian cancer [12], prostate cancer [13], and recently glioblastoma multiforme [14], but still, there is no information about the role of BRCA2 muta- tion in invasive urothelial bladder cancer [15]. In developed countries, the frequency rate of bladder cancer is about 9.5/100,000; the most common type of it is urothelial carcinoma, which represents around more than 90% [1]. In Egypt, urinary bladder cancers represent 30% of all cancer cases with an incidence rate of 13.5/ 100,000 individuals according to the National Cancer In- stitute in Egypt [2]. The incidence of bladder cancer is growing with the advance of the economy, and its recur- rence degree has developed a main economic burden on the health care systems [3]. The standardized mortality rate reported for bladder cancer was reported to be in males and females as 2–10/100,000 and 0.5–4/100000 per year, respectively, in 2016 [4]. Environmental factors, smoking, exposure to toxic industrial chemicals and gas- ses, and gene mutations of bladder cells are associated with increased incidence of bladder cancer [5]. The authors in this study aimed to investigate BRCA tumor suppressor gene mutations in bladder cancer- paired tissue and blood samples for patients who devel- oped secondary tumor after or during trimodal therapy. Khalifa et al. Journal of Genetic Engineering and Biotechnology (2021) 19:91 Case #2 A male patient, 53 years old, smoker presented with hematuria and blood clots; radiological investigations re- vealed a 5-cm bladder mass at the base of the bladder; the patient gave a past history of cystolithotomy, chole- cystectomy, and HCV treatment, no history of chronic medical diseases, and there was no family history of the same illness. TURBT revealed a high-grade muscle inva- sive urothelial carcinoma. After 1 month, radical cystec- tomy with ileal conduit diversion was done, and its histopathology revealed T2G3 with negative lymph node involvement. The patient was followed up, and at 8 months of follow-up, he developed a 4-cm left renal lower pole mass, for which partial nephrectomy was done, and its histopathology revealed papillary renal cell carcinoma (RCC). Library preparation and purification y p p p The required regions for the examined genes BRCA1 and BRCA2 were amplified by Oncomine BRCA1 & BRCA2 research kit (Life Technologies). Amplification process was carried out by means of Ion AmpliSeq Li- brary kit (Cat No# 4480441 Life technologies), according to the manufacturer’s protocol. After amplification, the primers were digested using FuPa Reagent; the samples were barcoded with Ion Xpress Barcode Adaptors. Bar- coded libraries were then purified using Agencourt beads, and libraries were measured using the Ion Library TaqMan Quantitation kit (Cat No# 4468802 Life tech- nologies). The quantified libraries were promoted to template preparation. Molecular analysis DNA extraction Genomic DNA was extracted from tissues and their matched blood samples using commercially available kits as follows: DNA extraction was done according to the manufacturer’s instructions of QIAamp DNA Mini blood kit (Cat No # 51104, Qiagen, Germany) based on spin column for DNA extraction method, while tissue DNA was extracted using Cat No # 51304, Qiagen, Germany from fresh tissue samples. Both purity and the concentration for extracted DNA were detected by nano-drop spectrophotometer (Qua- well, Q-500, Scribner, USA); then, extracted DNA was stored at −80 °C till further assessments. Case #3 A male, 47 years old, heavy smoker presented to out- patient clinic with complaints of hematuria with blood clots and no chronic medical diseases (CMD); he had no family history of the same illness. The patient had a past history of internal fixation for left femur fracture. Ac- cording to his imaging investigations, he was suffering from left bladder wall mass. TURBT was done, and its report revealed left lateral wall 4.5 cm mass high-grade transitional cell carcinoma (TCC) with muscle invasion. The patient was treated with trimodal therapy and followed up with CT with contrast. The patient responded to treatment, but his kidneys showed bilateral hypo-dense renal masses. Renal biopsy revealed Hodgkin lymphoma, and the patient was referred for chemotherapy. Clinical evaluation Case #1 A male patient, 65 years old, presented with urinary bladder mass invading the left ureteric orifice with mod- erate backpressure on the left kidney, and the patient gave a history of two transurethral resections of bladder tumors (TURB) 1 year before their pathology was non- muscular invasive bladder cancer (NMIBC). The patient was hypertensive with no family history of bladder can- cer, and serum creatinine was 5 mg/dl, total leukocyte count (TLC) 40 109/L, and platelets count 1.2.109/L. The patient underwent left nephrostomy for his renal insuffi- ciency, bone marrow aspiration for leukocytosis, and Khalifa et al. Journal of Genetic Engineering and Biotechnology (2021) 19:91 Page 3 of 8 Table 1 Demographic and clinicopathological characteristics for bladder cancer cases Characteristics Case 1 Case 2 Case 3 Case 4 Gender Male Male Male Male Age 65 years 53 years 47 years 57 years Family history of cancer Not reported Not reported Not reported Not reported Primary tumor stage T1 T2 T2 T2 Tumor grade G3 G3 G3 G3 Final histopathology T2G3 T2G3 T2G3 T2G3 Associated pathology Leukemia LT renal RCC Renal Hodgkin lymphoma DM Management TURB TURB and radical cystectomy with ileal conduit Trimodal therapy Trimodal therapy Table 1 Demographic and clinicopathological characteristics for bladder cancer cases history of diabetes mellitus on insulin treatment and ir- relevant family history. His radiology revealed multiple bladder masses; diagnostic cystoscopy revealed multiple bladder masses, and TURBT was done. Pathology report revealed high-grade transitional cell carcinoma (TCC) with muscle invasion. The patient was treated with tri- modal therapy. thrombocytopenia Then, the patient was deteriorated as his bone marrow aspiration reported the presence of leukemia, and he died while being treated for leukemia. Case #4 A male patient, 57 years old, presented to the outpatient clinic with complaints of hematuria with blood clots and The obtained libraries from the previous step were pooled on molar equivalent rations to yield at least Khalifa et al. Journal of Genetic Engineering and Biotechnology (2021) 19:91 (2021) 19:91 Khalifa et al. Journal of Genetic Engineering and Biotechnology Page 4 of 8 website, and the Oncomine BRCA-specific plugin was used to analyze the paired normal and tumor sample for each patient using the default plugin parameters. average 150x depth for coverage regarding every germ- line DNA sample and 750x for somatic samples. The as- sembled libraries were clonally amplified using Ion PGM Hi-Q view OT2 kit (Cat No# A29900 Life technologies) on the Ion OneTouch 2 instrument (Life technologies, USA) according to the manufacturer’s instructions. Then, the template ion sphere particles (ISP) were enriched using Ion PGM enrichment beads (Cat No# 4478525 Life technologies) by Ion OneTouch ES system (Life technologies, USA) according to the manufacturer’s instructions; the positive ISP Quality was assayed on Qubit 2.0 Fluorometer (Life technologies, USA) and then continued for accomplishing the sequencing process. Sequencing using ion torrent PGM platform Case #1 In case #1, there was an intronic BRCA1 mutation c.5050-104C>T along with three copies for the 17q21.31 (41197601-41276123) region. Regarding BRCA2, there was an exonic synonymous mutation rs397507876 in the tissue and germline DNA, while there was no detectable difference between the copy numbers in BRCA2. Subsequently, calibrations and adjustments of the pH were done according to the manufacturer’s instructions using the Ion PGM Hi-Q View Sequencing kit (Cat No# A30044 Life technologies). Entirely barcoded enriched samples were sequenced on the Ion Torrent PGM Plat- form (Ion Torrent PGM, Life technologies, USA) using Ion 318 Chip Kit V2 BC (Cat No# 4488150 Life technologies). Case #2 Only BRCA1 gene reported intronic mutation at vc.5050-104C>T, without any copy number variations. For BRCA2, no genetic alteration in the copy number or in the sequences was reported. Results Histopathological examination for the four enrolled samples was shown in Fig. 1a–d. Paired sample analyses for blood and tumor tissue were sequenced for BRCA genes (BRCA1 and 2). Analysis of NGS data output for investigated samples were presented in Fig. 2a–c; ac- cordingly, each case were analyzed and reported as follows. Discussion Bladder cancer is caused by many aspects. A number of genes perform an important role in the incidence and progression of the bladder cancer. The main mechanisms which result in bladder cancer are mu- tations of oncogenes and tumor suppressor genes [16]. Previous studies have exposed that E2F3, MMP, FGFR3, and HER-2 genes played a stimulating role in the occurrence of bladder cancer [17], while PTEN, p53, Rb, p27, and DMBT1 played an inhibi- tory task [18]. The status of DNA damage response (DDR) mutation of cancer is a significant predictive biomarker of im- mune checkpoint blockade (ICB) response [19]. Tumors with DDR mutations are immunologically hot and ap- proachable to ICB. Clinical examinations on urothelial cancer, NCT02553642, NCT02108652, and NCT01928394 (www.clinicaltrials.gov), have established that mutations in DDR genes are predictors of response to PD-1/PD-L1 ICB [20]. Furthermore, it is documented that tumor cells with DDR shortage show constitutive triggering of cellular IFN responses and emission of TIL conscripting chemokines, CCL5 and CXCL10 [21, 22]. DDR mutation status of a cancer could be shared with complementary biomarker methods for patient selection for ICB [23]. The influence of BRCA genes generally lies in re- pair of DNA and preservation of gene stability. Pre- viously, it was reported that the normal BRCA gene expression was of distinguished significance in the prevention of gene mutation such as BRCA1and BRCA2 which plays a major role in the DDR path- way. Mutations in these two genes and intragenic Case #4 Intronic BRCA1 mutation c.5050-104C>T was reported combined with three somatic mutations; two of them were found to be benign rs1064793056 and rs28897679 on the Clinivar database, and one was nonsense patho- genic variant rs80357006 while copy number was nor- mal. Regarding BRCA2, there were no detectable differences between the copy numbers; only one syno- nym mutation rs397507876 with an intronic variation rs2126042 was detected. [ ] Trimodal treatment is one of the choices for blad- der cancer patients; it comprises maximal trans- urethral resection of bladder tumors (TURBT) followed by altered regimens of combined radio and chemotherapy which achieved comparable results to radical cystectomy in many trials [27]. It has been reported that treatment with chemotherapy and radiotherapy in the management of a number of dif- ferent cancers, among bladder cancers, are one of the major treatment modalities. However, the treat- ment of urothelial cancer with either chemotherapy and radiotherapy or a combination of both has been considered as a double-edged solution, since it has been reported earlier that bladder cancer patients treated with chemotherapy followed by radiotherapy appeared to have leukemogenic effect due to the fact that some of the components of these treatment strategies have been responsible for long-term bone marrow toxicit y[28]; moreover, additional treatment with radiotherapy to attain a complete response may participate in a part of toxicity directing to acute myeloid leukemia (AML) or induce cancer after treatment [29]. In the present cases, three patients were tested positive for a pathogenic somatic muta- tion in BRCA genes; two of them developed another type of malignancy which might be consequences for the chemo and/or radiotherapy. Thus, the FDA ap- proved the number of PARP inhibitors drugs in breast, ovarian, and prostatic cancers for patients who test positive for pathogenic BRCA, so the same thing reflects the importance of BRCA testing in bladder cancer patients which might open new safer therapeutic strategy build on the ICB for those pa- tients [30]. Case #3 copy number variation, which was reported as a source of pathogenicity in these genes [24], played a diagnostic and prognostic role in breast and ovarian cancers [25]. The current study reported pathogenic BRCA copy number variations and SNV in bladder tissues which emphasize their relevance in the pathogenesis of bladder cancer. In a recent study based on next-generation sequencing for bladder cancer patients who underwent transurethral resec- tion and were treated with bacillus Calmette–Guérin (BCG) revealed the detection of some potential bio- markers and therapeutic targets and that DDR genes alterations were correlated with high tumor burden [26]. Case #3 In case #3, there was an intronic BRCA1 mutation c.5050-104C>T along with four copies for the 17q21.31 (41197601-41234616) and 17q21.31 (41249157-41276123) regions, while normal copy number for the rest of the gene was reported. Re- garding BRCA2, there was no detectable difference between the copy number or structural variations be- tween the blood and tissue. Data analysis y Generated BAM files were uploaded to the cloud-based Ionreporter server version 5.10 on ThermoFisher Fig. 1 Cross section of urothelial bladder tissues stained with H&E. a High-grade urothelial carcinoma infiltrating deep muscle layer (pT2) (H&E × 400), b papillary urothelial neoplasm of high malignant potential (H&E × 200) (pTa), c high-grade urothelial carcinoma infiltrating deep muscle layer (pT2) (H&E × 200), and d high-grade urothelial carcinoma infiltrating deep muscle layer (pT2) (H&E × 400) Fig. 1 Cross section of urothelial bladder tissues stained with H&E. a High-grade urothelial carcinoma infiltrating deep muscle layer (pT2) (H&E × 400), b papillary urothelial neoplasm of high malignant potential (H&E × 200) (pTa), c high-grade urothelial carcinoma infiltrating deep muscle layer (pT2) (H&E × 200), and d high-grade urothelial carcinoma infiltrating deep muscle layer (pT2) (H&E × 400) Khalifa et al. Journal of Genetic Engineering and Biotechnology (2021) 19:91 Page 5 of 8 Khalifa et al. Journal of Genetic Engineering and Biotechnology (2021) 19:9 (2021) 19:91 Page 5 of 8 Fig. 2 Analysis of NGS data report for the investigated samples. a Variant effect predictor diagram showing the detect variant effects in different BRCA transcripts. b Variant impact diagram showing the CNV heatmap. c Variant impact diagram showing the detected variants effects Fig. 2 Analysis of NGS data report for the investigated samples. a Variant effect predictor diagram showing the detect variant effects in different BRCA transcripts. b Variant impact diagram showing the CNV heatmap. c Variant impact diagram showing the detected variants effects Fig. 2 Analysis of NGS data report for the investigated samples. a Variant effect predictor diagram showing the detect variant effects in different BRCA transcripts. b Variant impact diagram showing the CNV heatmap. c Variant impact diagram showing the detected variants effects Fig. 2 Analysis of NGS data report for the investigated samples. a Variant effect predictor diagram showing the detect variant effects in different BRCA transcripts. b Variant impact diagram showing the CNV heatmap. c Variant impact diagram showing the detected variants effects Khalifa et al. Journal of Genetic Engineering and Biotechnology (2021) 19:91 (2021) 19:91 (2021) 19:91 Page 6 of 8 Page 6 of 8 Khalifa et al. Journal of Genetic Engineering and Biotechnology Availability of data and materials Not applicable Availability of data and materials Not applicable 13. Edwards SM, Evans DG, Hope Q, Norman AR et al (2010) Prostate cancer in BRCA2 germline mutation carriers is associated with poorer prognosis. Br J Cancer 103(6):918–924. https://doi.org/10.1038/sj.bjc.6605822 Acknowledgements The instruments listed in the current study were purchased through a grant from Science Technology Development Fund (STDF) through Capacity Building Grant Project (CBG) [No. 4940]. 9. Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM (2000) A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 10(15):886–895. https://doi.org/1 0.1016/S0960-9822(00)00610-2 Authors’ contributions This work was carried out in collaboration between all authors. Authors MS, NMB, and AR designed the study, performed the statistical analysis, wrote the protocol, and wrote the first draft of the manuscript. Authors MKK, NMB, AR, and AMN performed the practical work. Authors NMB and AR carried out the data acquisition; MKK, NMB, and AR managed the analyses of the study and literature searches. Authors KMA and ED provided samples with clinical reports. All authors have read and approved the final manuscript. 10. Davies AA, Masson JY, McIlwraith MJ, Stasiak AZ, Stasiak A, Venkitaraman AR, West SC (2001) Role of BRCA2 in control of the RAD51 recombination and DNA repair protein. Mol Cell 7(2):273–282. https://doi.org/10.1016/S1097-2 765(01)00175-7 11. Parvin S, Islam MS, Al-Mamun MM, Islam MS, Ahmed MU, Kabir ER, Hasnat A (2017) Association of BRCA1, BRCA2, RAD51, and HER2 gene polymorphisms with the breast cancer risk in the Bangladeshi population. Breast Cancer 24(2):229–237. https://doi.org/10.1007/s12282-016-0692-5 Abbreviations 6. Zhang X, Zhang Y (2015) Bladder cancer and genetic mutations. Cell Biochem Biophys 73:65–69 6. Zhang X, Zhang Y (2015) Bladder cancer and genetic mutations. Cell Biochem Biophys 73:65–69 BRCA: Breast cancer; FGFR3: Fibroblast growth factor receptor3; RB1: Retinoblastoma1; HRAS: Gene belong to RAS; TP53: Total p53; TSC1: Hamartin; H2AX: Histone A; dsDNA: Double stranded; TURBT: Transurethral resection of bladder tumor; NMIBC: Non-muscular invasive bladder cancer; TLC: Total leukocyte count; RCC: Renal cell carcinoma; CMD: Chronic medical diseases; TCC: Transitional cell carcinoma; EDTA: Ethylenediaminetetraacetic acid; DDR: DNA damage response; ICB: Immune checkpoint blockade; IFN: Interferon response; TILs: Tumor- infiltrating lymphocytes; CCL5: Chemokine (C-C motif) ligand 5 7. Nickerson ML, Dancik GM, Im KM, Edwards MG, Turan S, Brown J, Ruiz- Rodriguez C, Owens C, Costello JC, Guo G, Tsang SX, Li Y, Zhou Q, Cai Z, Moore LE, Lucia MS, Dean M, Theodorescu D (2014) Concurrent alterations in TERT, KDM6A, and the BRCA pathway in bladder cancer. Clin Cancer Res 20(18):4935–4948. https://doi.org/10.1158/1078-0432. CCR-14-0330 8. Font A, Taron M, Gago JL, Costa C, Sánchez JJ, Carrato C, Mora M, Celiz P, Perez L, Rodríguez D, Gimenez-Capitan A, Quiroga V, Benlloch S, Ibarz L, Rosell R (2011) BRCA1 mRNA expression and outcome to neoadjuvant cisplatin-based chemotherapy in bladder cancer. Ann Oncol 22(1):139–144. https://doi.org/10.1093/annonc/mdq333 Conclusion According to our knowledge, this is the first analysis on paired tumor and blood analysis from bladder cancer pa- tients on BRCA gene using NGS and shades the light on the mutation status of BRCA 1 and 2 clarifying that the Khalifa et al. Journal of Genetic Engineering and Biotechnology Page 7 of 8 (2021) 19:91 Page 7 of 8 Page 7 of 8 tumor tissues showed somatic events which were not present in the germline DNA which reflects the import- ance of BRCA1and 2 somatic mutation testing and how it might affect the treatment strategy selection and pre- dicting disease prognosis leading to better personalized medicine. 3. Sievert KD, Amend B, Nagele U, Schilling D, Bedke J, Horstmann M, Hennenlotter J, Kruck S, Stenzl A (2009) Economic aspects of bladder cancer: what are the benefits and costs? World J Urol 27(3):295–300. https:// doi.org/10.1007/s00345-009-0395-z 4. Gandomani HS, Tarazoj AA, Siri FH, Rozveh AK et al (2017) Essentials of bladder cancer worldwide: incidence, mortality rate and risk factors. Biomed Res Ther 4:1638 4. Gandomani HS, Tarazoj AA, Siri FH, Rozveh AK et al (2017) Essentials of bladder cancer worldwide: incidence, mortality rate and risk factors. Biomed Res Ther 4:1638 5. Jankovic S, Radosavljevic V (2007) Risk factors for bladder cancer. Tumori 93(1):4–12. https://doi.org/10.1177/030089160709300102 5. Jankovic S, Radosavljevic V (2007) Risk factors for bladder cancer. Tumori 93(1):4–12. https://doi.org/10.1177/030089160709300102 5. Jankovic S, Radosavljevic V (2007) Risk factors for bladder 93(1):4–12. https://doi.org/10.1177/030089160709300102 Competing interests No competing of interest to report. 17. Du X, Lin BC, Wang QR, Li H et al (2014) MMP- 1 and pro-MMP-10 as potential urinary pharmacodynamics biomarkers of FGFR3-targeted therapy in patients with bladder cancer. Clin Cancer Res 20(24):6324–6335. https:// doi.org/10.1158/1078-0432.CCR-13-3336 Ethics approval and consent to participate Ethics approval and consent to participate Ethics approval and consent to participate Ethical approval from Medical Committee at National Research Centre ID # 20028. Individuals who fulfilled the inclusion criteria signed their informed consent. 15. Nageeb AM, Mohamed MM, El Arab LRE, Khalifa MK, Swellam M (2020) Next generation sequencing of BRCA genes in glioblastoma multiform Egyptian patients: a pilot study. Arch Physiol Biochem. https://doi.org/10.1080/138134 55.2020.1729814 Consent for publication 16. El-Naggar M, Ebbing E, Bijnsdorp I, van den Berg J, Peters GJ (2014) Radiosensitization by thymidine phosphorylase inhibitor in thymidine phosphorylase negative and overexpressing bladder cancer cell lines. Nucleosides Nucleotides Nucleic Acids 33(4-6):413–421. https://doi.org/10.1 080/15257770.2014.892127 Declarations 14. Tutt A, Ashworth A (2002) The relationship between the roles of BRCA genes in DNA repair and cancer predisposition. Trends Mol Med 8(12):571– 576. https://doi.org/10.1016/S1471-4914(02)02434-6 Funding f 12. Manchan T, Phoolcharoen N, Tantbirojn P (2019) BRCA mutation in high grade epithelial ovarian cancers. Gynecol Oncol Rep 29:102–105. https://doi. org/10.1016/j.gore.2019.07.007 No funding received. Author details 1 Author details 1CSO at Omicsense, Cairo, Egypt. 2Biochemistry Department, Genetic Engineering and Biotechnology Research Division, National Research Centre, El-Bohouth Street, Dokki, Giza 12622, Egypt. 3High Throughput Molecular and Genetic Laboratory, Center for Excellences for Advanced Sciences, National Research Centre, El-Bohouth Street, Dokki, Giza 12622, Egypt. 4Urology Department, Zagazig University, Zagazig, Egypt. 18. Dodurga Y, Avci CB, Yilmaz S, Dogan ZO, Kesen Z, Tataroglu C, Satiroglu-Tufan N-L, Bushra T, Gunduz C (2011) Evaluation of deleted in malignant brain tumors 1 (DMBT1) gene expression in bladder carcinoma cases: preliminary study. Biomarkers 1–6. https://doi.org/10.3109/1354750X.2011.620627 19. 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https://openalex.org/W2283819300	https://discovery.ucl.ac.uk/id/eprint/1476269/1/acs%252Enanolett%252E5b03547.pdf	English	null	Mechanism of Exfoliation and Prediction of Materials Properties of Clay–Polymer Nanocomposites from Multiscale Modeling	Nano letters	2,015	cc-by	5,796	Mechanism of Exfoliation and Prediction of Materials Properties of Clay−Polymer Nanocomposites from Multiscale Modeling James L. Suter, Derek Groen,† and Peter V. Coveney* Centre for Computational Science, University College London, 20 Gordon Street, London, WC1H 0AJ, U utational Science, University College London, 20 Gordon Street, London, WC1H 0AJ, United Kingdom * S Supporting Information ABSTRACT: We describe the mechanism that leads to full exfoliation and dispersion of organophilic clays when mixed with molten hydrophilic polymers. This process is of fundamental importance for the production of clay−polymer nanocomposites with enhanced materials properties. The chemically speciﬁc nature of our multiscale approach allows us to probe how chemistry, in combination with processing conditions, produces such materials properties at the mesoscale and beyond. In general agreement with experimental observations, we ﬁnd that a higher grafting density of charged quaternary ammonium surfactant ions promotes exfoliation, by a mechanism whereby the clay sheets slide transversally over one another. We can determine the elastic properties of these nanocomposites; exfoliated and partially exfoliated morphologies lead to substantial enhancement of the Young’s modulus, as found experimentally. KEYWORDS: Multiscale modeling, clay−polymer nanocomposites, exfoliation dynamics, materials properties T T he search for new materials with speciﬁed performance properties continues apace. The incentives come not only from curiosity driven research but also from the pressure to meet important economic and societal needs. However, the history of innovation in materials design, and the path from initial discovery to implementation in engineering and manufacturing contexts, is a fraught one. Innovations frequently take decades to make their way into commercially viable applications. One of the central reasons for such problems stems from the diﬃculty of moving between the promise held out at the small (“microscopic”) scale by the entity discovered and the behavior of such substances on larger scales appropriate to their use in various applications. This has led to a vast amount of larger scale engineering and manufacturing research and development, almost all of which is conducted experimentally in a largely trial and error fashion. Here we describe a complementary approach, one based on a virtual materials modeling laboratory. Rather than simply synthesizing a great many samples of a material and testing these, we aim to predict materials properties from “ﬁrst-principles”, that is, based on the fundamental description of the starting ingredients in terms of their atomic and molecular composition and structure and the processing conditions. pubs.acs.org/NanoLett © 2015 American Chemical Society 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. 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. DOI: 10.1021/acs.nanolett.5b03547 Nano Lett. 2015, 15, 8108−8113 Nano Letters Thus, the results we report should also be applicable to much larger clays, such as those seen in the larger size distribution by Cadene et al.26 Initially, the tactoids are in their aggregated state with no polymer in the interlayer gallery. We vary the density from a very low charge montmorillonite clay (5% of Al3+ substituted with Mg2+, referred to as I in the remainder of this paper), via a typical Wyoming montmorillonite (11%, referred to as II) to a high charge density clay (17% substitution rate, referred to as III). These correspond to an approximate cation exchange capacity (CEC) of 0.31 (0.28 including clay edges) meq/g, 0.69 (0.62 including clay edges) meq/g, and 1.05 (0.98 including clay edges) meq/g, respectively. Figure 1. Top left: Na-montmorillonite is a layered mineral consisting of octahedrally coordinated aluminum oxide between two silica tetrahedral layers (the atoms are colored as followed: Al = pink, Ai = yellow, O = red, H = white, Na = blue, C = Cyan). Top right: exchanging the alkali ions in the interlayer for quaternary ammonium ions expands the clay interlayer spacing and gives the clay a hydrophobic character. Bottom: The clay layers can either remain aggregated (immiscible), or can penetrate the clay interlayer (intercalated), or the clay layers can fully disperse (exfoliated). Figure 1. Top left: Na-montmorillonite is a layered mineral consisting of octahedrally coordinated aluminum oxide between two silica tetrahedral layers (the atoms are colored as followed: Al = pink, Ai = yellow, O = red, H = white, Na = blue, C = Cyan). Top right: exchanging the alkali ions in the interlayer for quaternary ammonium ions expands the clay interlayer spacing and gives the clay a hydrophobic character. Bottom: The clay layers can either remain aggregated (immiscible), or can penetrate the clay interlayer (intercalated), or the clay layers can fully disperse (exfoliated). y g q g p y Our models were created by constructing a clay tactoid comprising four hexagonal layers, each composed of (110), (010), and (001) surfaces, with lateral dimensions measuring approximately 98 Å by 104 Å. Surfactant molecules were then added to this four clay-layer tactoid at the atomistic level, and subsequently relaxed using atomistic molecular dynamics at 500 K, leading to the initial d-spacings listed in Table 1. Nano Letters Letter comprise approximately 2−6% of the volume of the simulation cell, depending on whether we use the thickness of the clay sheet (1 nm) or the initial separation between clay layers (20− 28 Å) as the thickness of the platelets. The models are listed in Table 1. Each clay layer possesses an aspect ratio of approximately 10. an inﬁnite clay mineral system; such simulations give information on the interfacial behavior at the basal surface but do not describe the large-scale behavior (i.e., dispersion, aggregation) of clay layers.10,11 Simulations without molecular detail, such as self-consistent phase ﬁeld theory, provide predictions about the thermodynamic tendency for clay layers to exfoliate based on a mean ﬁeld approximation12−17 but, again, they cannot describe the chemically speciﬁc nature of the clays and polymers.18 For a comprehensive review of multiscale modeling of polymer nanocomposites, see Zeng et al.19 pp y Montmorillonite clays are often quoted as having an aspect ratio of between 10 and 1000;25 our models are therefore at the lower end of this distribution. Atomic force microscopy and photocorrelation spectroscopy studies indicated that the sizes of Na-montmorillonite clay exhibited a bimodal distribution with wide size distributions.26 The average dimensions of the ﬁrst population were typically 320−400 nm long/250 nm wide and 200−250 nm long/120 nm wide for natural and synthetic clays, respectively. The population with smaller sizes were, on average, 65 and 50 nm long and 35 and 25 nm wide for natural and synthetic clays, respectively. This is of the same order of magnitude as the lateral dimensions of the clay platelets in our simulations (10 nm), giving us conﬁdence that our models are representative of clay layers found experimentally. In a signiﬁcant advance on our previous multiscale modeling of pristine clays with hydrophilic polymers, which led to intercalated clay−polymer systems,4 in this study we examine the process of clay layer exfoliation using similar methods. To do so, we have chosen a clay, surfactant, and polymer combination that is known to produce at least partially exfoliated clay layers: montmorillonite clay, a quaternary ammonium dimethyldioctadecylammonium ionic surfactant, which has two alkyl chains (each of length C18), and hydrophilic poly(ethylene)-glycol (PEG) as the polymer matrix (Figure 1).20−23 This combination has been shown exper- With an aspect ratio of 10 for our models, the basal clay interactions will dominate, as will be the case for clays with higher aspect ratios. Mechanism of Exfoliation and Prediction of Materials Properties of Clay−Polymer Nanocomposites from Multiscale Modeling literature on the subject, the ﬁeld has failed to live up to expectations.1 There remains a very poor level of understanding of the mechanism of action of these materials, and no control over the outcome of mixing arbitrary polymers with clays.2,3 Models are therefore required to understand how large-scale dispersion of clay layers in polymers can be achieved and to determine structure−property relationships, thereby enabling the design of materials with desired properties. In this paper, we provide a clear and detailed, chemically speciﬁc, explanation for the mechanism of exfoliation of clays by suitable polymers and why, following the dispersion of the individual clay platelets, the resultant materials exhibit very favorable materials properties. Our multiscale scheme uses short time scale atomistic simulations to create interaction parameters for coarse-grained (CG) particles that represent several atoms.4−6 For clay polymer composites, we require a substantial number of atomistic simulations to deﬁne all interaction potentials. These simulations include matching to structural parameters using Iterative Boltzmann Inversion5,7 and to free-energy proﬁles for highly interacting CG particles8 to build up a complete set of chemically speciﬁc CG potentials in the vicinity of clay interfaces. For more details, see Supporting Information. This degree of coarse-graining produces speed-ups of order of 100 times, while retaining molecular speciﬁcity,9 allowing us to reach length and time scales approaching a hundred nanome- ters and microseconds, respectively. Almost all atomistic studies of clay−polymer nanocomposites have focused only on the interlayer region between the clay layers, eﬀectively simulating This is possible through a multiscale scheme that links these very short length (and time) scale descriptions to the larger ones that inform materials properties. We show how our scheme, thus far developed for the study of nanocomposite materials, can be successfully applied to understand and predict the behavior of exfoliating clay−polymer nanocomposites. Within four years of their initial discovery, Toyota was making car components from composites comprised of clay and nylon. However, since that early work, and notwithstanding a vast Received: September 3, 2015 Revised: November 11, 2015 Published: November 17, 2015 8108 DOI: 10.1021/acs.nanolett.5b03547 Nano Lett. 2015, 15, 8108−8113 DOI: 10.1021/acs.nanolett.5b03547 Nano Lett. 2015, 15, 8108−8113 Nano Letters (b) For model III, the clay layers have already separated and ultimately become fully exfoliated (see Figure 4c). the surfactant cation extend perpendicular to the clay layers accompanied by clay layer expansion. Once the monolayer of PEG molecules on the clay surface is ﬁlled, no further expansion occurs. We can infer that at this point the enthalpic interactions between the polymer and the clay are no longer enough to overcome the entropy loss through polymer conﬁnement.12 There is no further driving force for the clay layers to expand. The low density of PEG polymer molecules in the center of the interlayer conﬁrms that the interaction of the PEG polymer with the nonpolar alkyl chains of the surfactant is unfavorable, again lowering the tendency for further PEG polymer intercalation. step of 2 fs. To facilitate our simulations, we used our python- based toolkit, FabMD.30 Each simulation was run for >2 μs. Further computational details, including the procedure for generating the materials properties, are listed in the Supporting Information. In all the models listed in Table 1, we observe an initial expansion as the polymer intercalates between the clay layers over a short time period (approximately 2 ns). The intercalated d-spacings are shown in Table 1. Snapshots of this process are shown in Figure 2 for model II. The PEG polymer molecules intercalate into the gallery predominately adjacent to the clay surface, illustrating the favorable interactions between the hydrophilic PEG polymer molecules and the surface as compared to that of the alkyl chains. The rapid dynamics of polymer intercalation has been previously observed exper- imentally by Vaia and Giannelis and is attributed to the unhindered nature of the conﬁned polymers (there are fewer entanglements, unlike in the bulk).31 For models II and III, once the fast intercalation of PEG molecules has occurred we observe on longer time scales (>500 ns) the diﬀusion of the clay layers away from the clay platelets to form exfoliated morphologies. We show this mechanism in Figure 2 for model II and Figure 3 for model III. After more than 3 μs of simulation time, the structures of our eight tactoid models are shown for models I, II, and III in Figure 4. We see that for model II there is a mixture of partially and totally exfoliated clay layers while, and for model III all the clay tactoids have exfoliated into single dispersed layers. Nano Letters Figure 2. Snapshots from CG molecular dynamics simulations of a selected alkyl ammonium treated clay tactoid immersed in a melt of PEG polymer. (CG clay surface = pink, charge sites = green, edge sites = yellow, surfactant ammonium group = blue, alkyl chains = red.) The PEG polymers have been removed to aid visualization. (a) We show the organoclay model before interaction with the PEG polymer for model II. In (b), the PEG polymer has intercalated into the clay galleries and the separation between the clay layers has increased. In (c), we observe the uppermost two clay layers diﬀusing away from each other, eventually forming an exfoliated morphology, achieved by the upper clay layers translating in the plane of the clay tactoid layers. Figure 2. Snapshots from CG molecular dynamics simulations of a selected alkyl ammonium treated clay tactoid immersed in a melt of PEG polymer. (CG clay surface = pink, charge sites = green, edge sites = yellow, surfactant ammonium group = blue, alkyl chains = red.) The PEG polymers have been removed to aid visualization. (a) We show the organoclay model before interaction with the PEG polymer for model II. In (b), the PEG polymer has intercalated into the clay galleries and the separation between the clay layers has increased. In (c), we observe the uppermost two clay layers diﬀusing away from each other, eventually forming an exfoliated morphology, achieved by the upper clay layers translating in the plane of the clay tactoid layers. Figure 3. Snapshots from subdomains of simulations for (a) model I and (b) III after approximately 0.2 μs. We see that for the low charge system (a), the clay tactoid stack forms a tilted conﬁguration with a large number of alkyl ammonium chains interacting with the bulk PEG polymer (not shown). This conﬁguration appears to be stable on the time scale of our simulations. (b) For model III, the clay layers have already separated and ultimately become fully exfoliated (see Figure 4c). Figure 3. Snapshots from subdomains of simulations for (a) model I and (b) III after approximately 0.2 μs. We see that for the low charge system (a), the clay tactoid stack forms a tilted conﬁguration with a large number of alkyl ammonium chains interacting with the bulk PEG polymer (not shown). This conﬁguration appears to be stable on the time scale of our simulations. DOI: 10.1021/acs.nanolett.5b03547 Nano Lett. 2015, 15, 8108−8113 Nano Letters The partial charges on the edges of the clay sheet were determined through quantum mechanical simulations.27 This tactoid was sub- sequently coarse-grained, and 100 monomer PEG polymers, corresponding to a molecular weight of 4372 g mol−1, were built according to the protocol described by Suter et al.4 The simulations are replicated in a 2 × 2 × 2 array to form an 8 tactoid (32 clay layers) initial model (see Table 1). All simulations were performed at 500 K and at relatively high pressure (300 atm). The lattice parameters were allowed to vary under constant pressure and temperature conditions (NPT) for the ﬁrst 100 ps, after which each simulation was run with constant volume (NVT). The simulations were performed using the LAMMPS molecular dynamics code,28,29 using a time imentally to produce exfoliated clay layers, as demonstrated by the lack of peaks in X-ray diﬀractograms following melt intercalation.24 Although PEG-clay nanocomposites are often prepared using solution methods, melt processing is a more industrially convenient route of preparation; our simulations therefore take place at elevated temperatures and pressures (500 K and 300 atm) in the absence of water. We have simulated three models of diﬀering clay surface charge density (through isomorphic substitution) and hence surface density of quaternary ammonium surfactants. Each model is comprised of 8 tactoids with each tactoid composed of 4 layers (32 layers in total). Each clay layer is of hexagonal shape with a diameter of approximately 100 Å; in total, they Table 1. Organo-Modiﬁed Clay: PEG Models Studied in This Paper Table 1. Organo-Modiﬁed Clay: PEG Models Studied in This Paper system substitution rate simulation cell dimensions (Å)3 num. of CG atoms/all atoms equivalent initial/intercalated d-spacing Å I 5% 407 × 440 × 448 671200/4870912 20 Å/15−20 Å II 11% 415 × 415 × 449 667904/4829856 25 Å/32 Å III 17% 504 × 504 × 602 1363264/9568096 28 Å/32 Å DOI: 10.1021/acs.nanolett.5b03547 Nano Lett. 2015, 15, 8108−8113 8109 8109 8109 Letter Letter Nano Letters Nano Letters The separation between the clay layers increases by approximately 7 Å. This is consistent with an extra layer of PEG molecules resident on each clay surface. In Supporting Information Figure SI.20, we plot the density proﬁle perpendicular to the clay surface for species within the clay interlayer, which conﬁrms that the majority of the PEG molecules reside on the clay surfaces while the alkyl chains of We ﬁnd that the mechanism for the exfoliation of the clay layers does not involve the clay layers expanding perpendicular to the clay surface but rather involves the layers translating 8110 DOI: 10.1021/acs.nanolett.5b03547 Nano Lett. 2015, 15, 8108−8113 Letter Letter Nano Letters Figure 4. Snapshots from the CG molecular dynamics simulation, containing 8 tactoids (32 layers) of models (a) I, (b) II, and (c) III after approximately 3 μs of simulation. The PEG polymer and surfactant molecules have been removed to aid visualization. (d) The radial distribution function for the center-of-mass of the clay layers for model II (black) and model III (red) averaged over the last 10 ns of simulation, illustrating that model III is fully dispersed. Figure 3. For model II, or diﬀerent interlayers re 2 the inner-interlayer in other tactoids one of on and diﬀuses away. yers in model II may be same polymer molecule ent clay interlayers or same interlayer.4 Where cule are adsorbed on t interlayers, this serves e clay layers (see Figure istribution of exfoliated s. With much larger the greater number of ayers would resulting in ess, leading to a greater However, for model III, demonstrates, there is center of the interlayer, lar dynamics simulation, containing 8 tactoids (32 layers) of models (a) I, (b) II, and (c) III after polymer and surfactant molecules have been removed to aid visualization. (d) The radial distribution yers for model II (black) and model III (red) averaged over the last 10 ns of simulation, illustrating that Figure 5. An illustration of a PEG polymer (cyan) adsorbed within two diﬀerent clay layers in adjacent clay interlayers in model II. Such conﬁgurations are likely to inhibit the diﬀusive process leading to clay layer exfoliation. Figure 4. Snapshots from the CG molecular dynamics simulation, containing 8 tactoids (32 layers) of models (a) I, (b) II, and (c) III after approximately 3 μs of simulation. The PEG polymer and surfactant molecules have been removed to aid visualization. DOI: 10.1021/acs.nanolett.5b03547 Nano Lett. 2015, 15, 8108−8113 Nano Letters found that using the solvent casting method the Cloisite 15A nanocomposite showed better dispersion via transmission electron microscopy (TEM) and steady shear viscosity measurements, compared with the Cloisite 20A clay system.33 g p p We observe that exfoliated morphologies signiﬁcantly enhance the elastic properties of the composite and that even partially exfoliated clay morphologies provide a large enhance- ment. Ratna et al. reported a similar increase in Young’s modulus for PEG−clay nanocomposites formed through solution casting, which increased from approximately 0.1 GPa for pure PEG to 0.14 GPa for composites containing 2.5% clay and to 0.25 GPa for those containing 5% clay.34 The absolute values, although reasonably close, can not be compared due to the qualitative nature of the CG potentials at 100 K and the solution casting method used experimentally. The enhance- ment of the Young’s modulus of a factor of approximately 2 compared to the bulk polymer is also comparable to a 1.74 factor for the longitudinal stiﬀness, E11, for composites ﬁlled with unidirectional disklike particles using the Halpin−Tsai micromechanical model38 (see Supporting Information for details). In Figure 6 we show the stress−strain behavior for models II and III, calculated at 100 K, using a strain rate of 1 × 10−8 s. The ﬁnal snapshots of our simulations were rapidly cooled from 500 to 100 K over 4 ns to produce a quenched amorphous system in its glassy state from which stress−strain curves were computed. We have estimated the glass transition temperature To summarize, we have shown how organically treated clays promote exfoliation and report the ﬁrst ever multiscale modeling and simulation study that captures the full process, from the melt intercalation of suitable polymers into such organophilic clays, to the dispersion of individual clay platelets within the polymer matrix. At such longer time scales, we can compute the materials properties of the resultant nano- composite and ﬁnd that it exhibits substantially enhanced mechanical properties compared to the properties of its component parts. The predictions of our models are in generally good agreement with experimental observations. Nano Letters Our previous models of non- intercalated clay tactoids with a pristine montmorillonite clay immersed in PEG polymer produced a Young’s modulus of 0.137 GPa.4 Note that the calculations reported for the studies at 100 K are not expected to be quantitatively correct, as they employ potential parametrizations obtained at higher temper- atures. However, the qualitative trends are expected to remain intact, as previously found, for example, when examining the Young’s modulus of polystyrene−silica composites.37 interlayer. This is likely to be due to the combination of a greater number of ammonium groups on the clay surface (restricting the surface area available to the PEG molecules) and the greater initial d-spacing. As a result, there is no attraction between the clay layers and they exfoliate rapidly. y y y p y Experimentally, we can compare our structural observations with commercially available Cloisite 20A, an organically modiﬁed montmorillonite clay with dimethyl dihydrogenated tallow quaternary ammonium surfactants (alkyl chain lengths between C14 and C18) and a CEC of 0.95. In the work of Loyens et al.,24 when Cloisite 20A was blended with PEG using melt extrusion, a variety of nanostructures were found, depending on polymer molar mass and clay volume fraction: lower molecular mass PEG (100 000 g mol−1) at 5% clay vol. and small clay volume fractions produced a peak in the small- angle X-ray scattering measurements (SAXS) diﬀraction spectra at 37 Å as well as at higher spacings. The 32 Å d-spacing we have calculated via multiscale modeling for the intercalated structures is therefore in good agreement with experimental observations. In general, intercalation of PEG with molecular weight in the range 105−106 g mol−1 in organically modiﬁed clay composites results in reported d-spacings in the range of 34 ± 4 Å.32−36 Higher molecular weight PEGs were shown to produce exfoliated morphologies (no peaks in the SAXS diﬀraction spectra);24 we can assume, therefore, that in the lower molecular weight PEG models a combination of exfoliated and intercalated morphologies are present. The Cloisite 15A system is a similarly organically modiﬁed montmorillonite clay but has a higher CEC of 1.25; this provides a comparison with model III. Although no melt processing has been reported in the literature with PEG polymer, Hyun et al. Nano Letters (d) The radial distribution function for the center-of-mass of the clay layers for model II (black) and model III (red) averaged over the last 10 ns of simulation, illustrating that model III is fully dispersed. Figure 5. An illustration of a PEG polymer (cyan) adsorbed within two diﬀerent clay layers in adjacent clay interlayers in model II. Such conﬁgurations are likely to inhibit the diﬀusive process leading to clay layer exfoliation. parallel to one another, as illustrated in Figure 3. For model II, we observe this occurring randomly for diﬀerent interlayers within the tactoid; for example, in Figure 2 the inner-interlayer exhibits this translational motion, while in other tactoids one of the outer layers undergoes lateral motion and diﬀuses away. y g y The incomplete exfoliation of clay layers in model II may be in part due to diﬀerent sections of the same polymer molecule initially intercalating into either adjacent clay interlayers or bridging between clay layers within the same interlayer.4 Where various parts of a single PEG molecule are adsorbed on diﬀerent clay surfaces, including diﬀerent interlayers, this serves to resist any translational diﬀusion of the clay layers (see Figure 5). As a result, for model II we see a distribution of exfoliated and partially exfoliated clay platelets. With much larger platelets, it could be envisaged that the greater number of bridging conﬁgurations within the interlayers would resulting in full exfoliation becoming a slower process, leading to a greater number of partially exfoliated platelets. However, for model III, as Supporting Information Figure SI.15 demonstrates, there is much less PEG molecule density in the center of the interlayer, indicating that there are fewer PEG molecules bridging the Figure 5. An illustration of a PEG polymer (cyan) adsorbed within two diﬀerent clay layers in adjacent clay interlayers in model II. Such conﬁgurations are likely to inhibit the diﬀusive process leading to clay layer exfoliation. 8111 DOI: 10.1021/acs.nanolett.5b03547 Nano Lett. 2015, 15, 8108−8113 Letter DOI: 10.1021/acs.nanolett.5b03547 Nano Lett. 2015, 15, 8108−8113 Nano Letters (Tg) from the change in the slope of the volume versus temperature curve (see Supporting Information) to be approximately 220 K. We wish to examine the elastic properties in the glassy state of the composite; hence, we have chosen a temperature well below Tg such that we can determine the stiﬀness, as at this temperature the system will remain in a glassy state when strained. There is no evidence that crystallization of the PEG molecules is occurring (see Supporting Information for more details); we can be sure that we are observing the response of the quenched amorphous system. We ﬁnd that the stress−strain curve for partially exfoliated model II and totally exfoliated model III are almost identical, even though model III has a smaller clay volume fraction. We ﬁnd Young’s moduli of 0.284 GPa ± 0.001 and 0.266 GPa ± 0.001, respectively; this is over twice that of the neat polymer (0.126 GPa). Our previous models of non- intercalated clay tactoids with a pristine montmorillonite clay immersed in PEG polymer produced a Young’s modulus of 0.137 GPa.4 Note that the calculations reported for the studies at 100 K are not expected to be quantitatively correct, as they employ potential parametrizations obtained at higher temper- atures. However, the qualitative trends are expected to remain intact, as previously found, for example, when examining the Young’s modulus of polystyrene−silica composites.37 (Tg) from the change in the slope of the volume versus temperature curve (see Supporting Information) to be approximately 220 K. We wish to examine the elastic properties in the glassy state of the composite; hence, we have chosen a temperature well below Tg such that we can determine the stiﬀness, as at this temperature the system will remain in a glassy state when strained. There is no evidence that crystallization of the PEG molecules is occurring (see Supporting Information for more details); we can be sure that we are observing the response of the quenched amorphous system. We ﬁnd that the stress−strain curve for partially exfoliated model II and totally exfoliated model III are almost identical, even though model III has a smaller clay volume fraction. We ﬁnd Young’s moduli of 0.284 GPa ± 0.001 and 0.266 GPa ± 0.001, respectively; this is over twice that of the neat polymer (0.126 GPa). Nano Letters (16) Ginzburg, V. V.; Balazs, A. C. Macromolecules 1999, 32, 5681− 5688. based on the chemical speciﬁcity of the ingredients and their processing conditions. (17) Ginzburg, V. V.; Singh, C.; Balazs, A. C. 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(36) Lim, S.; Kim, J.; Chin, I.; Kwon, Y.; Choi, H. Chem. Mater. 2002, 14, 1989−1994. (37) Rahimi, M.; Iriarte-Carretero, I.; Ghanbari, A.; Böhm, M. C.; Müller-Plathe, F. Nanotechnology 2012, 23, 305702. Nano Letters In the future, we will investigate the role of processing on materials properties, such as shearing and extensional ﬂow, which are of particular importance in the context of hydrophobic polymers, for example, polystyrene.39 Moreover, our multiscale methods are at a very early stage of development and many improvements to their quantitative capabilities can be expected in the future, as well as their transferability between diﬀerent thermodynamic states. We anticipate that with forthcoming progress in both experimental observations and computational methods the latter will evolve into a powerful tool that will be able to guide materials property prediction Figure 6. Stress−strain curves for uniaxial compression and extension of our organoclay models II (black) and III (blue). The neat PEG polymer stress−strain curve is also shown (red). We calculate elastic properties (the Young’s modulus) from the gradient of the stress− strain curve. The starting conﬁgurations for these simulations are the ﬁnal simulation snapshots shown in Figure 4, cooled down to 100 K. Figure 6. Stress−strain curves for uniaxial compression and extension of our organoclay models II (black) and III (blue). The neat PEG polymer stress−strain curve is also shown (red). We calculate elastic properties (the Young’s modulus) from the gradient of the stress− strain curve. The starting conﬁgurations for these simulations are the ﬁnal simulation snapshots shown in Figure 4, cooled down to 100 K. 8112 DOI: 10.1021/acs.nanolett.5b03547 Nano Lett. 2015, 15, 8108−8113 Letter Present Address † (28) Plimpton, S. 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W4226301025.txt	https://link.springer.com/content/pdf/10.1007/s10531-022-02393-x.pdf	en		The need for local-adjusted Participatory Forest Management in biodiversity hotspots	Biodiversity and conservation	2,022	cc-by	6,504	Biodiversity and Conservation (2022) 31:1313–1328 https://doi.org/10.1007/s10531-022-02393-x ORIGINAL PAPER The need for local-adjusted Participatory Forest Management in biodiversity hotspots Joslyn Muthio Nzau1 · Werner Ulrich2 · Marco Rieckmann3 · Jan Christian Habel4 Received: 28 September 2021 / Revised: 14 February 2022 / Accepted: 21 February 2022 / Published online: 8 April 2022 © The Author(s) 2022 Abstract Numerous conservation activities in Africa have been of little effect. In this study, we investigate socio-economic trade-offs that might have been overlooked, yet may undermine conservation action in discret pathways. Data was collected in three study sites with fragile forest ecosystems in south-eastern Kenya, through locally adapted structured surveys and semi-structured expert guides. These analyses are drawn from 827 structured surveys and 37 expert interviews, which were done during 2016–2018. We found general coherences between age, gender, ethnicity, indigenous knowledge, formal education, and higher incomes, which shapes forest conservation attitudes. Indigenous knowledge is marginal, and most people with formal education in the rural setting are likely to be young without legal land rights or among the minority with off-farm employment. The reluctance to address historical land injustices and inequitable sharing of entitlements and management authority overrides positive attitudes and intentions towards forest conservation in all three study sites. However, we found considerable discrepancies among the three study sites. For Arabuko Sokoke forest, the awareness of forest conservation was relatively low when compared with the other two study sites. Forests play a major role against the backdrop of resource use in all three regions. But, different ecosystem services are used among the three study sites. For environmental education and communication, internet plays a comparatively minor role. Strategies to preserve forest differ among the three study sites: Reforestation is proposed in cloud forests of Taita Hills and riparian forests, whereas offfarm employment and alternative income sources plays a major role in Arabuko Sokoke forest. Our findings underline that locally specific conservation management is needed to conduct efficient nature conservation, particularly in countries with very heterogeneous ethnicities and environments. Keywords Biodiversity · Awareness · Perception · Attitude · Environmental communication · Conservation efficiency Communicated by Dirk Sven Schmeller. Extended author information available on the last page of the article 13 1314 Biodiversity and Conservation (2022) 31:1313–1328 Introduction The Anthropocene is characterized by rapid destruction and degradation of intact ecosystems (Jaeger, 2000; Hansen et al. 2020), and subsequent loss of biodiversity and ecosystem functions (Myers et al. 2000; Balmford et al. 2001). This may also undermine human livelihood quality (Büscher & Whande, 2007; Agrawal et al. 2008). Achieving a win-win situation between ecological conservation and human wellbeing remains an urgent global concern (Millennium Ecosystem Assessment, 2005). In the African context, the conservation of forests and woodlands is sporadic (Balmford et al. 2001; Miles et al. 2006) and deserves more attention (Riggio et al. 2019; Nzau et al. 2020; 2021a). An efficient nature conservation is a challenge, especially in rapidly shifting socio-economic landscapes (Igoe & Brockington, 2007; Githiru, 2007; Kavousi et al. 2020). Participatory Forest Management (PFM) is often considered as a fundamental approach towards reconciling biodiversity conservation and human livelihood needs (Kellert et al. 2000; Schreckenberg & Luttrell, 2009; Vyamana, 2009; Nzau et al. 2020). PFM encompasses diverse initiatives towards the co-management of natural resources by varied actors, including state agencies, civil society, and local people (Wily, 2002; Schreckenberg & Luttrell, 2009). Management of natural resources is understood as the right to regulate internal use patterns and transform the resource by making improvements (Ostrom & Schlager, 1996) wherein two or more social actors negotiate, define, and guarantee amongst themselves a fair sharing of the management functions, entitlements and responsibilities for a given area or set of natural resources (Borrini-Feyerabend et al. 2007). Although many different co-management principles exist within PFM schemes, the extent to which these principles are applicable in real-world scenarios depends on local conditions (Kellert et al. 2000; Brockington, 2004; Borrini-Feyerabend et al. 2007; Schreckenberg & Luttrell, 2009). Numerous case studies from Africa have revealed the shortcomings of PFM (Githiru, 2007; Ming’ate & Bollig, 2016). In this study, we analyze coherences and trade-offs that underpin the acceptance and legitimacy of conservation initiatives within PFM schemes in three different study sites with fragile forest ecosystems across south-eastern Kenya. We consider gallery forests in semiarid drylands in Kitui County, the dryland coastal Arabuko Sokoke forest, and the cloud forests of Taita Hills. While these three regions represent divergent agro-ecological, topographical, and geophysical conditions, they all are characterized by severe human demographic and rapid land-use pressure and a loss of natural forest with subsequent losses of natural habitats, species, and ecosystem functions (Habel et al. 2015). We conducted structured surveys among local smallholder farmers living in close vicinity of the respective forests (< 0.5 km distant). Additionally, we performed semi-structured interviews with representatives of different governmental and non-governmental agencies, working in the field of land management, forestry, agriculture, and/or nature and resource conservation. Data were collected during the years 2016–2019. Based on the results obtained we will answer the following questions: 1. Which socio-economic predictors impact the awareness, perceptions, and attitudes towards biodiversity for the respondents of the study sites? 2. Do environmental communication sources influence biases and conservation behaviors for the three study sites? 13 Biodiversity and Conservation (2022) 31:1313–1328 1315 Fig. 1 Study region in Kenya, Eastern Africa. The three study sites, gallery forests along Nzeeu River in Kitui County, the coastal dryland Arabuko Sokoke forest at the coast of the Indian Ocean, and the cloud forests of Taita Hills are display as squares 13 1316 Biodiversity and Conservation (2022) 31:1313–1328 3. Which socio-cultural conditions are the prerequisite for successful Participatory Forest Management? Materials and methods Study sites This study was carried out in three areas in south-eastern Kenya (Fig. 1). These areas include the gallery forests along Nzeeu River in Kitui County, the coastal dryland Arabuko-Sokoke forest (ASF) along the coastline of the Indian Ocean in Kilifi County, and the cloud forests of Taita Hills, in Taita-Taveta County. All these three areas harbor fragile ecosystems with high levels of biodiversity, including many endemic plant and animal species (see Burgess et al. 1998). Despite the partially strict protection (especially for ASF and some cloud forest fragments of Taita Hills), these forest ecosystems suffer extremely under illegal deforestation and the exploitation of natural resources (Burgess et al. 1998). All three areas differ in respect of abiotic conditions (semi-arid, high mountain, coastal region), forest types (gallery forest, dry coastal forest, cloud forest), and ethnicity of the local human population (Kamba in Kitui County, Giriama and Waatha around ASF, Taitas in Taita Hills). The dry coastal ASF is comparatively large forest fragment and officially protected, while the Taita Hills cloud forests and particularly the gallery forests along rivers in the semi-arid Kitui County are highly fragmented and anthropogenic modified, and invaded by exotic plant species (Teucher et al. 2018). A detailed description of the socio-economic status of each of the three study sites is given in Appendix S1. Data collection Data collection was done through a standardized questionnaire. The questionnaire was designed in English and subsequently translated into Swahili. In total, we obtained 827 filled questionairs (191 along the Nzeeu River in Kitui County, 336 around ASF, 300 around Taita Hill cloud forest fragments). We employed convenience sampling for the selection of survey participants. The sampling criteria included geographical proximity (< 0.5 km distant to the forest) to respective forests. The interviews were deliberately conducted exclusively with people living around the forest, as it can be assumed that these people benefit from the forest on the one hand (fruits, animals, wood, see REFS), but also suffer from humanwildlife conflicts (REFS). Further criterial were participant availability at the time of the survey, and their willingness to participate (Dörnyei, 2007). Household members nominated only one member per household, mainly the head of the household, to participate. A household encompassed all people who cooked together every evening. During expert interviews representatives of governmental and non-governmental organizations (NGOs) working in the field of land management, forestry, agriculture and conservation were asked an openended question regarding their ideas to protect the forest, namely reforestation, education and awareness, sustainable extraction of resources, streamlining and enforcement of environmental law, the providence of good quality water sources, co-management of natural resources, modern sustainable agriculture, alternative energy sources, nature-based livelihood opportunities and markets, and off-farm employment. These topics were used to judge 13 Biodiversity and Conservation (2022) 31:1313–1328 1317 the local priorities for forest conservation action. A detailed description of the questionnaire including the coding of answers is provided in Appendix S2. Data on the relevance and role of NGOs were only available for ASF and Taita, but not for Kitui County. Data analyses Qualitative answers were coded into themes using MAXQDA version 2020 (Appendix S2). We combined the answers to the questions about the benefits living close to the forest using principal component analysis (variance-covariance matrix) and used the dominant eigenvector EB as a proxy to the perceived benefits of near forest living. This eigenvector explained 33.9% of variance, higher values indicate more perceived benefits. Similarly, we calculated the dominant eigenvector EA (45.7% variance explanation) from the answers to the questions of whether plant and animals should be protected and to the willingness of keeping buffer zone and willingness of replanting trees. We interpreted this eigenvector as quantifying conservation awareness. For four paired questions regarding the awareness of ecological problems with respect to whole Kenya and own farm land (exotic tree plantations, land size, soil fertility, and erosion) we calculated the difference between the answer ranks for own farm and Kenya (ΔR = Rlocal – RKenya) as a relative proxy to perceived conservation problems. Negative values of ΔR indicate that the respondents see the local ecological problems of being small in comparison to the perceived status in whole Kenya. We used generalized linear nested models (Poisson error structure, identity link function) as implemented in STATISTICA 12.0 to link our quantitative (number of children, farm size) and categorical predictors (study site, level of education) to the benefit and awareness indices (response variables). As we were mainly interested in the differences between the three study sites, site identify was nested within the other predictors. We compared quantitative answers with one-way ANOVA and post hoc Tuckey tests. Fig. 2 Socio-economic comparisons of the participants in the three study sites. Except for children and land size (mean absolute values) mean codes according to the coding Table A1 are given. Error bars denote standard errors. The black lines denote the mean rank value. Gender: prevalence of female respondents, age classes: prevalence of older people in Kitui County, education and income: prevalence of lower education/income. Different letters above bars denote significant statistical differences between regions (one-way ANOVA, P < 0.01). The different colors of bars indicate the three different study sites. Red: ASF = Arabuko Sokoke forest; green: Kitui = riparian forests in Kitui County; blue: Taita = cloud forests of Taita Hills 13 1318 Biodiversity and Conservation (2022) 31:1313–1328 Results Environmental and sociodemographic characteristics Landscape and socioeconomic status of the local people strongly differed between the three study sites. Participants from ASF were comparably less educated, more often unemployed and had lower monthly incomes than people from Kitui County and Taita Hills (Fig. 2; Table 1). People from all three regions were comparatively poorly educated and had on average low income (Fig. 2). The mean number of people per household was 7.40 in the ASF study site, and 4.43 in Taita Hills (no data available for Kitui County). Ecological awareness The appreciation for the benefits of ecosystem services significantly differed between the study sites (Table 2). People from Kitui County perceived the socioeconomic and ecological benefits from close by forests highest, people from aSF lowest (Fig. 3a). Importantly, these differences were only marginally significant for people with higher education (Fig. 3a; Table 1 The critical socio-economic parameters of the participants according to the three study sites Variable Category Total numbers % Numbers Proportions ASF Kitui Taita ASF Kitui Participants N 827 336 191 300 0.41 0.23 Gender Male 319 38.57 109 67 143 0.34 0.21 Female 507 61.30 226 124 157 0.45 0.24 Education None 363 43.89 233 47 83 0.64 0.13 Primary 272 32.88 69 81 122 0.25 0.30 Secondary 167 20.19 29 57 81 0.17 0.34 Higher 25 3.02 5 6 14 0.20 0.24 Children 0 70 8.46 28 40 2 0.40 0.57 1 163 19.70 45 57 61 0.28 0.35 2–4 226 27.32 68 80 78 0.30 0.35 5–8 274 33.13 141 95 38 0.51 0.35 73 8.82 52 9 12 0.71 0.12 >8 Type of Income Farmer 468 56.59 174 177 117 0.37 0.38 Half-time farmer 148 17.89 73 48 27 0.49 0.32 Other income 166 20.07 59 60 47 0.36 0.36 Unemployed 45 5.44 30 15 0 0.67 0.33 Monthly income (ksh) < 5000 552 66.74 286 101 165 0.52 0.18 5000–10,000 176 21.28 32 55 89 0.18 0.31 10,000–15,000 40 4.83 12 8 20 0.30 0.20 15,000–20,000 23 2.78 2 11 10 0.09 0.48 33 3.99 3 14 16 0.09 0.42 > 20,000 Farm size (acr.) 0 21 2.53 18 3 0 0.86 0.14 184 22.24 38 132 14 0.21 0.72 <1 1–2 156 18.86 57 73 26 0.37 0.47 2–4 247 29.86 46 52 149 0.19 0.21 176 21.28 143 33 0 0.81 0.19 >4 13 Taita 0.36 0.45 0.31 0.23 0.45 0.49 0.56 0.03 0.37 0.35 0.14 0.16 0.25 0.18 0.28 0.00 0.30 0.51 0.50 0.43 0.48 0.00 0.08 0.17 0.60 0.00 Biodiversity and Conservation (2022) 31:1313–1328 1319 Table 2 Generalized linear nested model (study site nested in the other predictors, Poisson error structure, identity link function). Given are χ2 values with significances: : P < 0.05, : P < 0.001. N = 827 Variable Nested Number of Perceived Awarecategories benefits ness Study site 3 106.7* 14.6*** Education Study site 4 17.5* 1.08 Land size Study site 5 4.5 1.2 Gender Study site 2 2.5 2.9 Age Study site 6 0.7 < 0.01 Monthly income Study site 5 8.3* 0.25 Children Study site 5 1.1 0.47 Fig. 3 a), b) Differences between the three study sites (red: ASL, green: Kitui County, blue: Taita) in the degrees of the index of perceived benefits from living near a forest EB and conservation awareness EA in dependence on the level of education. c) Perceived socioeconomic benefits living close to forest: IS: infrastructure, LA: land availability, TR: tradition. In c) no data for Taita Hills are available. Higher levels indicate higher awareness/benefits. Error bars denote standard error. The different colors of bars indicate the three different study sites. Red: Arabuko Sokoke forest; green: Riparian forests in Kitui County; blue: Cloud forests of Taita Hills Table 2). Within each study site, gender, age, monthly income, and numbers of children did not significantly influence the levels of perceived benefits (Table 2). Water availability, availability of fertile soils, good soils for brick production, and good pastures for livestock 13 1320 Biodiversity and Conservation (2022) 31:1313–1328 were of high relevance for survey participants living along with the gallery forests (Appendix B). At ASF and Taita Hills, the availability of shade (cooler microclimate) was important. In turn, illegal logging was listed as the leading threat to forest conservation in all three study sites. Awareness for conservation significantly differed between the three study sites (Fig. 3b; Table 2). Awareness was highest at the Kitui County site, and lowest at the ASF site (Fig. 3b). Again, other socioeconomic predictors did not significantly enter the glm model (Fig. 3b; Table 2). Protection attitudes Participants of all three regions voted in the majority in favour of plant and animal protection (all average answer scores > 4) but preferred the protection of plants over animals (p(F1,812) < 0.001), most pronounced in Taita Hills. Irrespective of study site, participants saw the local ecological situation better than the perceived situation in whole Kenya (Fig. 4a). Soil erosion (average score 4.30 ± 0.10; mean ± standard error) and soil fertility (4.33 ± 0.09) were most often mentioned in Kitui County (least negative ΔR values, Fig. 4a), while not seen as a problem in Taita Hills (2.94 ± 0.07 and 3.48 ± 0.06, respectively) and ASF (1.73 ± 0.06 and 3.08 ± 0.08, respectively). Also, too small fields were not mentioned as a problem (all average scores < 3.5), while the planting of non-indigenous trees was even seen slightly positively (all average scores < 3.0), most so in Kitui County (2.28 ± 0.0.16). We found also highly significant differences between the study sites in respect of suggestions about conservation efforts (Fig. 4b). Reforestation was the major issue for respondents from Taita Hills, while in Kitui County sustainable resource extraction dominated. For local people from ASF, off-farm employment and alternative water sources dominated. Comparatively few mentions (< 10% respondents) gained alternative livelihoods, and water sources, as well as alternative energy sources (Fig. 4b). Rarely mentioned (≈ 10%) were also sustainable agriculture and participant resource management (Fig. 4b). Overall, participants below the age of 50 considered off-farm employment as a key strategy to reduce pressure on forest ecosystems (ANOVA: F2,343 = 5.4, p < 0.01). Participants with higher formal education emphasized the need for reforestation (F3,732 = 5.6, p < 0.001), while sustainable extraction of natural resources was most often mentioned by respondents with secondary education and with higher income although the latter trends were only marginally significant (F3,731 = 2.8, p = 0.03 and F2,252 = 2.9, p = 0.06, respectively). Finally, people from Kitui County were significantly more prone to do mixed cropping (F2,817 = 37.2, p < 0.001), while people from ASF were less prone to abandon livestock grazing (F2,812 = 158.7, p < 0.001). Environmental communication Communication of information by local authorities and mass media (radio / TV) were often mentioned, while internet sources and NGOs played only a minor role in environmental communication (Fig. 4c). Importantly, mass media and local authorities were also assessed as being most useful information sources (Fig. 4c). The respondents found internet sources and at ASF also NGO information of being less useful (Fig. 4c). These general trends were independent of study site although there were significant site-specific differences. Particu- 13 Biodiversity and Conservation (2022) 31:1313–1328 1321 Fig. 4 a) Perceived conservation problems ΔR of local people from Kitui County, ASF and Taita Hills. Personal ideas (b) and information sources (c) on how to improve forest conservation in their localities (proportions of mentioning with respect to the total number of answers to a given question). The vertical black line in c) denotes the average rescaled rank score indicating higher > 1) or lower (< 1) proportions of mentioning than expected from a random distribution around the mean score value. Errors denote standard errors. NGO data were not available for Kitui County. The different colors of bars indicate the three different study sites. Red: ASF = Arabuko Sokoke forest; green: Kitui = riparian forests in Kitui County; blue: Taita = cloud forests of Taita Hills 13 1322 Biodiversity and Conservation (2022) 31:1313–1328 larly, the local people at ASF tended to be more skeptical against all sources of environmental information (Fig. 4c: F2,824 = 95.7, P < 0.001) and prefer alternative dissemination channels like group meetings, so called barrazas. Discussion Environmental and sociodemographic characteristics We found that age, gender, ethnicity, indigenous knowledge, formal education, and higher incomes shapes attitudes towards the use of forest resources and forest conservation, across all three study sites. We found a rather low level of education and very low income for all three study sites. The people of ASF and Taita Hills positively affirm a general interest in forest conservation (Nzau et al. 2020; 2021a), although at different levels (with highest levels in Taita Hills). Most of the inhabitants record a historical interrelationship with conservation (Njogu, 2004; Shepheard-Walwyn, 2014). This becomes particularly clear when we take a closer look at the situation at ASF. This forest and region is currently experiencing accelerated urbanization, immigration influx, and lack of land-use planning (Bendzko et al. 2019). In this region, most people had lived for less than 20 years around the forest (REF). These factors accelerates parceling of land per capita (Bendzko et al. 2019; Schürmann et al. 2020; Nzau et al. 2020). Indigenous and local ecological knowledge systems are hardly integrated into the forest management of ASF, as is evident by the marginalization of the Waatha indigenous community there (Nzau et al. 2020). Residents surrounding ASF demonstrated the most negligible spatial bias yet showed the highest indifference towards forest conservation (Nzau et al. 2020; Nzau, 2021). The relatively high protection status and large size of the forest may contribute to illusions that the forest resources are finite (Nzau et al. 2020; Nzau, 2021). Furthermore, ASF still host large megafauna, such as elephants. The residents around the forest acknowledged the aesthetic (and touristic) value of wildlife, unlike in Kitui County and Taita Hills (Nzau et al. 2020; 2021a; Nzau, 2021). In comparison to ASF, the Taita Hills inhabitants showed a higher awareness of the threats to their forests due to their place-based experiences and longer relationship with their environment (Njogu, 2004; Hohenthal, 2018, Nzau, 2021). We found that age, gender and education shape behavior and attitudes towards forest and nature protection. In ASF, men and long-term residents were likely to be involved in making rules of forest use, while in Taita Hills, people with higher incomes and education likely to be involved. In both cases, many people were thereby excluded from decision-making processes, such as women, recent settlers, and full-time farmers (Nzau, 2021). Relevance of forest ecosystem services The fact that the forest is of great relevance for the quality of life of people was clear to most of the respondents. The appreciation for tangible ecosystem services provided by the forest was highest in Kitui County, a semi-arid region, where riparian river zones are often a lifeline for sustaining human livelihoods (Habel et al., 2018). Ecosystem services awareness differed among the three regions, and different services have been mentioned as being of relevance, such as soil i.e. soil fertility in Kitui County, and mesoclimatic conditions i.e. 13 Biodiversity and Conservation (2022) 31:1313–1328 1323 shade in Taita Hills and ASF. Awareness was highest in Kitui County and lowest in ASF. Surprisingly, there was only little consideration payed on water springs and access to water, which is of relevance to most of the regions, particularly the Taita Hills, where the planting of exotic eucalyptus trees caused the drying out of many springs (Hohenthal, 2018). Surprisingly, the planting of exotic trees is considered positive by the local people across all three study sites. The establishment of so-called wood-lots (tree plantations consisting of exotic, fast-growing tree species) is widespread in Kenya and provides a basic supply of timber and firewood, and is highly appreciated by the local population and authorities. In Kitui County, educated people notably showed a deep bias to protect plants over animals. Instead, people with no formal education in Kitui County, who were also likely to be older, showed heightened support for the protection of animals (this could be traced back to the histories of the inhabitants of these as revered hunters with intricate human-wildlife interrelationships (Steinhart, 2000; Nzau, 2021). On the contrary, older people in Taita (also likely not to have formal education) showed the least protection attitudes. Similarly, this negative relationship can be traced back to the wildlife conservation histories of the Taita Hills region, which is home to one of the oldest and largest National Parks in Kenya, The Tsavo (Njogu, 2004; Rülke et al. 2020; Nzau et al. 2021a). Awareness and attitudes towards forest conservation Historical land injustices and recent unequal benefit-sharing combats positive attitudes towards nature conservation (Njogu, 2004; Githiru, 2007; Nzau et al. 2020; 2021a). The promise of PFM to address these inequalities (see Kellert et al. 2000) has so far achieved mixed results in improving people’s livelihoods around ASF (Ming’ate & Bollig, 2016; Busck-Lumholt & Treue, 2018) and Taita Hills (Hohenthal, 2018; Rülke et al. 2020), as well as in ecological conservation outcomes (Cuadros-Casanova et al. 2018; Bendzko et al. 2019; Schürmann et al. 2020; Teucher et al. 2020). We recorded low awareness on biodiversity for all three study sites, despite the fact that ASF and Taita Hills are listed as global biodiversity hotspots and still hold a large number of endemic endangered plant and animal species (Nzau et al. 2020; Rülke et al. 2020; Nzau et al. 2021a). Logging is ranked as major threat to the forest and was associated with the exclusion of local people, corruption, and pervasive lack of benefit-sharing arrangements (Nzau et al. 2020; 2021a). Generally, men of intermediate education showed higher awareness than women, who are closely associated with place-based knowledge and experiences in the context of local labour migration and marriage patterns (Nzau et al. 2021a; Nzau, 2021). Income and the level of poverty (land availability) also affects attitudes towards forest conservation. Most of the participants were full-time small-holder farmers, with minimal monthly incomes (< 50 USD). We observed that wealthier households tended to purchase charcoal, firewood, poles, and timber from more impoverished farmers, who in turn sourced these products directly from the forest (in ASF and Taita Hills) or their private land properties (in Kitui County). Consequently, resource overexploitation pressures may be shifted from wealthier to the more impoverished landowners (Stern, 2004; Mills & Waite, 2009; Vyamana, 2009). This scenario is especially evident in Kitui County, where landholdings per household were relatively small (≤ 1 acre) (Nzau et al. 2018) and in the Taita Hills, where landholdings per household are highly fragmented into small distinctive parcels (Hohenthal, 2018; Teucher et al. 2020). 13 1324 Biodiversity and Conservation (2022) 31:1313–1328 Environmental communication Radio and TV are the most important media for communicating information to the population. The internet, on the other hand, played a subordinate role. Overall, there was a high spatial bias among educated people, which correlated positively with access to mass media, the internet, and NGOs (Nzau, 2021). Spatial biases have been linked to environmental communication when global environmental concerns are given dominance over immediate problems (Schultz et al. 2014; Nzau et al. 2018). Spatial biases impact conservation action in that when people underestimate the magnitude of environmental problems at their local scales, they are unlikely to implement timely solutions (Gifford et al. 2009; Schultz et al. 2014). In Kitui County and Taita Hills, the highest spatial bias was among part-time farmers and participants with higher incomes which may be explainable by their higher purchasing power (Stern, 2004; Mills & Waite 2009) for consistent access to mass media and the internet as well as circular migration which constantly exposes them to comparative environmental at locally and elsewhere (Nzau et al. 2021a). Full-time farmers notably showed lesser trust for all external environmental information (Nzau et al. 2021a). This trend can be tied to long-term experiences with disenfranchisement (Brockington, 2004; Githiru, 2007; Rülke et al. 2020). In general, environmental information from governmental- and non-governmental organizations was met with distrust (Nzau et al. 2020; 2021a), while local chiefs were regarded as important sources of environmental information, even though they were hardly trained as environmental experts (Nzau, 2021). This distrust may arise from past alienation experiences with conservation agencies, and creates communication anomalies that derail the success of co-management conservation initiatives (Hohenthal, 2018; Busck-Lumholt & Treue, 2018; Nzau et al. 2020; 2021a). Although the usage of all sources of environmental information was prevalent in Taita Hills, it coincided with a general distrust for all external sources of environmental information, which increased with age (Nzau et al. 2021a). In ASF, the distrust for environmental information from government agencies increased with higher formal education. In contrast, trust for governmental information increased with the education level in Kitui County. Simultaneously, distrust for all external sources of information was pronounced among full-time farmers in Kitui County (Nzau, 2021). Suggestions for forest protection Activities to protect the forest differed considerably among the three forest regions. The proposals for forest protection and conservation and the corresponding measures differed among the three regions. For example, reforestation was proposed for the Taita Hills, and more sustainable use of resources was proposed for Kitui County. Sustainable agriculture did not play a central relevance in any of the three regions as a possible measure to conserve nature and the forest. Furthermore, other different concrete solutions were presented, depending on age and education level. People with intermediate to higher formal education levels considered reforestation, and those with higher incomes favoured sustainable extraction of natural resources. These trends can be tied again to increased purchasing powers among the educated people who can negotiate off-farm employment and thereby less reliant on immediate ecosystem services (Stern, 2004; Vyamana, 2009). Other prominent local opinions included the water availability for agriculture, education and awareness, enforce- 13 Biodiversity and Conservation (2022) 31:1313–1328 1325 ment of environmental law and the co-management of environmental resources (Nzau, 2021). Tree planting was the most common alternative livelihood strategy among people living in Taita Hills, with men likely to engage in it. On the other hand, butterfly farming was common in ASF, irrespective of gender (Gordon & Ayiemba, 2003). The long-term residents surrounding ASF were also highly likely to keep bees (Nzau, 2021). While young people demonstrated the highest willingness to implement good environmental practices, they were less likely to have the resources, especially land rights (Bendzko et al. 2019; Schürmann et al. 2020) and the decision-making authority to release their good intentions (Nzau, 2021). Young people, therefore, held the opinion that off-farm employment would reduce resource extraction pressure from forests which was in parallel to the ideas of alternative income strategies such as butterfly farming and beekeeping that are often advanced by governmental and non-governmental agencies (Nzau et al. 2020; 2021a). Conclusions The principles of co-management and Participatory Forest Management (PFM) offer a valuable framework for conserving tropical forests. However, the degree to which these principles can be implemented is dependent on distinct and discreet social-ecological factors that can contribute and reinforce inverse trade-offs and significantly undermine positive conservation outcomes. We found various discrepancies among the three study sites, which needs to be considered when developing and implementing efficient conservation strategies. In the ASF forest, many people live there for less than 20 years; at the same time, the awareness of forest conservation was relatively low there compared to the other two study sites. Forests play a major role against the backdrop of resource use in all three regions, with significant differences in the ecosystem services used in the three different regions. Likewise, the proposals for efficient forest conservation differ: Reforestation is proposed in Taita Hills and Kitui County, whereas off-farm employment plays a major role in ASF. The proposed conservation measures also differed according to age and education level. For environmental education and communication, the internet plays a comparatively minor role. Based on our findings, we argue that the implementation of locally specific conservation action needs to be developed and applied, particularly in countries with very heterogeneous ethnicities and environments. Overall, the lack of transparency and equity in natural resource management overrides positive conservation attitudes, intentions, and willingness with negative implications for governmental and non-governmental conservation experts’ legitimacy and ultimately for the conservation agenda. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1007/s10531-022-02393-x. Acknowledgements We thank the local populations and representatives of governmental and non-governmental organizations of the three study sites for participating in our study. We are grateful to Lozi Maranga for the collection of data in the field. We thank Mike Teucher for producing Fig. 1. The German Academic Exchange Service (DAAD) granted a PhD fellowship to JMN. We are grateful for two very valuable and critical reviews about a draft version of our manuscript. Author contribution JMN and JCH designed the study, JMN performed data collection, WU run analyses, all contributed while data interpretation and writing. 13 1326 Biodiversity and Conservation (2022) 31:1313–1328 Funding Funding is reported in the Acknowledgement section. Open access funding provided by Paris Lodron University of Salzburg. Data Availability All data are provided as electronic appendix. Declarations Conflict of interest There is not conflict of interest. Animal Research Not applicable. Consent to Participate Not applicable. Consent to Publish All approved to publish this data and article. Plant Reproducibility Not applicable. Clinical Trials Registration Not applicable. 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/. References Agrawal A, Chhatre A, Hardin R (2008) Changing Governance of the World’s Forests. 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Authors and Affiliations Joslyn Muthio Nzau1 · Werner Ulrich2 · Marco Rieckmann3 · Jan Christian Habel4 Jan Christian Habel Janchristian.habel@sbg.ac.at 1 Terrestrial Ecology Research Group, Department of Ecology and Ecosystem Management, Technische Universität München, D-85354 Freising, Germany 2 Department of Ecology and Biogeography, Nicolaus Copernicus University Torun, PL-87-100 Toruń, Poland 3 Higher Education Development Research Group, Department of Education, Faculty of Education and Social Sciences, University of Vechta, D-49377 Vechta, Germany 4 Evolutionary Zoology, Department of Environment and Biodiversity, University of Salzburg, A-5020 Salzburg, Austria 13
https://openalex.org/W3210408815	https://josr-online.biomedcentral.com/track/pdf/10.1186/s13018-021-02807-6	English	null	Factors influencing early and long-term survival following hip fracture among nonagenarians	Journal of orthopaedic surgery and research	2,021	cc-by	6,690	© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Weinberg et al. J Orthop Surg Res (2021) 16:653 https://doi.org/10.1186/s13018-021-02807-6 Weinberg et al. J Orthop Surg Res (2021) 16:653 https://doi.org/10.1186/s13018-021-02807-6 Open Access Abstract Background: The outcomes of nonagenarian patients undergoing orthopaedic surgery are not well understood. We investigated the 30-day mortality after surgical treatment of unilateral hip fracture. The relationship between postop‑ erative complications and mortality was evaluated. Methods: We performed a single-centre retrospective cohort study of nonagenarian patients undergoing hip frac‑ ture surgery over a 6-year period. Postoperative complications were graded according to the Clavien–Dindo classifica‑ tion. Correlation analyses were performed to evaluate the relationship between mortality and pre-specified mortality risk predictors. Survival analyses were assessed using Cox proportional hazards regression modelling. Results: The study included 537 patients. The 30-day mortality rate was 7.4%. The mortality rate over a median follow-up period of 30 months was 18.2%. Postoperative complications were observed in 459 (85.5%) patients. Both the number and severity of complications were related to mortality (p < 0.001). Compared to patients who survived, deceased patients were more frail (p = 0.034), were at higher ASA risk (p = 0.010) and were more likely to have preop‑ erative congestive heart failure (p < 0.001). The adjusted hazard ratio for mortality according to the number of com‑ plications was 1.3 (95% CI 1.1, 1.5; p = 0.003). Up to 21 days from admission, any increase in complication severity was associated significantly greater mortality [adjusted hazard ratio: 3.0 (95% CI 2.4, 3.6; p < 0.001)]. Conclusion: In a nonagenarian cohort of patients undergoing hip fracture surgery, 30-day mortality was 7.4%, but 30-month mortality rates approached one in five patients. Postoperative complications were independently associ‑ ated with a higher mortality, particularly when occurring early. Keywords: Anaesthesia, Complication, Nonagenarian, Fracture, Surgery and mortality [1–3]. In Australia, hip fracture-related hospital admissions account for approximately 0.5% of all hospitalisations and result in more than 579,000 bed days or 1.9% of annual hospitalisations [4]. Despite reduc- tions in hospitalisation rates among ageing and at-risk populations with hip fractures, these patients still present a significant clinical burden [4]. Hip fractures are over- represented in the elderly, with a median age of hospitali- sation in Australia being 84 years [4]. When compared to younger populations, nonagenarians (patients aged Factors influencing early and long‑term survival following hip fracture among nonagenarians Laurence Weinberg1,2,5* , Bobby Ou Yang1, Luka Cosic1, Sarah Klink1, Peter Le1, Jasun Kai Li1, Anoop Ninan Koshy3, Daryl Jones4, Rinaldo Bellomo4,5, Chong Oon Tan1 and Dong‑Kyu Lee6 Introduction In both absolute numbers, and as a proportion of many Western populations, the number of people in their eighth, ninth and tenth decades continues to increase [1, 2]. Hip fractures are similarly becoming more common and carry an associated increased burden of morbidity Correspondence: laurence.weinberg@austin.org.au 1 Department of Anaesthesia, Austin Health, 145 Studley Rd, Melbourne, VIC 3084, Australia Full list of author information is available at the end of the article Correspondence: laurence.weinberg@austin.org.au 1 Department of Anaesthesia, Austin Health, 145 Studley Rd, Melbourne, VIC 3084, Australia Full list of author information is available at the end of the article Weinberg et al. J Orthop Surg Res (2021) 16:653 Page 2 of 11 capture and retrieval of patient health information in the perioperative setting from September 2014 onwards. 90–99 years) are highly vulnerable to hip fractures and experience increased rates of postoperative morbidity, mortality and adverse functional outcomes [2, 5, 6]. Surgical osteosynthesis is considered the gold standard of care for hip fracture, with nearly all patients receiv- ing operative management in Australia [4]. While some international literature has reported on the preoperative, surgical and anaesthetic factors affecting postoperative outcomes in nonagenarian patients [2, 7, 8], no com- parative data exist in the Australian context. Moreover, the majority of existing data focus on preoperative fac- tors affecting post-surgical outcomes, including time to surgery, patient comorbidity and advanced age [2, 7, 8]. Additionally, there remains a dearth of international data assessing the impact of complications and periop- erative factors on short- and long-term mortality among nonagenarians. Objectivesh The primary endpoint of this study was to quantify the rate of perioperative mortality at 30 postoperative days. Secondary endpoints were the number and severity of postoperative complications and their association with short- and long-term mortality. We also evaluated the perioperative risk factors associated with long-term sur- vival. Mortality was assessed using Austin Health’s elec- tronic medical record system or by contacting patients’ general practitioners to obtain out-of-hospital mortality status. Methods Following Human Research Ethics Committee approval (No: HREC/21/Austin/30), we performed a single-centre retrospective cohort study of nonagenarian patients who underwent post-fracture restorative surgery over a six- year period between 1 September 2014 and 31 August 2020. Perioperative outcomes for the period ending 28 February 2021 were collected, providing a minimum of 6-month postoperative data following the final surgical episode. The study was performed at the Austin Hospi- tal, a tertiary university teaching hospital in Melbourne, Australia. Inclusion criteria were patients aged > 90 years and < 100 years presenting with a unilateral femoral neck, intertrochanteric or subtrochanteric femoral fracture (collectively termed ‘hip fracture’), who required opera- tive management. All operative techniques were con- sidered. Patients were excluded if they were managed conservatively or died prior to operative management. Four independent investigators extracted data from the electronic medical records. The above dates were cho- sen a priori because Austin Health Cerner® electronic medical records allowed comprehensive electronic data Definitions Medical comorbidities were collected from electronic medical records, including anaesthetic records. Charl- son comorbidity index (CCI) scores were calculated for each patient [11]. Frailty was defined as a score of 5 or more on the Canadian Study of Health and Ageing Clini- cal Frailty Scale [12]. Time to surgery was defined as the length of time from hospital presentation until the com- mencement of anaesthesia. Surgery performed out-of- hours was defined as surgery performed between 6 p.m. and 8 a.m. on Monday to Friday, and on Saturdays, Sun- days and public holidays. Return to theatre was defined as any second or subsequent surgery performed as a consequence of the original surgical management of hip fracture during the index admission. Complications were defined as any deviation from the normal postoperative course, guided by the European Perioperative Clinical Outcome definitions [13]. Complications were recorded by two independent clinicians and graded according to the Clavien–Dindo (CVD) classification [14]. The CVD classification is a validated approach to surgical outcome assessment that assigns severity grades to surgical com- plications. In case of disagreement on grading by two assessors, the case was decided with reference to the clas- sification guide by a third assessor. Instances of review by the hospital Medical Emergency Team (MET) were obtained from the dedicated electronic database, com- pleted by the intensive care registrar at the conclusion of the MET call. Length of stay was determined by the period between presentation and discharge. Readmission was defined as an unplanned readmission to the hospital within a 30-day follow-up period. Given the strong association between increasing age and mortality secondary to hip fractures [5, 9, 10], and the significant burden that these injuries place on health- care systems, it is valuable to identify perioperative fac- tors that may present opportunities to reduce morbidity and mortality among nonagenarians suffering from them. Therefore, we investigated the overall mortality in patients who, in their tenth decade, underwent sur- gery for hip fracture. The primary outcome was 30-day mortality. Secondary outcomes included the number and severity of postoperative complications and their associa- tion with short- and long-term mortality. Statistical analysis Statistical analysis was performed using R software 4.0.2 (R Development Core Team, Vienna, Austria, 2020). Data were deidentified, the variable names were encrypted, and data were coded with numerical values to blind the Weinberg et al. J Orthop Surg Res (2021) 16:653 Weinberg et al. J Orthop Surg Res (2021) 16:653 Page 3 of 11 Baseline associations with 30‑day mortality Baseline associations with 30‑day mortality collected variables’ characteristics to the statistician. Data are presented as mean ± standard deviation, median (1st–3rd quartiles) [Max/Min], or the number of cases (percentile) for descriptive statistics. Estimated values are described with 95% confidence intervals (CI). Statis- tical results are presented with p values. Any two-sided p value below 0.05 or Bonferroni’s corrected significance levels was considered statistically significant. Baseline patient characteristics are presented in Table 1 and in Additional file 1. Several associations with mortal- ity were identified. Males made up 25% of all patients and 36.7% of all deceased patients (p = 0.019); however, the effect of sex differences was small (Cramér’s V = 0.10). The age difference between surviving and deceased patients was not statistically significant. Frailty was asso- ciated with a higher unadjusted mortality rate, although the estimated effect size was small. Finally, deceased patients had higher ASA classification scores (p = 0.010) and a higher incidence of congestive heart failure on admission (p < 0.001). i Before statistical analysis, normality was assessed for continuous variables using the quantile–quantile plot. If normality criteria were violated, nonparametric statistical methods were applied for that variable. The homogeneity of variance assumption was applied where appropriate. Standard statistical methods identified extreme values, which were then reconciled by interrogating the clinical notes and the context of the value. Perioperative associations with 30‑day mortality As shown in Table 2, preoperative and intraoperative differences between survivors and deceased patients were small and did not reveal any clear risk factor. The postoperative course, however, was characterised by a higher frequency of medical emergency team (MET) calls in deceased patients (p = 0.019, common effect size r = –0.10). Variables with a missing rate of more than 5% were identified, and missing value patterns were analysed to determine missingness mechanisms. Data that were missing completely at random were excluded from the statistical analysis. Student’s t test, Mann–Whitney rank- sum test, Chi-squared test and Fisher’s exact test were applied to determine the differences between survived and deceased cases. Statistical analysis Correlation analysis was performed to evaluate the relationship between mortality and other variables. According to these results and knowledge based on their clinical impacts, several factors were identified as independent parameters for the following survival analysis. Considering the longitudinal character- istics, Cox proportional hazard regression modelling was used for survival analysis. The goodness-of-fit test for constant proportional hazard assumption was assessed using Schoenfeld residuals. In case of violated constant proportional hazard assumption, time-dependent Cox regression was applied according to the split observation duration using a step function. The splitting point was determined by the interpretation of Schoenfeld residuals. Association between postoperative complications and mortality y As shown in Table 3, both the number and severity of postoperative complications were associated with over- all mortality (p < 0.001). Complications were observed in 459 patients (85.5%, 95% CI 82.5, 88.0). The observed complication rate was 84.5% (95% CI 81.1, 87.9) in sur- viving patients and 89.8% (95% CI 83.8, 95.8) in deceased patients. The number of postoperative complications was higher in the deceased compared to surviving patients (p < 0.001). Similarly, the severity of complications was higher in deceased patients (p < 0.001, Table 3). Gender, frailty, ASA classification, congestive heart failure, pre- anaesthesia hypertension, the number of intraoperative hypotensive episodes, the number of MET calls and the number or severity of complications were selected covar- iates for the Cox regression. In addition to these, weight and intra- and postoperative opioid dose were included in the analysis as covariates in view of their traditional statistical threshold as potential predictors [15]. Given their clinical importance, CCI [11], time to surgery from hospital admission [16], surgery performed out-of-hours [17, 18] and combined regional anaesthesia [19] were also considered covariates for the subsequent survival analysis. Details of cohorthi The final analysis included data from 537 patients. There were two variables with a missing value rate of more than 5%: height (51.2%) and preoperative albumin concentra- tion (35.2%). The pattern of omission for both was ran- dom, but because neither variable carried a statistically significant relationship to mortality, these omissions were tolerated. Over the study follow-up of 30 (12–53) [1:77] months, 98 deaths were recorded (18.2%, 95% CI 15.0, 21.5%). Of the cases of mortality, 40 patients (7.4%, 95% CI 5.2, 9.7%) died within 30 days from admission. Cox regression was performed separately for the number or severity of complications. The proportional hazard assumption was violated in both cases of the number and severity of complications. According to Weinberg et al. J Orthop Surg Res (2021) 16:653 Page 4 of 11 Table 1 Demographic data for nonagenarian patients undergoing surgery for hip fracture Values are expressed as mean ± SD, median (IQR) [Max/Min], or number (%) Effect size: Cohen’s d for t test, common effect size r for Mann–Whitney test, Cramér’s V for the Chi-squared test. Correlation coefficient: Spearman’s rho and corresponding p value Time to surgery: time from admission to surgery start Opioid doses are presented as a total amount of all kinds of opioids used as a morphine- Category and variables Survived (n = 439) Deceased (n = 98) p value Effect size Correlation coefficient (p value) Demographics Sex (Male)+ 110 (25.1) 36 (36.7) 0.019* 0.10 0.101 (0.019)* Age (years)‡ 92.97 ± 2.70 93.26 ± 2.61 0.339 0.11 0.056 (0.197) Frailty+ 327 (74.7) 83 (84.7) 0.034* 0.09 0.091 (0.034)* Weight (kg)‡ 59.23 ± 11.93 61.68 ± 12.39 0.068 0.20 0.071 (0.101) ASA classification§ II 40 (9.1) 5 (5.1) 0.010* – 0.135 (0.002)* III 248 (56.5) 43 (43.9) IV 150 (34.2) 49 (50.0) V 1 (0.2) 1 (1.0) Diabetes mellitus+ 73 (16.6) 19 (19.4) 0.512 0.03 0.028 (0.513) Chronic kidney disease+ 111 (25.3) 30 (30.6) 0.279 0.05 0.047 (0.279) Congestive heart failure+ 97 (22.1) 40 (40.8) < 0.001* 0.17 0.166 (< 0.001)* Chronic obstructive airways disease+ 44 (10) 15 (15.3) 0.130 0.07 0.065 (0.131) Cerebrovascular accident/transient ischaemic attack+ 84 (19.1) 13 (13.3) 0.172 0.06 –0.059 (0.173) Dementia+ 166 (37.8) 39 (39.8) 0.715 0.02 0.016 (0.716) Charlson comorbidity index 6 (5–7) [4:14] 6 (5–8) [4:15] 0.297 –0.05 0.045 (0.297) Table 1 Demographic data for nonagenarian patients undergoing surgery for hip fracture Effect size: Cohen’s d for t test, common effect size r for Mann–Whitney test, Cramér’s V for the Chi-squared test. Correlation coefficient: Spearman’s rho and corresponding p value. Time to surgery: time from admission to surgery start. Opioid doses are presented as a total amount of all kinds of opioids used as a morphine- i t t d ’s d for t test, common effect size r for Mann–Whitney test, Cramér’s V for the Chi-squared test. Correlation coefficient: Spearman’s rho and value. Time to surgery: time from admission to surgery start. Opioid doses are presented as a total amount of all kinds of opioids used as a ASA classification American Society of Anesthesiologist physical status classification, CCI Charlson’s comorbidity index ct size: Cohen’s d for t test, common effect size r for Mann–Whitney test, Cramér’s V for the Chi-squared test. Correlation coefficient: Spear responding p value. Time to surgery: time from admission to surgery start. Opioid doses are presented as a total amount of all kinds of op ipotent dose Values are expressed as mean ± SD, median (IQR) [Max/Min], or number (%) Discussion Key findings the Schoenfeld residuals, the observed period was split at 12 days after admission for the number of complica- tions and at 21 days and 17 months after admission for the severity of complications. A time-dependent coef- ficient Cox regression was then used to analyse overall mortality with respect to the remaining covariates. We investigated the short-term and long-term outcome of nonagenarians undergoing hip fracture surgery and assessed the relationship of complications with mortal- ity in this cohort [1, 3, 20]. We found that short-term mortality affected one in fifteen patients and that long- term mortality affected one in five patients. Moreover, we also found that preoperative and intraoperative characteristics and events did not show a clear associa- tion with outcome. However, we also found that post- operative complications affected almost 90% of patients and that their number and severity were associated with increased risk of death. y g As shown in Table 4 and Figs. 1 and 2, the adjusted hazard ratio for the number of complications was 1.3 (95% CI 1.1, 1.5; p = 0.003) during the observed period for these patients (Fig. 1). Increasing Clavien–Dindo grade of complication was associated with increased adjusted risk of mortality (HR 3.0 (95% CI 2.4, 3.6; p < 0.001 per 1 unit rise in CVD) until 21 days from admission (Fig. 2). After 21 days, the effects of post- operative complications on mortality were not sta- tistically significant. A summary of the postoperative complications is presented in Additional file 2. Weinberg et al. Discussion Key findings J Orthop Surg Res (2021) 16:653 Page 5 of 11 Table 2 Surgical management, perioperative and anaesthetic clinical information amongst nonagenarian patients undergoing surgery for hip fracture Category and variables Survived (n = 439) Deceased (n = 98) p value Effect size Correlation coefficient (p value) Surgical factors Time to surgery from admission to hospital (hours)‡ 34.25 ± 37.49 38.28 ± 45.92 0.358 0.10 0.042 (0.328) Operation time (min)‡ 131.92 ± 137.71 138.77 ± 145.15 0.660 0.05 − 0.004 (0.921) Surgery performed out- of-hours+ 237 (54) 61 (62.2) 0.137 0.06 0.064 (0.137) Emergency§ 432 (98.4) 95 (96.9) 0.400 – − 0.042 (0.332) Femur neck fracture+ 367 (83.6) 78 (79.6) 0.341 0.04 − 0.041 (0.342) Preoperative block+ 235 (53.5) 59 (60.2) 0.230 0.05 0.052 (0.231) Combined other surgery+ 25 (5.7) 8 (8.2) 0.358 0.04 0.040 (0.358) Preoperative conditions Preoperative transfusion+ 23 (5.2) 5 (5.1) 0.956 0.001 − 0.002 (0.956) Anaesthesia factors Regional anaesthesia combined+ 200 (45.6) 45 (45.9) 0.948 0.003 0.003 (0.948) Invasive monitoring+ 311 (71.2) 73 (74.5) 0.509 0.03 0.029 (0.510) Intraoperative vasopressors used+ 371 (84.5) 87 (88.8) 0.281 0.05 0.047 (0.282) Intraoperative fluid management Total fluid amount (ml)¶ 1000 (1000–1000) [0:3000] 1000 (1000–1000) [0:2250] 0.729 − 0.01 0.015 (0.729) No. of patients who received an intraoperative transfusion+ 23 (5.2) 9 (9.2) 0.136 0.06 0.064 (0.136) Intraop‑ eratively trans‑ fused red blood cell units 1 (1‒1) [1:2], N = 23 1 (1–1.5) [1:3], N = 9 0.273 − 0.194 – No. of events of Intraoperative hypotension¶ 0 (0–2) [0:21] 0 (0–1) [0:13] 0.038* − 0.09 − 0.090 (0.038)* No. of events of Intraoperative hypotension, severe 0 (0–0) [0:28] 0 (0–0) [0:16] 0.246 − 0.05 − 0.050 (0.246) Opioid Preoperative opioid used+ 323 (73.6) 79 (80.6) 0.147 0.06 0.063 (0.147) Intraoperative opioid use+ 352 (80.2) 85 (86.7) 0.132 0.07 0.065 (0.132) Intraoperative opioid dose (mg)‡ 10.1 ± 12.57 12.88 ± 21.19 0.087 0.19 0.039 (0.365) Postoperative opioid used+ 172 (39.2) 43 (44.8) 0.310 0.04 0.044 (0.311) Table 2 Surgical management, perioperative and anaesthetic clinical information amongst nonagenarian patients undergoing surgery for hip fracture Category and variables Survived (n = 439) Deceased (n = 98) p value Effect size Correlation coefficient (p value) Table 2 Surgical management, perioperative and anaesthetic clinical information amongst nonagenarian patients undergoing surgery for hip fracture Table 2 Surgical management, perioperative and anaesthetic clinical information amongst nonagenarian patients undergoing f hi f t Weinberg et al. p Effect size: Cohen’s d for t test, common effect size r for Mann–Whitney test, Cramér’s V for the Chi-squared test. Correlation coefficient: Spearman’s rho and corresponding p value. Time to surgery: time from admission to surgery start. Opioid doses are presented as a total amount of all kinds of opioids used as a morphine- equipotent dose Two-sided p value < 0.025 Cochran–Armitage test for trend and Spearman correlation analysis Values are expressed as mean ± SD, median (IQR) [Max/Min], or number (%) Discussion Key findings J Orthop Surg Res (2021) 16:653 Page 6 of 11 Table 2 (continued) Category and variables Survived (n = 439) Deceased (n = 98) p value Effect size Correlation coefficient (p value) Patient controlled analgesia+ 131 (29.8) 24 (25) 0.344 0.04 − 0.041 (0.345) Postoperative opioid dose (mg)‡ 183.59 ± 448.78 323.52 ± 788.17 0.096 0.27 0.052 (0.228) Postoperative management Postoperative hypotension episodes 0 (0–1) [0:26] 0 (0–0) [0:15] 0.362 − 0.04 − 0.039 (0.362) Postoperative vasopressor use+ 31 (7.1) 10 (10.3) 0.276 0.05 0.047 (0.277) ICU care§ 12 (2.7) 4 (4.1) 0.509 – 0.031 (0.479) Return to theatre+ 20 (4.6) 8 (8.2) 0.146 0.06 0.063 (0.147) Readmission+ 11 (2.5) 5 (5.1) 0.187 0.06 0.059 (0.172) No. of medical emergency team activations¶ 0 (0–0) [0:7] 0 (0–1) [0:5] 0.019 − 0.10 0.101 (0.019)* Two sided p value < 0.050 + Chi-squared test ‡ T test § Fisher’s exact test ¶ Mann–Whitney test Table 3 Postoperative complications in surviving and deceased patients Table 3 Postoperative complications in surviving and deceased patients C h A it t t f t d d S l ti l i Complications Survived (n = 439) Deceased (n = 98) p value Common effect size r Correlation coefficient (p value) Number of complications No complication 68 (15.5) 10 (10.2) < 0.001* 0.19 0.16 (< 0.001)* 1 complication 85 (19.4) 15 (15.3) 2 complications 98 (22.3) 10 (10.2) 3 complications 66 (15.0) 15 (15.3) 4 or more complications 122 (27.8) 48 (49.0) Clavien Dindo grade No complication 68 (15.5) 10 (10.2) < 0.001* 0.49 0.22 (< 0.001)* I 92 (21.0) 17 (17.3) II 248 (56.5) 35 (35.7) IIIa 6 (1.4) 2 (2.0) IIIb 15 (3.4) 3 (3.1) IVa 9 (2.1) 4 (4.1) IVb 1 (0.2) 0 (0.0) V 0 (0.0) 27 (27.6) Weinberg et al. J Orthop Surg Res (2021) 16:653 Page 7 of 11 Table 4 Estimated hazard ratios of selected covariates for overall mortality in nonagenarian hip surgery patients Hazard ratios are estimated using constant proportional hazard ratio assumption validated Cox regression for number of complications and time-dependent coefficient Cox regression for severity of complications. Estimated hazard ratios are presented with 95% CI Two-sided p value below 0.025, a Bonferroni’s corrected significance level No. Discussion Key findings of complications Severity of complications Hazard ratio p value Hazard ratio p value Sex 1.4 (0.9–2.2) 0.197 1.5 (0.9–2.4) 0.095 Frailty 1.5 (0.8–2.6) 0.174 1.4 (0.8–2.5) 0.241 Weight 1.0 (1.0–1.0) 0.791 1.0 (1.0–1.0) 0.490 ASA classification 1.3 (0.5–3.4) 0.534 1.2 (0.5–3.0) 0.731 Congestive heart failure 1.8 (1.2–2.8) 0.008 1.7 (1.1–2.7) 0.027 Charlson comorbidity index 1.0 (0.9–1.2) 0.640 1.0 (0.9–1.2) 0.524 Time to surgery 1.0 (1.0–1.0) 0.391 1.0 (1.0–1.0) 0.154 Surgery performed out-of-hours 1.5 (1.0–2.2) 0.084 1.7 (1.1–2.6) 0.017* Hypertensive response immediately before anaesthesia induction 1.7 (0.9–3.3) 0.093 1.1 (0.5–2.3) 0.773 Combined regional anaesthesia 1.1 (0.7–1.7) 0.557 1.2 (0.8–1.9) 0.378 Number of intraoperative hypotensive episodes 0.9 (0.8–1.0) 0.039 0.9 (0.8–1.0) 0.116 Intraoperative opioid dose 1.0 (1.0–1.0) 0.119 1.0 (1.0–1.0) 0.206 Postoperative opioid dose Day of admission to 12 days 1.0 (1.0–1.0) 0.265 1.0 (1.0–1.0) 0.006* After 12 days 1.0 (1.0–1.0) 0.005* No. of medical emergency team activations 1.0 (0.8–1.3) 0.802 0.9 (0.7–1.2) 0.609 Number of complications 1.3 (1.1–1.5) 0.003* – – Clavien Dindo severity Day of admission to 12 days – – 3.0 (2.4–3.6) < 0.001* 12 days to 7 months – – 1.2 (0.9–1.6) 0.145 After 7 months – – 1.0 (0.6–1.6) 0.982 Table 4 Estimated hazard ratios of selected covariates for overall mortality in nonagenarian hip surgery patients Hazard ratios are estimated using constant proportional hazard ratio assumption validated Cox regression for number of complications and time-dependent coefficient Cox regression for severity of complications. Estimated hazard ratios are presented with 95% CI *Two-sided p value below 0.025, a Bonferroni’s corrected significance level Relationship to literature not increase the risk of 30-day or inpatient mortality or postoperative complications, and that considera- tion should be given to performing hip fracture surgery out-of-hours to meet national guidelines (< 48 h) [28]. In the present study, we did not observe any significant differences in mortality when surgery was performed out-of-hours. Possible reasons for this finding are that our institution has a dedicated ‘out-of-hours’ consult- ant-led emergency orthopaedic service that facilitates operating room availability and allows early and timely access to surgery. The overall mortality rate for nona- genarians having hip fracture surgery at our institution was comparable to that reported in the previous litera- ture [2, 3, 29].h Preoperative optimisation remains a challenge in this cohort. We did not identify time to surgery as an inde- pendent risk factor for increased postoperative mortal- ity. The average time to surgery in our cohort was 34 h in patients who survived and 38 h in deceased patients. These times are aligned with international guidelines [21], systematic reviews, meta-analysis, meta-regression [16, 22–24] and large database registries [25, 26], which demonstrate reduced mortality and intraoperative com- plications if hospitals operate on patients within 48 h after fracture [27]. Some studies have also reported that surgery per- formed after hours significantly increases general complication rates [27]. Potential contributing factor for these increased risks include surgeon fatigue, sur- geon experience, and the potential for more severe or emergent injuries occurring after-hours. Other large- scale studies have refuted such findings [28]. In a sys- tematic review and meta-analysis of 13 studies with 177,090 patients, Kim et al. reported that performing hip fracture surgery after hours or on the weekend did There is disagreement within the literature regard- ing the impact of comorbidity among nonagenarians on mortality. Some studies [3, 29] corroborate our findings, while others [2, 3, 5, 20, 30, 31] have reported increasing comorbidity to be associated with increased mortality. Our findings indicate postoperative complications have the greatest significance regarding mortality following hip fracture surgery. Weinberg et al. J Orthop Surg Res (2021) 16:653 Page 8 of 11 Fig. 1 Survival curves according to the number of complications in nonagenarian hip surgery patients using time-dependent coefficient Cox regression Fig. 1 Survival curves according to the number of complications in nonagenarian hip surgery patients using time-dependent coefficient Cox regression mortality for the study cohort. Relationship to literature The extended follow-up time frames also allowed us to quantify the period for which surgical complications increased the risk of post- surgical mortality. We acknowledge several limitations of this study. The retrospective design inherently lim- its the quality of its findings. Despite this, the missing data points were small in number and did not statisti- cally affect our findings. Given that data were collected retrospectively, the follow-up period varied for patients depending on the year of surgery. This may have affected the long-term mortality data because, at the time of data collection, patients with more recent sur- geries had shorter postoperative timeframes. This may have led to an underestimation of mortality; however, the large sample size mitigates this effect. Data on the causes of delays in receiving surgery were not collected. A better understanding of these details may provide further insights into why this variable had no impact on Study implicationsi Our findings imply that short-term mortality after hip surgery in nonagenarians is relatively low. However, it also implies that, as expected, once follow-up is extended to 30 months, one in five such patients have died. It also implies that preoperative characteristics and intraop- erative events are not major risk factor for such postop- erative mortality. However, it implies that postoperative complications are extremely common and are a major risk factor for mortality, especially when occurring in the early postoperative period. Strengths and limitations Our study presents new data regarding the impact of complications on mortality among nonagenarians having hip fracture surgery. The Clavien–Dindo clas- sification allowed us to demonstrate a close correla- tion between complication severity and increasing Weinberg et al. J Orthop Surg Res (2021) 16:653 Page 9 of 11 Weinberg et al. J Orthop Surg Res Fig. 2 Survival curves according to the Clavien–Dindo grade of complications in nonagenarian hip surgery patients using time-dependent coefficient Cox regression Fig. 2 Survival curves according to the Clavien–Dindo grade of complications in nonagenarian hip surgery patients using time-dependent coefficient Cox regression be enhanced by assessing functional outcome measures such as the World Health Organisation Disability Assess- ment Schedule (WHODAS). mortality despite previous studies being strongly sug- gestive of such an effect.hf f The CCI has been shown to effectively predict mortal- ity following hip fracture; however, the index only col- lects data on selected comorbidities [31]. This specificity of the CCI may explain why our findings did not iden- tify comorbidity as a predictor of mortality. We did not assess functional outcomes. Functional outcomes in the nonagenarian cohort are arguably more important than objective measures of mortality, and future studies may Abbreviations Abbreviations ASA: American Society of Anesthesiologists; CI: Confidence interval; CVD: Cla‑ vien–Dindo; HR: Hazard ratio; MET: Medical Emergency Team; WHODAS: World Health Organisation Disability Assessment Schedule. 4. Australian Institute of Health and Welfare 2018. Hip fracture incidence and hospitalisations in Australia 2015–16. Cat. no. PHE 226. Canberra: AIHW. https://www.aihw.gov.au. Accessed 14 April 2021. Availability of data and materialsi The complete deidentified dataset analysed during the current study is avail‑ able from the corresponding author on reasonable request. Funding Not applicable. 13. Jammer I, Wickboldt N, Sander M, Smith A, Schultz MJ, Pelosi P, European Society of Anaesthesiology (ESA) and the European Society of Intensive Care Medicine (ESICM); European Society of Anaesthesiology; European Society of Intensive Care Medicine, et al. Standards for definitions and use of outcome measures for clinical effectiveness research in perioperative medicine: European Perioperative Clinical Outcome (EPCO) definitions: a statement from the ESA-ESICM joint taskforce on perioperative outcome measures. Eur J Anaesthesiol. 2015;32:88–105. Declarations 14. Dindo D, Demartines N, Clavien P. Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. Ann Surg. 2004;240:205–13. Ethics approval and consent to participate The study was approved by the Austin Health Human Research Ethics Com‑ mittee (No: HREC/21/Austin/30). Participant consent was waivered due to the retrospective nature of the research. 15. Chowdhury MZI, Turin TC. Variable selection strategies and its impor‑ tance in clinical prediction modelling. Fam Med Community Health. 2020;8(1):e000262. https://doi.org/10.1136/fmch-2019-000262. Competing interests 16. Simunovic N, Devereaux P, Sprague S, Guyatt G, Schemitsch E, DeBeer J, et al. Effect of early surgery after hip fracture on mortality and complica‑ tions: systematic review and meta-analysis. CMAJ. 2010;182:1609–16. The authors declare that they have no competing interests. Acknowledgements Not applicable. 9. Barangan J. Factors that influence recovery from hip fracture during hospitalization. Orthop Nurs. 1990;9:19–30. 9. Barangan J. Factors that influence recovery from hip fracture during hospitalization. Orthop Nurs. 1990;9:19–30. 10. Cobey JC, Cobey JH, Conant L, Weil UW, Greenwald WF, Southwick WO. Indicators of recovery from fractures of the hip. Clin Orthop Relat Res. 1976;117:258–62. 10. Cobey JC, Cobey JH, Conant L, Weil UW, Greenwald WF, Southwick WO. Indicators of recovery from fractures of the hip. Clin Orthop Relat Res. 1976;117:258–62. References 1. Bokshan S, Marcaccio S, Blood T, Hayda R. Factors influencing survival following hip fracture among octogenarians and nonagenarians in the United States. Injury. 2018;49:685–90. y 2. Hapuarachchi K, Ahluwalia R, Bowditch M. Neck of femur fractures in the over 90s: a select group of patients who require prompt surgical intervention for optimal results. J Orthop Traumatol. 2014;15:13–9. 2. Hapuarachchi K, Ahluwalia R, Bowditch M. Neck of femur fractures in the over 90s: a select group of patients who require prompt surgical intervention for optimal results. J Orthop Traumatol. 2014;15:13–9. 3. Lin WT, Chao CM, Liu HC, Li YJ, Lee WJ, Lai CC. Short-term outcomes of hip fractures in patients aged 90 years old and over receiving surgical intervention. PLoS ONE. 2015;10(5):e0125496. Authors’ contributions LW contributed to study conception/design; BOY, SK, PL and JL were involved in data acquisition; DKL, LW, LC, ANK, DJ, RB and COT contributed to data analysis/interpretation; DKL and LW were involved in statistical analysis; and LW, DKL, ANK, DJ, COT and RB contributed to drafting of article. All authors read and approved the final manuscript. 11. Charlson ME, Pompei P, Ales KL, et al. A new method of classifying prog‑ nostic comorbidity in longitudinal studies: development and validation. J Chronic Dis. 1987;40:373–83. 12. Rockwood K, Song X, MacKnight C, Bergman H, Hogan D, McDowell I, et al. A global clinical measure of fitness and frailty in elderly people. CMAJ. 2005;173:489–95. Supplementary Information 5. Holt G, Macdonald D, Fraser M, Reece A. Outcome after surgery for fracture of the hip in patients aged over 95 years. J Bone Jt Surg Br. 2006;88:1060–4. The online version contains supplementary material available at https://doi. org/10.1186/s13018-021-02807-6. The online version contains supplementary material available at https://doi. org/10.1186/s13018-021-02807-6. 6. Vochteloo A, Borger van der Burg B, Tuinebreijer W, de Vries M, Nigge‑ brugge A, Bloem R, et al. Do clinical characteristics and outcome in nonagenarians with a hip fracture differ from younger patients? Geriatr Gerontol Int. 2013;13:190–7. Additional file 1. Detailed demographic data, laboratory findings and clinical information for nonagenarian patients with a hip fracture under‑ going surgical treatment. 7. de Groot R, Nijmeijer W, Folbert E, Vollenbroek-Hutten M, Hegeman J. ‘Nonagenarians’’ with a hip fracture: is a different orthogeriatric treatment strategy necessary?’ Arch Osteoporos. 2020;15:19. Additional file 2. The number of postoperative complications after hip fracture surgery among nonagenarians. Most patients had more than one complication. 8. Mayordomo-Cava J, Abásolo L, Montero-Fernandez N, Ortiz-Alonso J, Vidán-Astiz M, Antonio S-R. Hip fracture in nonagenarians: characteristics and factors related to 30-day mortality in 1177 patients. J Arthroplasty. 2020;35:1186–93. Conclusion Patients aged between 90 and 99 years undergo- ing elective or emergent hip fracture surgery had a 30-day mortality rate of 7.4% and mortality within 30 months after surgery approached 20%. Almost nine out of ten patients developed at least one postoperative Weinberg et al. J Orthop Surg Res (2021) 16:653 Page 10 of 11 Page 10 of 11 of Anesthesiology and Pain Medicine, Dongguk University Ilsan Hospital, Ilsandong‑gu, Goyang 10326, Republic of Korea. complication. The development of any postopera- tive complication was independently associated with a higher mortality throughout the entire observation period, especially when they occurred in the earlier postoperative period. Our findings suggest that strat- egies to minimise postoperative complications may improve postoperative survival in this patient age group. Further study and efforts to reduce complica- tions among nonagenarians having hip fracture surgery are warranted. Received: 11 June 2021 Accepted: 20 October 2021 Weinberg et al. J Orthop Surg Res (2021) 16:653 Am J Med Qual. 2011;26:461–7. 31. Kirkland L, Kashiwagi D, Burton C, Cha S, Varkey P. The Charlson Comor‑ bidity Index Score as a predictor of 30-day mortality after hip fracture surgery. Am J Med Qual. 2011;26:461–7. Weinberg et al. J Orthop Surg Res (2021) 16:653 Page 11 of 11 Page 11 of 11 19. Neuman M, Silber J, Elkassabany N, Ludwig J, Fleisher L. Comparative effectiveness of regional versus general anesthesia for hip fracture sur‑ gery in adults. Anesthesiology. 2012;117:72–92. of 73,557 patients reported to the Norwegian Hip Fracture Register. Bone Jt J. 2019;101-B(9):1129–37. 26. Tran Z, Hsiue PP, Pan C, Verma A, Rahimtoola R, Stavrakis A, et al. Impact of delayed intervention on clinical outcomes following traumatic hip fracture in the elderly: a national analysis. J Orthop. 2021;27:74–8. 20. Liu Y, Peng M, Lin L, Liu X, Qin Y, Hou X. Relationship between Ameri‑ can Society of Anesthesiologists (ASA) grade and 1-year mortality in nonagenarians undergoing hip fracture surgery. Osteoporos Int. 2015;26:1029–33. 27. Halvachizadeh S, Teuber H, Cinelli P, Allemann F, Pape HC, Neuhaus V. Does the time of day in orthopedic trauma surgery affect mortality and complication rates? Patient Saf Surg. 2019;5(13):8. https://doi.org/10. 1186/s13037-019-0186-4. 21. Hip fracture: management. National Institute for Health and Care Excel‑ lence. Clinical guideline [CG124]. Published 22 June 2001, Updated 10 May 2017. https://www.nice.org.uk/guidance/cg124/chapter/recom mendations. 28. Kim RG, An VVG, Petchell JF. Hip fracture surgery performed out-of- hours—a systematic review and meta-analysis. Injury. 2021;52:664–70. 22. Shiga T, Wajima Z, Ohe Y. Is operative delay associated with increased mortality of hip fracture patients? Systematic review, meta-analysis, and meta-regression. Can J Anaesth. 2008;55:146–54. 29. de Leur K, Vroemen J, Vos D, Elmans L, van der Laan L. Outcome after osteosynthesis of hip fractures in nonagenarians. Clin Interv Aging. 2014;9:41–9. 30. Porter C, Moppett I, Juurlink I, Nightingale J, Moran C, Devonald M. Acute and chronic kidney disease in elderly patients with hip fracture: preva‑ lence, risk factors and outcome with development and validation of a risk prediction model for acute kidney injury. BMC Nephrol. 2017;14(18):20. 23. Klestil T, Röder C, Stotter C, Winkler B, Nehrer S, Lutz M, et al. Impact of timing of surgery in elderly hip fracture patients: a systematic review and meta-analysis. Sci Rep. 2018;8(1):13933. https://doi.org/10.1038/ s41598-018-32098-7. 24. Moja L, Piatti A, Pecoraro V, Ricci C, Virgili G, Salanti G, et al. Timing matters in hip fracture surgery: patients operated within 48 hours have better outcomes. A meta-analysis and meta-regression of over 190,000 patients. PLoS ONE. 2012;7(10):e46175. https://doi.org/10.1371/journal.pone.00461 75. 31. Kirkland L, Kashiwagi D, Burton C, Cha S, Varkey P. The Charlson Comor‑ bidity Index Score as a predictor of 30-day mortality after hip fracture surgery. Author details 1 17. Sheehan KJ, Sobolev B, Villán Villán YF, Guy P. Patient and system fac‑ tors of time to surgery after hip fracture: a scoping review. BMJ Open. 2017;7(8):e016939. https://doi.org/10.1136/bmjopen-2017-016939. 1 Department of Anaesthesia, Austin Health, 145 Studley Rd, Melbourne, VIC 3084, Australia. 2 Department of Surgery, The University of Melbourne, Austin Health, Melbourne, VIC 3084, Australia. 3 Department of Cardiology, Austin Health, Melbourne, VIC 3084, Australia. 4 Department of Intensive Care, Austin Health, Melbourne, VIC 3084, Australia. 5 Department of Critical Care, The University of Melbourne, Melbourne, VIC 3084, Australia. 6 Department 18. Keren Y, Sailofsky S, Keshet D, Barak M. The effect of “Out of hours surgery Service” in Israel on hip fracture fixation outcomes: a retrospective analysis. Isr J Health Policy Res. 2017;6(1):27. https://doi.org/10.1186/ s13584-017-0150-7. 18. Keren Y, Sailofsky S, Keshet D, Barak M. The effect of “Out of hours surgery Service” in Israel on hip fracture fixation outcomes: a retrospective analysis. Isr J Health Policy Res. 2017;6(1):27. https://doi.org/10.1186/ s13584-017-0150-7. Weinberg et al. J Orthop Surg Res (2021) 16:653 Publisher’s Note 25. Leer-Salvesen S, Engesæter LB, Dybvik E, Furnes O, Kristensen TB, Gjertsen JE. Does time from fracture to surgery affect mortality and intraoperative medical complications for hip fracture patients? An observational study Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 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https://openalex.org/W4390448200	https://www.qeios.com/read/2OBD72/pdf	English	null	Review of: "Post-Pandemic Reflections from Sub-Saharan Africa: What We Know Now That We Wish We Knew Then"	null	2,023	cc-by	156	Qeios, CC-BY 4.0 · Review, December 30, 2023 Review of: "Post-Pandemic Reflections from Sub-Saharan Africa: What We Know Now That We Wish We Knew Then" Helena Belchior Rocha1 1 ISCTE-Instituto Universitário de Lisboa Helena Belchior Rocha1 Potential competing interests: No potential competing interests to declare. Qeios ID: 2OBD72 · https://doi.org/10.32388/2OBD72 Potential competing interests: No potential competing interests to declare. This article is a very good reflection on the post-pandemic situation in Sub-Saharan Africa. Although it is not an empirical study and only refers to 2 countries, the courage to say things that should be considered in future similar situations is remarkable. Also, separating politics and business from situations of public health, although always linked, is a recommendation that everybody should read about. Therefore, I find this article very pertinent, and, in my perception of what should be a scientific contribution to society, it should be accepted for publication. Best regards, Qeios ID: 2OBD72 · https://doi.org/10.32388/2OBD72 1/1
https://openalex.org/W3026916565	https://www.researchsquare.com/article/rs-4671/v3.pdf?c=1631845941000	English	null	Adapting a nurse-led primary care initiative to cardiovascular disease control in Ghana: a qualitative study	BMC public health	2,020	cc-by	12,381	Adapting a nurse-led primary care initiative to cardiovascular disease control in Ghana: A qualitative study Adapting a nurse-led primary care initiative to cardiovascular disease control in Ghana: A qualitative study Aurelia Abapali Navrongo Health Research Centre Aurelia Abapali Navrongo Health Research Centre Elliasu Yakubu Navrongo Health Research Centre Elliasu Yakubu Navrongo Health Research Centre Edith Dambayi Navrongo Health Research Centre Edith Dambayi Navrongo Health Research Centre Elizabeth Jackson Columbia University Mailman School of Public Health Elizabeth Jackson Columbia University Mailman School of Public Health Raymond Aborigo Navrongo Health Research Centre Raymond Aborigo Navrongo Health Research Centre Denis Awuni Navrongo Health Research Centre Background In Ghana, which has fewer than one physician per 10,000 people [26], the national CHPS program is staffed by CHOs, community health nurses who have been trained for 18 months in the provision of primary health care services and subsequently oriented for six months in community engagement and outreach. Although these CHOs practice predominantly at CHPS clinic compounds, most are also responsible for home visits and community health education. CHPS’ model for community-level primary care in Ghana is the outcome of a process of implementation science that commenced in 1994 and continues to the present [27, 28, 29, 30, 31]. Following the 1978 Declaration of Alma Ata, the Ghanaian government embraced the goal of “Health for All by the Year 2000,” setting out to address primary causes of death and disability, such as infectious diseases of children under age five [32]. To develop a strategy for addressing the “Health for All” goal, an 18 month, three village participatory pilot study was convened to explore culturally compatible means of nurse deployment and support [28, 31]. Based on results of the pilot, a factorial experiment was convened that involved redeploying nurses from sub-district and district clinics to live and work in satellite Community Health Compounds (CHCs) located in remote rural communities and provide door-to-door health screening and primary care to community members as CHOs [33]. Launched as a district-wide trial of the Navrongo Health Research Centre (NHRC), results showed that nurse deployment could reduce childhood mortality by 50% in only three years [33], which led to a national policy to scale-up the Navrongo model in nearly 4,500 CHCs that are dispersed across all districts in Ghana [30, 33, 35]. CHPS operations are supervised by sub-district leaders (SDLs), who are midwives accountable to the Ghana Health Service. Since its inception as a national program in 2000, implementation science has been directed to testing replicability [30], accelerating the pace of scale-up [36], developing emergency referral services [37], improving family planning effectiveness [38], and improving systems support for CHPS operations [36]. The current study is a component of a more general implementation science agenda for improving CHPS functionality and effectiveness [39]. However, further reform and development of CHPS is needed. Background CVD is the leading global cause of morbidity and mortality [1, 2], and the prevalence of CVD is rising in low- and middle-income countries (LMIC) [3, 4, 5, 6]. Non-communicable diseases (NCDs) like CVD caused 70% of all global deaths in 2017, with CVD the leading contributor [2, 7]. These increases are due to demographic, epidemiologic, and nutritional transitions caused by the development and urbanization of LMICs [8]. Consequently, the burden of CVD is most acute in LMICs, where some 74% of global CVD morbidity and mortality occurs [1, 9]. Moreover, in both high- and low-income countries, CVD and other NCDs disproportionately affect the lowest-income populations [10]. In many LMICs, weak health infrastructure further undermines control of these conditions [11]. Recognizing the detrimental impact of NCDs on human health and sustainable development [6, 12], the World Health Organization (WHO) has developed strategies for their control, such as the 2010 Package of Essential NCD Interventions (WHO-PEN) [13]. In 2013, the WHO released a Global Action Plan for the Prevention and Control of NCDs, with a goal to achieve a 25% relative reduction in worldwide premature mortality from the estimated prevalence of NCDs in 2013 by 2025, via six action objectives [14]. Objective 4 of this Global Action Plan calls for adapting existing health systems by improving NCD primary health care prevention and services. The WHO next released the HEARTS intervention package in 2016, which builds on WHO-PEN by detailing cost-effective interventions to prevent and treat CVD and its risk factors using community-based primary health workers [15, 16]. This global agenda is particularly relevant in sub-Saharan Africa, where CVD prevalence is increasing dramatically [17, 18]. Adjusted for age, West African countries such as Ghana have among the highest prevalence of CVD and its risk factors globally [19]. Hypertension, for example, affects up to 32% of adults in Ghana [20] and 24.5% of individuals in the Kassena-Nankana East and West districts [21]. The rising tide of CVD in this region has prompted several initiatives to implement select elements of the WHO-PEN and HEARTS protocols [22, 23], including for the control of hypertension in Ghana. However, none have attempted to build the complete HEARTS CVD care model into an existing primary care system. In regions where physicians are scarce, non-physician health workers (NPHWs) including nurses, pharmacists, and community health officers (CHOs) can effectively provide primary care [24, 25]. Research article 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 BMC Public Health on May 24th, 2020. See the published version at https://doi.org/10.1186/s12889-020-08529-4. Page 1/13 Page 1/13 Page 1/13 Abstract Background : Cardiovascular Disease (CVD) is a growing cause of morbidity and mortality in Ghana, where rural primary health care is provided mainly by the Community-based Health Planning and Services (CHPS) initiative. CHPS locates nurses in community-level clinics for basic curative and preventive health services and provides home and outreach services. But CHPS currently lacks capacity to screen for or treat CVD and its risk factors. Methods : In two rural districts, we conducted in-depth interviews with 21 nurses and 10 nurse supervisors to identify factors constraining or facilitating CVD screening and treatment. Audio recordings were transcribed, coded for content, and analyzed for key themes. Results : Respondents emphasized three themes: community demand for CVD care; community access to CVD care; and provider capacity to render CVD care. Nurses and supervisors noted that community members were often unaware of CVD, despite high reported prevalence of risk factors. Community members were unable to travel for care or afford treatment once diagnosed. Nurses lacked relevant training and medications for treating conditions such as hypertension. Respondents recognized the importance of CVD care, expressed interest in acquiring further training, and emphasized the need to improve ancillary support for primary care operations. Conclusions : CHPS staff expressed multiple constraints to CVD care, but also cited actions to address them: CVD-focused training, provision of essential equipment and pharmaceuticals, community education campaigns, and referral and outreach transportation equipment. Results attest to the need for trial of these interventions to assess their impact on CVD risk factors such as hypertension, depression, and alcohol abuse. Sample We conducted our study in the Kassena-Nankana East and West districts in the UER of Ghana. Study participants were NPHWs working in the CHC setting. Data were collected between April and August 2017. In-depth interviews (IDIs) were conducted at CHCs, and the providers sampled were CHOs and SDLs. Recruitment and Data Collection We used convenience and purposive sampling to select CHOs and SDLs from CHCs throughout the study area. We purposefully contacted respondents across a geographically diverse list of CHPS locations to minimize bias. We excluded providers who were not fluent in English. We recruited NPHWs through letters to the Kassena-Nankana East and West districts’ health directorate and SDLs, as well as through a stakeholder engagement meeting at the NHRC, both of which described the aims of the study to prospective participants. We planned to recruit 20 NPHWs to allow for adequate data saturation [54, 55], as previous work suggests that new themes emerge infrequently after analysis of twelve qualitative interviews, and that these subsequent themes tend to be variations on existing themes rather than novel ones [56]. Researchers include research staff at the NHRC with experience living and conducting field research in the community served [AA, EY, ED, RA, DA, EN, AO], researchers at the University of Ghana [AB], a physician and medical students at the Icahn School of Medicine at Mount Sinai [DH, LH, JF], and researchers at Columbia University [EJ, JP]. Both female [LH, AA, ED, EJ] and male [JF, EY, RA, DA, EN, AO, AB, DH] researchers participated in the study. All participants had baccalaureate degrees, and several had additional professional or graduate degrees. y [ ], p y [ , , ], researchers at Columbia University [EJ, JP]. Both female [LH, AA, ED, EJ] and male [JF, EY, RA, DA, EN, AO, AB, DH] researchers participated in the study. All participants had baccalaureate degrees, and several had additional professional or graduate degrees. The semi-structured IDI guides were designed to evaluate providers’ clinical experiences with CVD, barriers to CVD care, training of CHOs, health literacy, community CVD burden, CVD risk factors, CVD prevention and treatment, CHPS resources, and CHO capabilities, as well as their suggestions for improvement. The final IDI guides can be found in the appendices. Researchers [LH, JF, AA, EY, ED] conducted the interviews in English after obtaining written consent. Background Since the conception of CHPS, CVD has emerged as a major cause of morbidity and mortality in Ghana [40, 41], and CHPS compounds designed primarily for management of maternal health and childhood illnesses remain poorly equipped to respond to this epidemic. Moreover, the feasibility of leveraging the CHPS model of nurse-led preventive and curative primary care for CVD and other NCDs has yet to be established as a coherent component of their work. Page 2/13 Recent work demonstrates that NPHWs are effective in the assessment and treatment of CVD and other NCDs. CHOs in multiple settings can screen patients for CVD risk with similar accuracy to nurses and doctors [42], which can improve identification of patients at high risk compared to usual care [43]. Furthermore, recent systematic reviews demonstrate that NPHWs can prevent and treat CVD by prescribing medications for risk factors such as hypertension and diabetes [44, 45, 46]. Our study seeks to explore perceptions of NPHWs regarding their capacity to manage CVD and barriers to implementing the WHO HEARTS package at CHPS facilities in the Upper East Region (UER) of Ghana. Presently, CHPS nurses do not manage CVD, despite working in areas where CVD risk factors such as hypertension [47] and obesity [41] are increasingly common. Study Design and Theoretical Framework This qualitative research is a component of a mixed-methods implementation science study that aims to develop a protocol for leveraging CHPS nurses to provide CVD care through the WHO-PEN protocol. Our previous work aimed to quantify and trend the burden of CVD mortality among adults in the UER as a function of age, gender, and socioeconomic status to identify high-risk groups [48]. We then undertook the current qualitative evaluation of barriers to provision and acquisition of CVD care at CHPS facilities to inform the practical design of a modified CVD prevention program in this region. Due to the lack of baseline data on perceptions of CVD in this population, we employed grounded theory to identify codes and themes. Briefly, grounded theory expressly avoids a priori theoretical frameworks for analyzing data, and instead involves collecting data first and then subsequently building codes and themes “grounded in data systemically gathered and analyzed.” [49]. Specifically, we employed a situational analysis approach to grounded theory [50], reviewing these data not as specific elements causing or effecting each other but rather as a contextual whole. This approach allowed our research team to theorize based on the data during and after its analysis, rather than employing an a priori theoretical framework. It also allowed us to examine the data as a whole (its entire discourse, as well as specific statements) [50] to shape theme development and analysis, rather than imposing our own preexisting beliefs and assumptions on individual fragments or narratives. Because this analytic approach focused heavily on social and cultural context (in addition to logistical and biomedical factors), the social-ecological model [51] emerged as optimal for framing these data and their underlying themes. This theoretical framework seeks to understand the multifaceted and interrelated effects of individual and environmental factors, in order to identify opportunities for health promotion [51]. This model aligned best with use of situational analysis because it acknowledges the multiple levels at which societal (and other situational) factors influence individual beliefs and behavior, such as interpersonal, institutional, community, and sociocultural factors, many of which relate to provider and patient beliefs on NCD control in low-income settings [52, 53]. Methods Study Design and Theoretical Framework Sample In accordance with grounded theory, we avoided assigning this or any other model or framework to the data until after coding was complete. We performed all analysis using NVIVO software (version 11) [58]. Sample Care was taken to ensure privacy to allow participants to speak freely and to reduce social desirability bias. We supplemented the IDI guides with questions based upon respondents’ initial responses. No repeat interviews were performed, and no field notes were taken. The IDIs, which ranged from 19 to 49 minutes in length, were audio recorded. Members of the research team transcribed the IDIs, with the quality of transcripts checked by other members of the team. Transcripts were corrected only to better adhere to the content of the audio recordings. Data were deidentified prior to analysis. Page 3/13 We developed codes and themes for the study by iterative review, rooted in the grounded theory approach detailed above [49]. Multiple reviewers [LH, JF] separately reviewed initial transcripts, individually introducing codes. They then met to compare these codes with other reviewers [RA, EJ, DH] in order to agree upon which existing codes best captured the data and to create new codes jointly in order to classify uncoded content, representing constant comparative analysis [49]. After a set of 15 codes emerged, reviewers coded all transcripts. Reviewers [LH, DH] then examined these codes to identify emerging themes and analyze the broader implications of these themes [57]. As described above, our analysis employed the social ecological model, allowing for exploration of the interrelationships between the experiences of individual providers and their broader environmental contexts. In accordance with grounded theory, we avoided assigning this or any other model or framework to the data until after coding was complete. We performed all analysis using NVIVO software (version 11) [58]. We developed codes and themes for the study by iterative review, rooted in the grounded theory approach detailed above [49]. Multiple reviewers [LH, JF] separately reviewed initial transcripts, individually introducing codes. They then met to compare these codes with other reviewers [RA, EJ, DH] in order to agree upon which existing codes best captured the data and to create new codes jointly in order to classify uncoded content, representing constant comparative analysis [49]. After a set of 15 codes emerged, reviewers coded all transcripts. Reviewers [LH, DH] then examined these codes to identify emerging themes and analyze the broader implications of these themes [57]. As described above, our analysis employed the social ecological model, allowing for exploration of the interrelationships between the experiences of individual providers and their broader environmental contexts. Results Our sample included 21 CHOs and 10 SDLs, for a total of 31 participants across 23 CHPS sub-districts. Respondents discussed barriers to CVD care – and solutions to these challenges – across three themes: community demand for CVD care; community access to CVD care; and provider capacitity to render CVD care. A coding tree, which can be found in the appendices, depicts these three themes as well as ten sub-themes – each directly informed by our (fifteen) main codes and their sub-codes. Themes and sub-themes were grounded in the content of the IDIs via those codes, developed in turn by the constant comparative analysis of data detailed above. All analysts found that the sample achieved a high degree of thematic saturation. I. Community Member (Demand-Side) Engagement Barriers to CVD Care This theme refers to challenges NPHWs face with rendering CVD care. These challenges include high risk factor burdens, poor health literacy, and other community member factors. Many participants believe that the communities that they serve experience a high burden of CVD risk factors. These risk factors include stress, alcohol and tobacco use, diet, and sedentary lifestyle. Most SDLs say that stress, particularly family-related stress, is a leading cause of CVD in their communities. Participants pointed to several common stressors, such as family problems, work, overthinking, and emotional triggers. Common family problems included being “abandoned” when one’s children move to cities, being childless and unable to support oneself, having children who are “useless,” bad marriages where one is commonly insulted, and not being able to afford school tuition for children. Respondents spoke often about stress due to the migration of youth to nearby cities and towns. This has created an unprecedented problem in rural Ghana as traditional support for the elderly has declined. These social dynamics are described by an SDL: “Some people have got a lot of problems.… Like you have delivered your children, all of them have run away and left you alone. You are old, and you are still doing everything for yourself. You see, that is a problem that can give you heart disease. And at times, some people don’t even have children. If you don’t have a child, how do, you see, you are old, and you needed somebody to support you, there is nobody to support you. Results So that, all that will lead to heart disease.” (SDL) Participants were also worried about the degree of alcohol consumption that they see in their communities, and its implications for CVD and community health more generally. As one SDL describes, To me, the biggest one is the alcoholism…. Anywhere you move you see that everyone is just drunk, drunk, drun “To me, the biggest one is the alcoholism…. Anywhere you move you see that everyone is just drunk, drunk, drunk.” (SDL) This concern was compounded by the belief that patients are not forthcoming in speaking about their alcohol use with CHOs. Some CHOs have attempted to intervene on patients who use alcohol excessively, either by creating a therapeutic alliance with them or by involving their families, with varying levels of success. Respondents reported that many community members smoke tobacco, and that this behavior is particularly Respondents reported that many community members smoke tobacco, and that this behavior is particularly common among men and youths: “This is a tobacco farming community, and we all know that smoking is a very high-risk factor. And almost all the youth in this community smoke excessively, so actually it’s a problem here. The smoking. Almost all the youth, even teenagers, smoke here.” (CHO) “This is a tobacco farming community, and we all know that smoking is a very high-risk factor. And almost all the youth in this community smoke excessively, so actually it’s a problem here. The smoking. Almost all the youth, even teenagers, smoke here.” (CHO) Several CHOs say that they are encouraged by their SDLs to counsel patients regarding the risks associated with smoking and the benefits of smoking cessation. Some CHOs found, however, that patients often try to conceal their smoking status from NPHWs. Several CHOs say that they are encouraged by their SDLs to counsel patients regarding the risks associated with smoking and the benefits of smoking cessation. Some CHOs found, however, that patients often try to conceal their smoking status from NPHWs. Several CHOs say that they are encouraged by their SDLs to counsel patients regarding the risks associated with smoking and the benefits of smoking cessation. Some CHOs found, however, that patients often try to conceal their smoking status from NPHWs. Finally, numerous participants feel that community members’ diets and sedentary lifestyles put them at high risk for CVD. Results The diets described were heavy in salt, fat, meat, and soda, and deficient in vegetables. Some respondents say that they often counsel patients on the benefits of diet and exercise. One SDL describes that these conversations can be difficult, however, due to conflicting cultural attitudes regarding diet, exercise, and weight: “We don’t see the need to exercise. We don’t see the need to diet. You feel that growing big is something that is good in our society, that’s what they think. When you are big, it’s a sign of good living.” (SDL) “We don’t see the need to exercise. We don’t see the need to diet. You feel that growing big is something that is good in our society, that’s what they think. When you are big, it’s a sign of good living.” (SDL) In addition to having many CVD risk factors, a preponderance of participants feel that community members lack a basic understanding of the symptoms and health consequences of CVD. As a result, individuals who develop CVD symptoms sometimes eschew care and as several respondents describe, Page 4/13 Page 4/13 instead attribute these symptoms to other diseases, such as malaria, or to divine judgment. Many note that due to a combination of these factors, individuals who are afflicted with CVD are often unaware of their illness. instead attribute these symptoms to other diseases, such as malaria, or to divine judgment. Many note that due to a combination of these factors, individuals who are afflicted with CVD are often unaware of their illness. Medical screening of asymptomatic individuals for NCDs is exceedingly rare in Ghana [59], which likely contributes to this low level of health literacy. In fact, many community members are unfamiliar with the concept of preventative screenings and asymptomatic treatment. This lack of familiarity was described by numerous respondents: “It’s lack of knowledge…. If you do not visit them, they don’t know what check-ups are…. So, they sit at home and then develop the pressure without managing it.” (CHO) “Some of them, when it is better, they don’t take the medication. That is one problem we can face. They don’t take the medication because they think they are fine.” (CHO) Several respondents note that once diagnosed with CVD, community members do not feel comfortable discussing their health problems, representing a barrier to care. Results Referral centers are often far away from the communities in which patients live; even when geographically close, many patients lack the means of transport to get there. Some patients, particularly the elderly, require help getting to referral centers, but do not have anyone to accompany them. “Especially, the elderly one. Those who don’t also have someone to support them…. So, if the person cannot walk to Navrongo, there is no money to transport to Navrongo, so the patient will not go.” (CHO) Cost is a large barrier to receiving care at referral centers. Many patients will pay others for the fuel or vehicle needed for transport. But as referral centers often draw from low resource areas, this cost can be too great for some, as outlined by numerous participants: Cost is a large barrier to receiving care at referral centers. Many patients will pay others for the fuel or vehicle needed for transport. But as referral centers often draw from low resource areas, this cost can be too great for some, as outlined by numerous participants: “Sirigu is nearer, but before the person even gets there, maybe that person will need a motor rider, that person will need fuel, and that person has no money. And to talk of Navrongo, when you mention Navrongo, they even get frightened. So actually, it’s very difficult for them.” (SDL) “Sirigu is nearer, but before the person even gets there, maybe that person will need a motor rider, that person will need fuel, and that person has no money. And to talk of Navrongo, when you mention Navrongo, they even get frightened. So actually, it’s very difficult for them.” (SDL) “You know when she came, and I mentioned referral, the mother went back to the house…. I’m sure they have to go to the market to look for the money, but when they get there, by all means, they will not buy medicines, they will have to eat and other things. So, the poverty level here is very high so when you are referring.” (SDL) “You know when she came, and I mentioned referral, the mother went back to the house…. I’m sure they have to go to the market to look for the money, but when they get there, by all means, they will not buy medicines, they will have to eat and other things. Results Participants mentioned patients who do not inform friends and family of their CVD because they do not want to burden them with the costs of their treatment. Others do not want to discuss their CVD because they do not understand the serious implications of their illness. Some CHOs also mentioned stigma surrounding CVD: “They think when they share it out and you also tell others. Maybe this house gets to know that that house has someone suffering from this condition it means they will lose respect or something like that, so they will not even like others to know.” (CHO) Once connected to care, some patients demonstrate poor adherence to CVD treatment. CHOs routinely survey medication adherence both at CHPS compounds and on home visits. They say that community members often become ill because they discontinue their medications when they are feeling better. However, the reasons for community members’ poor adherence are likely multifactorial, including fear of potential side effects, cost, misunderstanding, polypharmacy, and lack of symptoms [60]. II. Poor Access to CVD Care at CHPS This theme refers to community members’ difficulty obtaining care for CVD. Respondents described difficulty due to cost, distance, and other barriers to reaching a CHPS site, independent of the logistics of CHPS itself. CHPS workers say that they most often encounter heart disease patients on home visits, because these patients generally do not come to CHPS to receive care. As described by a CHO: T]hey don’t come into the facility with that condition. Unless when we do home visiting and we identify them. ( Several participants said that patients with heart disease used to come to CHPS compounds more often. However, they do not anymore, because CHPS compounds no longer carry CVD medications. Several participants said that patients with heart disease used to come to CHPS compounds more often. However, they do not anymore, because CHPS compounds no longer carry CVD medications. “Some of the treatments for the other diseases, we don’t have them here. So, somebody will say ‘Why do I waste my time to come to this clinic, come and expose myself to them, after all they are not going to give me any treatment.’.... So, it’s better that I go to Navrongo than come to this facility.” (CHO) At present, CVD care is largely provided at referral centers. However, patients experience barriers to receiving care at these centers. Results So, the poverty level here is very high so when you are referring.” (SDL) “You see, this village like this is a deprived village. They don’t have money. So, some of them, it is really a problem to them.” (CHO) “You see, this village like this is a deprived village. They don’t have money. So, some of them, it is really a pro Respondents describe significant barriers to accessing CVD care, namely CHPS-related limitations, lack of transportation, distance to referral centers, and high costs. As a result of these barriers, many patients with CVD or CVD risk factors such as hypertension are not able to successfully access care. III. Community Health Center (Supply-Side) Barriers to CVD Care Respondents describe significant barriers to accessing CVD care, namely CHPS-related limitations, lack of transportation, distance to referral centers, and high costs. As a result of these barriers, many patients with CVD or CVD risk factors such as hypertension are not able to successfully access care. Respondents describe significant barriers to accessing CVD care, namely CHPS-related limitations, lack of transportation, distance to referral centers, and high costs. As a result of these barriers, many patients with CVD or CVD risk factors such as hypertension are not able to successfully access care. p g g y p high costs. As a result of these barriers, many patients with CVD or CVD risk factors such as hypertension are not able to successfully access care. III. Community Health Center (Supply-Side) Barriers to CVD Care III. Community Health Center (Supply-Side) Barriers to CVD Care Page 5/13 Page 5/13 This theme referred to challenges that CHOs and the CHPS program face with rendering CVD care. We found that these challenges include gaps in training, logistics, and other structural factors. While respondents highlighted strengths with regards to the CHOs’ training, most participants, particularly SDLs, expressed that CHOs lack important knowledge about CVD. CHOs were confident in their knowledge of topics such as CVD risk factors and detection. However, in a quiz administered at 21 CHCs, CHOs showed gaps in knowledge about CVD risk factors and causes, as well as about the diagnosis and treatment of CVD. Due to CHPS’ origins as an organization primarily focused on improving maternal and child health, CVD has not historically been at the core of CHO training. During their schooling, CHOs receive training about CVD that focuses primarily on prevention and diagnosis. “After school scarcely will you open the book again…. But with the refresher training, it reminds you of the things you have forgotten, then you are refreshed to do the work better.” (CHO) “After school scarcely will you open the book again…. But with the refresher training, it reminds you of the things you have forgotten, then you are refreshed to do the work better.” (CHO) As such, almost every CHO said that more in-service trainings would help them better understand and care for CVD. Participants felt that trainings should focus on CVD prevention, symptoms, management, and treatment. In addition to enhanced training, participants expressed that they also require materials including medications, blood pressure monitors, motorbikes, and fuel in order to provide adequate care for CVD and its risk factors at CHPS compounds. Presently, most CHPS compounds do not carry essential medications for CVD risk factors such hypertension, hyperlipidemia, and diabetes. This is because the National Health Insurance Scheme (NHIS) does not reimburse CHPS compounds for disbursing medications such as antihypertensives, statins, and antihyperglycemics. As a result, patients are forced to travel to larger regional health centers for treatment. As outlined previously, because these facilities are farther away and transportation to them is poor, patients often choose not to go, and instead live with untreated CVD or CVD risk factors. For these reasons, participants feel that NHIS’ decision to not reimburse CHPS for these medications is making their communities sicker. While CHOs may not dispense medications, they are currently able to screen for, diagnose, monitor, and manage straightforward cases of CVD, as well as to refer to regional health centers. Some participants felt that this level of care was not adequate to help patients with conditions such as hypertension. Others say that they turn CVD patients away because they know that they are unable to treat them. At times, patients do not understand the limitations placed on CHPS compounds, and this can be discouraging for CHOs. As one CHO states, “They ask you questions like, ‘Now you come and stand and talk like this. How come when I tell you people, well I’m having BP, my drugs, you tell me I should go to hospital? Then what are you people doing?’ You see that? So sometimes those things discourage us. We feel bad. We just wish that we could be able to do it.” (CHO) Most CHOs want to be able to treat CVD pharmacologically at CHPS compounds. Many participants felt that with more training, CHOs would be capable of treating straightforward CVD cases with medications. Results However, many SDLs felt that this training is insufficient. Further, many CHOs expressed that they had not learned enough about CVD during their training. This sentiment was expressed by an SDL: “[CHO knowledge of heart disease symptoms is] not adequate per my own assessment. Because some of them, they can’t even tell you the treatment you give to maybe a patient with high blood pressure, diabetic, stroke.” (SDL). Contributing to these knowledge gaps is lack of exposure. Participants felt that knowledge gained during training is lost over time because CHOs do not often see CVD patients at CHPS facilities. Discussion Most respondents also feel that their facilities also require more BP cuffs, motorbikes, and fuel, for which the HEARTS package suggests potential funding sources [16]. Although CHOs and SDLs voiced concern over community members’ lack of awareness of CVD risk factors despite a high community CVD burden, they expressed optimism that individual counseling coupled with community durbars, radio broadcasts, and informative posters in local dialects could disburse health information. The HEARTS package provides a protocol that can be adapted to guide individual counseling, durbars, and other modes of communication to counsel patients on behaviors that promote cardiovascular health [16]. The distance and cost associated with travel to referral centers are major barriers to seeking care for many patients with CVD. Atuoye et al. (2015) underscore the need for public policy to address rural transport problems in Ghana in order to improve health and call for sustainable transport services driven by community participation [69, 70]. Access to hospital-level CVD management could also be increased through further expansion of the National Ambulance Service [71]. Lastly, CHOs and SDLs spoke to the high prevalence of CVD risk factors such as stress, alcohol use, smoking, poor diet, and sedentary lifestyle in their communities. Each of these risk factors contributes to multiple other chronic conditions: for example, anxiety is highly associated with depression, musculoskeletal pain, and gastrointestinal disorders in addition to CVD [72]; alcohol and other substance abuse similarly contributes to each of these conditions. Future work to target these underlying CVD risk factors through CHPS using the approaches proposed in this paper – namely improved access to medications, provider training, community member education, and transportation – could therefore have downstream effects on other diseases processes as well. Previous interventions in Ghana have addressed CVD risk factors in isolation, such as hypertension, but none have successfully impacted multiple risk factors [46, 60]. A pilot initiative to counsel vulnerable community members on these factors – for example, through counseling on alcohol and tobacco cessation – could therefore impact outcomes such as depression in addition to blood pressure control, especially if supplemented by medication, community outreach, and improved care accessibility as described above. To this end, we are currently developing an intervention based on the results of this study involving the joint screening and treatment of depression and hypertension by NPHWs within the CHPS system. Discussion To our knowledge, our study is the first to explore the qualitatively perceptions of nurses within the CHPS program regarding implementation of the HEARTS protocol in Ghana. Our use of IDIs allowed for a nuanced understanding of barriers to implementing the HEARTS package, which include inadequate CHO training; poor health literacy among community members; lack of access to CVD medications and other necessary resources; a high burden of CVD risk factors; and difficulty accessing CHPS compounds and referral centers. Although previous pilot studies have implemented elements of the HEARTS protocol through CHPS [61], our work suggests means to adapt CHPS itself from a program primarily focused on infectious disease and child and maternal health to that which functions across the lifespan to accommodate all aspects of this integrated CVD care model, as well as the care of other NCDs. These approaches include, among others, improved 1) access to essential medications; 2) provider training; 3) community member education; and 4) means of transportation to referral sites, as detailed below. While this study specifically examined the perceptions of NPHWs in the UER of Ghana, its findings regarding barriers and facilitators to implementing the HEARTS package have implications for other LMICs and low-resource settings. This work also supports a growing body of evidence [42, 43, 44, 45] that the scope of NPHWs’ practice can effectively be expanded to address CVD and other NCDs in areas of physician scarcity, building on the demonstrated efficacy of NPHWs in treating and preventing infectious diseases [62, 63, 64]. CHPS’ current infrastructure struggles to provide the 23 medications that HEARTS considers essential for primary care CVD interventions [16], in large part due to lack of timely reimbursement by the NHIS; furthermore, CHOs are prohibited from prescribing them when available. However, CHOs expressed confidence in their capacity to prescribe and monitor patients taking these medications, consistent with evidence from other contexts that nurses can safely render such care [65, 66, 67, 68]. Abdel-All et al. (2017) found that training was effective in raising pre-training scores in assessments of knowledge about CVD, with moderate decline in scores six to eight months post-training, which could be minimized with refresher trainings [65]. The HEARTS package provides evidence-based resources that should be used to design such trainings [16]. Further work should test this hypothesis by permitting CHOs to prescribe hypertension medications under physician oversight. “After school scarcely will you open the book again…. But with the refresher training, it reminds you of the things you have forgotten, then you are refreshed to do the work better.” (CHO) These are some of the things hampering the work. If those things are improved, it will go a long way to help.” (CHO) “After school scarcely will you open the book again…. But with the refresher training, it reminds you of the things you have forgotten, then you are refreshed to do the work better.” (CHO) They feel that this effort would be beneficial to patients by making these medications more accessible. “They can treat by sometimes giving some of the health centers, the smaller clinics, the opportunity to also treat…. Because some of us, we don’t have access to treatments. We only monitor, refer. And here, like this one, you tell them to go to Navrongo, it’s like you cursed them or something. They don’t want to go.” (CHO) Many CHOs want a return to the “old system” in which NHIS reimbursed CHPS compounds for CVD medications. They feel that this would reduce the burden on low-income patients and increase medication adherence. “Most of the hypertensive cases who are put on drugs as they grow older, some today is on the drug, tomorrow the person is not. When asked why, they go ‘Oh, I don’t have money to buy.’ But if GHS is able to come out with drugs, that will cover health insurance, people will get with it.” (CHO) In addition to medications, most participants claimed that their facilities lack equipment and materials necessary for managing CVD. Notably, they held that more blood pressure apparatuses are needed for adequate care. Several CHOs reported one apparatus was available at their facility; some CHPS compounds had none. Their absence prevents routine blood pressure surveillance during outreach and home visits, necessitating referral of patients to hospitals or CHCs for routine blood pressure readings. Non-adherence with such referrals thus impedes the provision of even the most basic of CVD interventions, as described by a SDL: “Even to go around with the common BP apparatus, they don’t have. For the facility, we have one and it breaks down very often. So, they don’t have. They only refer per symptoms…. It’s the BP apparatus that you use to measure, and if it’s broken down, you can’t tell whether the person’s BP has risen, Page 6/13 Page 6/13 Page 6/13 whether it’s gone down.” (SDL) Participants also lack the motorbikes and fuel required to go on home visits and to take patients to the hospital. Some say that due to NHIS underfunding, they currently use their personal motorbikes and fuel to transport patients, because many patients cannot afford their own transport: “We go into the communities such cases, we interact with people, attend to old, the aged and all those things. Now we don’t have motor bikes, we don’t have fuel…. Discussion This work could generate models for integrated chronic disease care beyond the HEARTS protocol, as a framework potentially adaptable to contexts outside the CHPS model. Our approach has several limitations. While we sought to minimize bias in interview analysis through codes derived from the independent agreement of four analysts and validated by achievement of thematic saturation, our findings are subjective given the qualitative nature of this study. Additionally, our data analysts and several interviewers were external observers from the United States with prior beliefs regarding CVD care – especially regarding CVD treatment and risk factor management – that may have biased interpretation. Although all participants spoke English, not all learned English as a first language, and as such, language barriers may have impeded the accurate gathering of data. As this study represents only 31 participants from across the Kassena Nankana East and West districts, NPHWs who were not interviewed may have differing perceptions that were not captured. While NPHWs from Page 7/13 Page 7/13 Page 7/13 all CHCs in the Kassena Nankana East and West districts were contacted for participation in this study, the group that chose to participate may non- randomly differ from non-participants. We sought to ask broad, open-ended questions about CVD care and its barriers, but our interviews omitted some potentially relevant topics, such as how CVD care was previously provided in the region, the role of health volunteers in providing counseling about CVD, and community members’ behaviors related to CVD. Nevertheless, our findings represent an important first evaluation of the perception of NPHWs regarding barriers to implementing the WHO HEARTS package at CHPS facilities in the UER of Ghana. all CHCs in the Kassena Nankana East and West districts were contacted for participation in this study, the group that chose to participate may non- randomly differ from non-participants. We sought to ask broad, open-ended questions about CVD care and its barriers, but our interviews omitted some potentially relevant topics, such as how CVD care was previously provided in the region, the role of health volunteers in providing counseling about CVD, and community members’ behaviors related to CVD. Nevertheless, our findings represent an important first evaluation of the perception of NPHWs regarding barriers to implementing the WHO HEARTS package at CHPS facilities in the UER of Ghana. Conclusion We found that NPHWs in the UER of Ghana hold many strong beliefs regarding how to reform the CHPS program to provide care for CVD. While NPHWs voiced many concerns regarding obstacles to providing such care, they also listed several feasible interventions to address them, including enhanced training of CHOs and education of community members about CVD, more accessible methods for disbursement of CVD medications, increased funding for equipment, and improved means of transportation to referral sites – each of which could improve CHPS’ care for CVD as well as other chronic diseases. Although further formative work to develop these solutions remains, an intervention to address these gaps in care could constitute a novel step towards adapting HEARTS to the CHPS model in the UER of Ghana. List Of Abbreviations List Of Abbreviations CHC: Community health compound CHO: Community health officer CHPS: Community-Based Health Planning and Services CVD: Cardiovascular disease IDI: In-depth interview LMIC: Low- and middle-income country NCD: Non-communicable disease NHIS: National Health Insurance Scheme NHRC: Navrongo Health Research Centre NPHW: Non-physician health worker PEN: Package of Essential Non-Communicable Disease Interventions SDL: Sub-district leader UER: Upper East Region WHO: World Health Organization CHC: Community health compound CHO: Community health officer WHO: World Health Organization Declarations Ethics Approval and Consent to Participate Authors’ Contributions AO, AB, JP, and DH conceived of the study and developed the interview instrument. LH, JF, AA, EY, and ED conducted the qualitative interviews, with DA acting as an advisor. LH and JF coded the interview transcripts and performed a constant comparative analysis, with assistance from EJ, RA, and DH. LH wrote the manuscript, and made revisions based on comments from JF, ED, RA, EN, JP, and DH. All authors reviewed and approved of the final manuscript. Acknowledgements Acknowledgements We acknowledge and appreciate our study participants, without whom this study would not be possible, as well as research staff from the Navrongo Health Research Centre for their academic contributions and assistance with study coordination. We acknowledge and thank Khadija Jones for her help with manuscript formatting, and Allison Squires for her expertise regarding qualitative research theory and methods. We also acknowledge the Fogarty International Center at the US National Institutes of Health, and Teva Pharmaceuticals, for their financial support for this work. References 1. Cardiovascular diseases [Internet]. Geneva: World Health Organization; 2017 [cited 2018 Mar 18]. Available from: http://www.who.int/mediacentre/factsheets/fs317/en/. 2. Noncommunicable Diseases Progress Monitor [Internet]. 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https://openalex.org/W2609006221	https://europepmc.org/articles/pmc5401914?pdf=render	English	null	Comparative Genomic Analysis of the Class Epsilonproteobacteria and Proposed Reclassification to Epsilonbacteraeota (phyl. nov.)	Frontiers in microbiology	2,017	cc-by	17,433	Comparative Genomic Analysis of the Class Epsilonproteobacteria and Proposed Reclassiﬁcation to Epsilonbacteraeota (phyl. nov.) David W. Waite1, Inka Vanwonterghem1, Christian Rinke1, Donovan H. Parks1, Ying Zhang2, Ken Takai3, Stefan M. Sievert4, Jörg Simon5, Barbara J. Campbell6, Thomas E. Hanson7, Tanja Woyke8, Martin G. Klotz9,10 and Philip Hugenholtz1* 1 Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia, 2 Department of Cell and Molecular Biology, College of the Environment and Life Sciences, University of Rhode Island, Kingston, RI, USA, 3 Department of Subsurface Geobiological Analysis and Research, Japan Agency for Marine-Earth Science and Technology, Yokosuka, Japan, 4 Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA, USA, 5 Microbial Energy Conversion and Biotechnology, Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany, 6 Department of Biological Sciences, Life Science Facility, Clemson University, Clemson, SC, USA, 7 School of Marine Science and Policy, College of Earth, Ocean, and Environment, Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA, 8 Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA, 9 Department of Biology and School of Earth and Environmental Sciences, Queens College of the City University of New York, New York, NY, USA, 10 State Key Laboratory of Marine Environmental Science, Institute of Marine Microbes and Ecospheres, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China ORIGINAL RESEARCH published: 24 April 2017 doi: 10.3389/fmicb.2017.00682 Edited by: Svetlana N. Dedysh, Winogradsky Institute of Microbiology (RAS), Russia Reviewed by: Barny Whitman, University of Georgia, USA Craig Lee Moyer, Western Washington University, USA The Epsilonproteobacteria is the ﬁfth validly described class of the phylum Proteobacteria, known primarily for clinical relevance and for chemolithotrophy in various terrestrial and marine environments, including deep-sea hydrothermal vents. As 16S rRNA gene repositories have expanded and protein marker analysis become more common, the phylogenetic placement of this class has become less certain. A number of recent analyses of the bacterial tree of life using both 16S rRNA and concatenated marker gene analyses have failed to recover the Epsilonproteobacteria as monophyletic with all other classes of Proteobacteria. In order to address this issue, we investigated the phylogenetic placement of this class in the bacterial domain using 16S and 23S rRNA genes, as well as 120 single-copy marker proteins. Single- and concatenated-marker trees were created using a data set of 4,170 bacterial representatives, including 98 Epsilonproteobacteria. Phylogenies were inferred under a variety of tree building methods, with sequential jackkniﬁng of outgroup phyla to ensure robustness of phylogenetic afﬁliations under differing combinations of bacterial genomes. Based on the assessment of nearly 300 phylogenetic tree topologies, we conclude that the continued inclusion of Epsilonproteobacteria within the Proteobacteria is not warranted, and that this group should be reassigned to a novel phylum for which we propose the name Epsilonbacteraeota (phyl. nov.). We further recommend the reclassiﬁcation of the order Desulfurellales (Deltaproteobacteria) to a novel class within this phylum and a number of subordinate changes to ensure consistency with the genome-based phylogeny. Phylogenomic analysis of 658 genomes belonging to the newly proposed Epsilonbacteraeota suggests that the ancestor of this phylum *Correspondence: Philip Hugenholtz p.hugenholtz@uq.edu.au Specialty section: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology Citation: Waite DW, Vanwonterghem I, Rinke C, Parks DH, Zhang Y, Takai K, Sievert SM, Simon J, Campbell BJ, Hanson TE, Woyke T, Klotz MG and Hugenholtz P (2017) Comparative Genomic Analysis of the Class Epsilonproteobacteria and Proposed Reclassiﬁcation to Epsilonbacteraeota (phyl. nov.). Front. Microbiol. 8:682. doi: 10.3389/fmicb.2017.00682 April 2017 \| Volume 8 \| Article 682 1 Frontiers in Microbiology \| www.frontiersin.org Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. was an autotrophic, motile, thermophilic chemolithotroph that likely assimilated nitrogen from ammonium taken up from the environment or generated from environmental nitrate and nitrite by employing a variety of functional redox modules. The emergence of chemoorganoheterotrophic lifestyles in several Epsilonbacteraeota families is the result of multiple independent losses of various ancestral chemolithoautotrophic pathways. Our proposed reclassiﬁcation of this group resolves an important anomaly in bacterial systematics and ensures that the taxonomy of Proteobacteria remains robust, speciﬁcally as genome-based taxonomies become more common. Keywords: Epsilonproteobacteria, taxonomy, classiﬁcation, genome, phylogenomics, Epsilonbacteraeota, evolution Keywords: Epsilonproteobacteria, taxonomy, classiﬁcation, genome, phylogenomics, Epsilonbacteraeota, evolution INTRODUCTION et al., 1994; Eisen, 1995; Ludwig et al., 1995; Klenk et al., 1999), while others did not resolve this association at all (Gupta, 2000; Reysenbach et al., 2000; Sheridan et al., 2003; Yarza et al., 2014). More recent phylogenomic evidence based on multiple marker proteins and greater outgroup representation have largely failed to recover the Epsilonproteobacteria as reproducibly monophyletic with the rest of the Proteobacteria, further suggesting that taxonomic revision is required at the phylum level (Wu et al., 2009; Di Rienzi et al., 2013; Dodsworth et al., 2013; McLean et al., 2013; Rinke et al., 2013; Zhang and Sievert, 2014; Hug et al., 2016; Yeoh et al., 2016). The class Epsilonproteobacteria currently comprises two orders, Campylobacterales and Nautiliales, encompassing a number of species with ambiguous placement. Particularly problematic are the genera Nitratifractor and Nitratiruptor. The SILVA and LPSN taxonomies (Quast et al., 2013; Parte, 2014; Yilmaz et al., 2014) currently list these organisms as members of the Nautiliaceae, but this classiﬁcation is not universally accepted (Nakagawa and Takai, 2014) and phylogenetic evidence suggests that they may represent novel families (Nakagawa and Takaki, 2009; Anderson et al., 2011). In a review of the Epsilonproteobacteria, Campbell et al. (2006) used the placeholder family Thiovulgaceae to group the Nitratifractor with Sulfurovum and Sulfurimonas, and the family Nitratiruptoraceae to describe the divergent nature of Nitratiruptor from other families. An identical taxonomy was proposed with recent phylogenomic evidence (Zhang and Sievert, 2014), which also revealed a stable monophyletic clade of Epsilonproteobacteria with the deltaproteobacterial genus Hippea, a member of the order Desulfurellales. Both genera belonging to this order, Desulfurella and Hippea, have low 16S rRNA gene sequence identity to other members of the Deltaproteobacteria and frequently form a clade with members of the Epsilonproteobacteria (Haddad et al., 1995; Moyer et al., 1996; Miroshnichenko et al., 2002; Kersters et al., 2006; Florentino et al., 2016), suggesting that they should be transferred from the Deltaproteobacteria to this group. The Epsilonproteobacteria were ﬁrst described in the early 1990s as the ﬁfth subclass of the Proteobacteria (Tenover et al., 1992) and subsequently assigned class status within this phylum (Garrity et al., 2005). The group is widely known for its pathogenic genera Campylobacter, Helicobacter and, to a lesser extent Arcobacter. 1https://gold.jgi.doe.gov/ Frontiers in Microbiology \| www.frontiersin.org INTRODUCTION However, other members of this class are known to play ecologically important roles across a diverse range of environments in which they thrive as mesophiles or moderate thermophiles (Nakagawa and Takaki, 2009). Epsilonproteobacteria are important chemolithotrophic primary producers in deep-sea hydrothermal vent systems, where they are often the dominant bacterial lineage in vent plumes and deposits (Huber et al., 2010; Flores et al., 2011), and surrounding microbial mats (Moussard et al., 2006; Opatkiewicz et al., 2009; Rassa et al., 2009). On vent chimneys, Epsilonproteobacteria can account for up to 85% of the microbial biomass (Nakagawa et al., 2006). Their metabolic capacity to perform sulfur oxidation coupled to N-oxide reduction while ﬁxing carbon via the reverse TCA cycle (Hügler et al., 2005; Campbell et al., 2009) enables them to be early colonizers of uninhabited vent ecosystems (Alain et al., 2004; Campbell et al., 2006; Gulmann et al., 2015). Non- pathogenic relatives of Campylobacter such as Sulfurospirillum and Thiovulum are often detected in sulﬁde-rich sediments while others show an aﬃnity for hydrocarbon-rich environments (Hubert et al., 2012). Host-association is also common in this class: Campylobacter, Helicobacter, and some Arcobacter species are known opportunistic pathogens of vertebrates while members of the Caminibacter, Nautilia, and Sulfurospirillum have been reported in association with deep-sea hydrothermal vent fauna. Recent metatranscriptomic data highlighted the role of Sulfurimonas-like bacteria in hydrogenase-driven sulfur oxidation and denitriﬁcation on the gills of a deep vent sea snail (Sanders et al., 2013). The Epsilonproteobacteria were ﬁrst described in the early 1990s as the ﬁfth subclass of the Proteobacteria (Tenover et al., 1992) and subsequently assigned class status within this phylum (Garrity et al., 2005). The group is widely known for its pathogenic genera Campylobacter, Helicobacter and, to a lesser extent Arcobacter. However, other members of this class are known to play ecologically important roles across a diverse range of environments in which they thrive as mesophiles or moderate thermophiles (Nakagawa and Takaki, 2009). Epsilonproteobacteria are important chemolithotrophic primary producers in deep-sea hydrothermal vent systems, where they are often the dominant bacterial lineage in vent plumes and deposits (Huber et al., 2010; Flores et al., 2011), and surrounding microbial mats (Moussard et al., 2006; Opatkiewicz et al., 2009; Rassa et al., 2009). On vent chimneys, Epsilonproteobacteria can account for up to 85% of the microbial biomass (Nakagawa et al., 2006). Keywords: Epsilonproteobacteria, taxonomy, classiﬁcation, genome, phylogenomics, Epsilonbacteraeota, evolution Genome Data An ingroup comprising 619 Epsilonproteobacteria, four Hippea species and Desulfurella acetivorans were obtained from NCBI RefSeq and GenBank (Supplementary Table S1), and 33 Epsilonproteobacteria population genomes (Supplementary Table S2) were recovered from public metagenomic datasets2. The genome of H. thermophila was sequenced using the Illumina HiSeq 2500 platform (2 × 150 bp chemistry). Raw sequence data (2.4 M reads) were quality ﬁltered using trimmomatic v0.33 (Bolger et al., 2014) in paired end mode, requiring an average quality score of Q ≥20 over a sliding window of four bases, and a minimum sequence length of 36 nucleotides. A draft genome was assembled using SPAdes v3.8.1 (Bankevich et al., 2012) with a kmer size range of 35–75 (step size = 4) and automatic coverage cutoﬀ. The genome was then scaﬀolded using FinishM v0.0.93, and scaﬀolds assessed for assembly errors using ReﬁneM v0.0.134. Maximum likelihood inference of the multiple sequence alignment was performed using the Jones-Taylor-Thornton (JTT), Whelan and Goldman (WAG), and Le and Gascuel (LG) models for amino acid evolution with gamma distributed rate heterogeneity (+0) (Jones et al., 1992; Whelan and Goldman, 2001; Le and Gascuel, 2008) implemented in FastTree v2.1.9 (Price et al., 2009). Neighbor joining (NJ) was performed using the Jukes-Cantor and Kimura distance corrections, and with an uncorrected distance matrix implemented in Clearcut v1.0.9 (Sheneman et al., 2006). Under each model/correction, tree building was performed with all sequences included, then once with each phylum or singleton lineage removed, with the exception of Proteobacteria and ingroup genomes (a total of 186 trees). All trees were bootstrap-resampled 100 times to assess the stability of tree topologies. Robustness and reproducibility of the tree topology and association between the Epsilonproteobacteria, Desulfurellales, and Proteobacteria was assessed by manual examination of all tree topologies in ARB (Ludwig et al., 2004). Three partial Thioreductor genomes were obtained by single cell genome sequencing (Supplementary Table S2). Raw sequence data (41 M reads) were quality ﬁltered as per H. thermophila. Quality-ﬁltered sequences were digitally normalized using khmer v2.0 (Crusoe et al., 2015) using the default two-pass approach. Normalized sequences were assembled using SPAdes, and the resulting contigs were scaﬀolded and reﬁned using ReﬁneM and FinishM as for H. thermophila. The taxonomic identity of each Thioreductor genome was conﬁrmed by screening high-quality reads for 16S rRNA gene sequence fragments using GraftM5. INTRODUCTION Their metabolic capacity to perform sulfur oxidation coupled to N-oxide reduction while ﬁxing carbon via the reverse TCA cycle (Hügler et al., 2005; Campbell et al., 2009) enables them to be early colonizers of uninhabited vent ecosystems (Alain et al., 2004; Campbell et al., 2006; Gulmann et al., 2015). Non- pathogenic relatives of Campylobacter such as Sulfurospirillum and Thiovulum are often detected in sulﬁde-rich sediments while others show an aﬃnity for hydrocarbon-rich environments (Hubert et al., 2012). Host-association is also common in this class: Campylobacter, Helicobacter, and some Arcobacter species are known opportunistic pathogens of vertebrates while members of the Caminibacter, Nautilia, and Sulfurospirillum have been reported in association with deep-sea hydrothermal vent fauna. Recent metatranscriptomic data highlighted the role of Sulfurimonas-like bacteria in hydrogenase-driven sulfur oxidation and denitriﬁcation on the gills of a deep vent sea snail (Sanders et al., 2013). While the Epsilonproteobacteria constitute a stable monophyletic group within the bacterial tree of life, a number of studies suggest that they do not reproducibly aﬃliate with other Proteobacteria, with the exception of the Desulfurellales, which are presently classiﬁed as an order of the Deltaproteobacteria (see below). Early conserved marker gene-based studies showing Epsilonproteobacteria branching immediately basal to other Proteobacteria often had inadequate outgroups and/or bootstrap values supporting this placement (Tenover et al., 1992; Trust The widespread adoption of high-throughput sequencing technologies has resulted in the number of sequenced genomes from bacteria exceeding 70,000 in recent years (Mukherjee et al., 2017)1. Additionally, advances in obtaining high-quality draft genomes from metagenomic data (population genomes; April 2017 \| Volume 8 \| Article 682 2 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. Wrighton et al., 2012; Albertsen et al., 2013) and single cells (Marcy et al., 2007; Rinke et al., 2013) greatly augments genomic coverage of microbial diversity and provides the opportunity to supplant the 16S rRNA gene as the basis for microbial classiﬁcation. Here, we report a phylogenomic characterization of 624 publicly available Epsilonproteobacteria and Desulfurellales isolate genomes supplemented with 33 Epsilonproteobacteria population genomes. As part of this study, we also sequenced a near-complete genome of Hydrogenimonas thermophila, and analyzed three partial genomes of single cells belonging to the genus Thioreductor. Based on our results, we propose reclassifying the Epsilonproteobacteria and Desulfurellales as a new phylum, the Epsilonbacteraeota (phyl. nov.), together with a number of subordinate changes and additions at the order and family levels. NCBI. INTRODUCTION Completeness and contamination of all genomes was estimated using CheckM v1.0.6 with default settings (Parks et al., 2015). Phylogenetic Inference Ingroups for phylogenetic analyses were selected from the 653 Epsilonproteobacteria (including H. thermophila and the 33 population genomes) and ﬁve Desulfurellales genomes. The three partial Thioreductor genomes were only included in a reduced concatenated gene analysis due to their low estimated completeness (see below). To resolve the placement of the ingroup in the bacterial domain, 98 ingroup genomes representative at the species-level were selected and combined with the 4,072 outgroup genomes described above. Phylogenetic inference was performed on the 4,170 genomes using a concatenation of 120 conserved protein marker sequences (Ormerod et al., 2016). Protein sequences in each genome were identiﬁed and aligned to reference alignments using hmmer v3.1 (Eddy, 1998). Aligned markers were then concatenated and poorly aligned regions removed using Gblocks v0.91b (Castresana, 2000; Talavera and Castresana, 2007). 2http://gtdb.ecogenomic.org/downloads 3https://github.com/wwood/ﬁnishm 4https://github.com/dparks1134/ReﬁneM 5https://github.com/geronimp/graftM Genome Data Putative 16S rRNA gene fragments were aligned using the SINA web aligner (Pruesse et al., 2012) and inserted into the SILVA SSU non-redundant database v123.1 using the parsimony insertion tool in ARB. To resolve the internal structure of Epsilonbacteraeota relationships, a slightly larger data set of 110 ingroup genomes were selected (106 Epsilonproteobacteria, four Desulfurellales) and maximum likelihood inference performed using RAxML v8.1.11 (Stamatakis, 2014). An outgroup of 10 genomes representing six bacterial phyla, including Proteobacteria and Aquiﬁcae was used to root this tree. Phylogenetic inference was performed with the WAG+0 model and 100 bootstrap resamples. To further evaluate the robustness of Epsilonbacteraeota relationships, single gene trees were constructed using FastTree with the WAG+0 model on the individual protein alignments and tree topologies compared to that of the concatenated alignment using the phytools and ape packages in the R software environment (Paradis et al., 2004; Revell, 2012; R Core Team, 2016). An outgroup of 4,072 publicly available genomes representing unique species of 24 bacterial phyla were also obtained from 2http://gtdb.ecogenomic.org/downloads 3https://github.com/wwood/ﬁnishm 4https://github.com/dparks1134/ReﬁneM 5https://github.com/geronimp/graftM Manual inspection of the three partial Thioreductor genomes identiﬁed 14 protein families common to all three. Phylogenetic analysis of Thioreductor was performed using the above set April 2017 \| Volume 8 \| Article 682 Frontiers in Microbiology \| www.frontiersin.org 3 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. of 110 ingroup genomes and associated outgroup, using only these 14 protein markers. Phylogenetic inference was performed using RAxML as described above. To assess the placement of species for which genome data is not available, 16S rRNA gene analysis was performed. Epsilonbacteraeota sequences were obtained from the SILVA Living Tree Project v123 (Yilmaz et al., 2014). As this database does not possess a representative for the genus Thiovulum, a 16S rRNA sequence for this lineage was obtained from NCBI GenBank. Full length 16S rRNA gene sequences of Thiofractor thiocaminus, Candidatus Thioturbo danicus, Cetia paciﬁca, and Thioreductor species were aligned using the SINA web aligner (Pruesse et al., 2012). An outgroup comprising members of the Proteobacteria, Aquiﬁcae, and four other phyla was used to root the tree. The sequence alignment was masked using the LTP 50% SSU conservation ﬁlter prior to tree construction. Phylogenetic inference of the masked alignment was performed using RAxML with the general time reversible model with gamma distributed rate heterogeneity and 1,000 bootstrap resamples. Genome Data A total of 619 Epsilonproteobacteria and ﬁve Desulfurellales genomes were obtained from RefSeq version 76 and GenBank version 213 (Supplementary Table S1). Genomes were assessed for completeness and contamination by scoring the presence of conserved single-copy marker genes within each genome using CheckM (Parks et al., 2015). The median estimated genome completeness for this dataset is 99.4% and the minimum is 81.9%. Genomes were estimated to be less than 10% contaminated, with all but eight under 5% (Supplementary Table S1). The taxonomic annotation of the type strain Campylobacter geochelonis (GCA_900063025.1) was manually modiﬁed as the NCBI record for this genome incorrectly labels it as C. fetus (Piccirillo et al., 2016). Thirty-three draft population genomes (median completeness 93.8%, contamination 1.1%) belonging to the Epsilonproteobacteria were recovered from publicly available Sequence Similarity Comparisons q y p In order to compare our taxonomic proposals to previously proposed sequence similarity-based thresholds for taxonomic ranking, we performed 16S rRNA gene sequence and amino acid identity (AAI) comparisons between members of reclassiﬁed or newly proposed families. 16S rRNA gene sequences belonging to Epsilonproteobacteria and Desulfurellales type strains were extracted from the SILVA Living Tree Project v123 database. Sequences were aligned and hypervariable regions removed using the Lane mask (Lane, 1991). Pairwise sequence distances were calculated for 16S rRNA sequences belonging to the same family but diﬀerent genera using mothur v1.39.1 (Schloss et al., 2009). AAIs of all best bi-directional diamond (Buchﬁnk et al., 2015) hits between pairwise comparisons of the 110 ingroup genomes were made using CompareM v0.0.216. AAI scores were obtained for genome pairs belonging to the same family, but diﬀerent genera. Sequence similarity results for each family were visualized using R and compared to previously proposed taxonomic rank boundaries (Konstantinidis and Tiedje, 2005; Yarza et al., 2014). 6https://github.com/dparks1134/CompareM 7https://github.com/Ecogenomics/mingle 8gtdb.ecogenomic.org Genome Data Short sequences (<1,000 bp) belonging to Candidatus Thioturbo danicus and Thioreductor sp. Shim25-G were inserted into the resulting topology using the ARB parsimony insert tool. All tree ﬁgures were edited for publication in Inkscape v0.48. et al., 2016). Genomes were partitioned into host-associated or ‘environmental’ and indicator analysis was performed using the package indicspecies (De Cáceres and Legendre, 2009; De Cáceres et al., 2011). KO groups that were signiﬁcantly associated with either the host-associated or environmental lifestyle were grouped into their functional pathway, and ﬁtted to the PCA ordination using the envﬁt function in vegan. Additional annotation of hydrogenase enzymes was performed using BLAST (Altschul et al., 1990) against a manually curated database (Greening et al., 2016). Homologous sequences were deﬁned as greater than 30% AAI over at least 70% of the target protein length. Annotation of the reference proteins ACM93230, ACM93747, and ACM93557 of the pathway proposed to facilitate nitrite reduction to ammonium in Nautilia profundicola (Campbell et al., 2009; Hanson et al., 2013) was performed with the same BLAST parameters as for hydrogenases. Phylogenetic analyses of genes involved in carbon ﬁxation, nitrogen and sulfur cycling, and ﬂagella structure and formation were performed using mingle v0.0.187. Protein markers for marker genes (Supplementary Table S3) were downloaded from UniProt and used for initial homolog discovery against the Genome Taxonomy Database (GTDB)8. Putative protein homologs were manually inspected for false positive matches and genes below the identity threshold or with inconsistent annotations were removed. Putative citrate lyase alpha/beta subunits sequences were also removed if a homolog of each protein in the pair was not detected in a given genome to ensure paralogs were not being directly compared. A similar strategy was applied to the Sox thiosulfate oxidation proteins (SoxA and SoxB). For each data set, protein sequences were aligned using MAFFT v7.221 using the L-INS-i algorithm (Katoh et al., 2002; Katoh and Standley, 2013). The alignment was then masked using Gblocks and phylogenetic inference performed with RAxML as described above. Functional Proﬁling of Epsilonbacteraeota Functional gene predictions for all Epsilonbacteraeota genomes were performed using Prodigal v2.6.3 (Hyatt et al., 2010). Amino acid translations of predicted genes were annotated using diamond v0.8.10.72 (Buchﬁnk et al., 2015) against the Uniref 100 database (downloaded October 2015) and the accessions of target sequences mapped to their KEGG Orthology (KO) group. Annotations were transformed into an abundance matrix using a custom perl script and principal component analysis was performed using the R package vegan v2.3 (Oksanen April 2017 \| Volume 8 \| Article 682 Frontiers in Microbiology \| www.frontiersin.org 4 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. metagenomic data sets as part of a larger study (Parks et al., submitted) and included in our analysis. In addition to the public genomes, we sequenced the type strain of H. thermophila, sole representative of the genus Hydrogenimonas (Takai et al., 2004) and three single cells belonging to the genus Thioreductor (Supplementary Table S2). For H. thermophila, an Illumina-based assembly produced a draft genome of 96 contigs with a predicted completeness of 99.6 and 1.8% contamination. Thioreductor single cells ampliﬁcations were assembled into partial genomes with completeness estimates between 27.7 and 36.5%, and with low contamination estimates (0.3–1.2%) (Supplementary Table S2). Owing to their low completeness Thioreductor genomes were excluded from the majority of analyses, resulting in an ingroup comprising 658 quality-ﬁltered genomes (119 complete and 539 draft) for comparative analysis. Outgroup genomes broadly representative of the bacterial domain were selected from a total of 60,258 quality controlled reference genomes available from the Genome Taxonomy Database. might have contributed to the observed association. Importantly, removal of the Aquiﬁcae in the jackknife analysis did not aﬀect the apparent separation of the Epsilonproteobacteria from the other proteobacterial classes. Proposal for a New Phylum Based on our phylogenetic analyses, we propose to reclassify the Epsilonproteobacteria and Desulfurellales as a single phylum called the Epsilonbacteraeota (phyl. nov.), consistent with a recently proposed naming convention for the phylum rank (Oren et al., 2015). The Epsilonproteobacteria constitute a class-level lineage within this phylum (Figure 2 and Table 2), but we propose to rename it as Campylobacteria (class. nov.) to remove any remaining association with the phylum Proteobacteria. The existing Epsilonproteobacteria orders Campylobacterales and Nautiliales are maintained within this proposed taxonomy as they represent robustly monophyletic groupings and have no direct link to the Proteobacteria in their names (Figure 2 and Supplementary Figure S1 and Table S4). Similarly, the taxon Desulfurellales is retained as the sole order within a new class, the Desulfurellia (class. nov.) (Figure 2 and Supplementary Figure S1). Family and genus level groupings are mostly consistent with existing taxonomic authorities (Table 2), and reclassiﬁed or newly proposed families fall within proposed guidelines for 16S rRNA gene similarity (Yarza et al., 2014) and AAI (Konstantinidis and Tiedje, 2005) thresholds (Supplementary Figure S2). Proposed changes to family membership are detailed below. Proposed Genome-Based Taxonomy Proposed Genome Based Taxonomy Phylogenetic aﬃliation(s) of the ingroup (Epsilonproteobacteria and Desulfurellales, 98 genomes) to species-level representatives of the outgroup (4,072 genomes) were assessed using two diﬀerent datasets. The ﬁrst dataset was a concatenation of 120 single-copy marker proteins (Parks et al., submitted) and the second was a concatenation of the 16S and 23S rRNA gene sequences (Williams et al., 2010; Abby et al., 2012; Kozubal et al., 2013; Guy et al., 2014; Ochoa de Alda et al., 2014; Sen et al., 2014). Note that the 3,144 genomes contributing to the second dataset are a subset of the ﬁrst as most genome sequences derived from metagenomic data lack complete rRNA gene sequences (Hugenholtz et al., 2016), and is used here primarily to validate the concatenated protein tree. Based on these datasets, phylogenetic trees were inferred using Maximum Likelihood (ML) with the JTT, WAG, and LG models of amino acid substitution (Jones et al., 1992; Whelan and Goldman, 2001; Le and Gascuel, 2008) as well as NJ with Jukes-Cantor and Kimura distance corrections (Jukes and Cantor, 1969; Kimura, 1980). Robustness of tree topologies was analyzed with a combination of bootstrapping and taxon resampling, implemented by removal of one phylum at a time from the outgroup dataset. The consensus of these analyses indicate that the Epsilonproteobacteria and Desulfurellales are robustly monophyletic and not reproducibly aﬃliated with any other phyla (Figure 1 and Table 1), which is consistent with recent reports also using concatenated protein markers (Zhang and Sievert, 2014; Hug et al., 2016). The phylum-level jackknife analysis suggests a speciﬁc association of the ingroup with the Aquiﬁcae, which is also supported by bootstrap resampling of this dataset (Figure 1). Tree topologies which suggest a common ancestry between Aquiﬁcae and Epsilonproteobacteria have been reported for several marker genes (Gruber and Bryant, 1998; Klenk et al., 1999; Iyer et al., 2004); however, this association is often not statistically robust. Phylogenomic evidence suggests that Aquiﬁcae genomes have been shaped by extensive lateral gene transfer from lineages including the Epsilonproteobacteria (Eveleigh et al., 2013), a phenomenon that Frontiers in Microbiology \| www.frontiersin.org Changes to the Campylobacteraceae g py The currently deﬁned family Campylobacteraceae, comprised of the genera Arcobacter, Campylobacter, and Sulfurospirillum, is polyphyletic in the concatenated protein tree (Figure 2), although it is monophyletic in the concatenated rRNA gene tree (Supplementary Figure S1). Inspection of individual protein tree topologies revealed that only two of the 120 markers resolve the Campylobacteraceae as monophyletic (Supplementary Figure S3). For this reason, we propose to transfer the Arcobacter and Sulfurospirillum into their own families, the Arcobacteraceae (fam. nov.) and Sulfurospirillaceae (fam. nov.), respectively. Campylobacter is a deeply divergent genus which likely requires reclassiﬁcation into a number of distinct genera (indicated in Figure 2 by alphabetical suﬃxing). Note also that Dehalospirillum multivorans and Geospirillum barnesii have been formally transferred to the genus Sulfurospirillum (Finster et al., 1997; Luijten et al., 2003), and Thiomicrospira denitriﬁcans has been reclassiﬁed to Sulfurimonas (Takai et al., 2006), although these names persist in some online archives. Changes to the Helicobacteraceae Changes to the Helicobacteraceae The currently deﬁned Helicobacteraceae do not form a monophyletic group to the exclusion of the Campylobacteraceae and Arcobacter; therefore, the genera Thiovulum, Sulfuricurvum, and Sulfurimonas have been removed from the Helicobacteraceae into their own family, the Thiovulaceae (fam. nov.). Monophyly of the Helicobacteraceae is also not robustly supported in the concatenated rRNA gene tree (Supplementary Figure S1). Species in the Sulfurovum and Nitratifractor genera are either April 2017 \| Volume 8 \| Article 682 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 5 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. FIGURE 1 \| Maximum likelihood phylogenetic inference (WAG + 0 model) of Epsilonproteobacteria and Desulfurellales in the context of bacterial genomes and based on 120 concatenated protein sequences. Robustness of the placement of the Epsilonbacteraeota was assesse bootstrap support and per-phylum jackkniﬁng. Symbols above each branch root (circles) represent bootstrap support of >90% (black) or ≥75% (hollo below each root reﬂect jackknife reproducibility of a node, with black square representing nodes recovered with ≥90% support in ≥90% of tree topol squares indicate ≥75% support in ≥90% of topologies. Under every tree topology explored Epsilonproteobacteria and Desulfurellales were recovered supported monophyletic group with no association to the remaining Proteobacteria. Key ﬁndings of tree topologies under alternate tree building mode summarized in Table 1. FIGURE 1 \| Maximum likelihood phylogenetic inference (WAG + 0 model) of Epsilonproteobacteria and Desulfurellales in the context of 4,170 bacterial genomes and based on 120 concatenated protein sequences. Robustness of the placement of the Epsilonbacteraeota was assessed using bootstrap support and per-phylum jackkniﬁng. Symbols above each branch root (circles) represent bootstrap support of >90% (black) or ≥75% (hollow). Symbols below each root reﬂect jackknife reproducibility of a node, with black square representing nodes recovered with ≥90% support in ≥90% of tree topologies. Hollow squares indicate ≥75% support in ≥90% of topologies. Under every tree topology explored Epsilonproteobacteria and Desulfurellales were recovered as a strongly supported monophyletic group with no association to the remaining Proteobacteria. Key ﬁndings of tree topologies under alternate tree building models are summarized in Table 1. FIGURE 1 \| Maximum likelihood phylogenetic inference (WAG + 0 model) of Epsilonproteobacteria and Desulfure bacterial genomes and based on 120 concatenated protein sequences. Proposal for New Families We further propose that the genus Nitratiruptor be placed into its own family Nitratiruptoraceae (fam. nov.) within the order Campylobacterales, a move that, while inconsistent with taxonomic authorities placing this genus in the Nautiliaceae (Table 2), is supported by 16S rRNA gene-based phylogenies (Nakagawa and Takaki, 2009; Anderson et al., 2011; Nakagawa and Takai, 2014). Analysis of the three partial single cell Thioreductor genomes conﬁrms the 16S rRNA-based analysis that this genus is a member of the family Nautiliaceae (Supplementary Figures S4, S5). In addition to the proposed revisions in the class Campylobacteria, we propose that Hippea be transferred to its own family Hippaceae (fam. nov.) within the class Desulfurellia to reﬂect the depth of its relationship with the genus Desulfurella (Figure 2). A complete summary of proposed changes within the Epsilonbacteraeota are provided in Supplementary Table S3. Genera Not Yet Represented by Complete Genome Sequences currently unclassiﬁed at the family level, or classiﬁed into the families Helicobacteraceae and Nautiliaceae, respectively (Table 2). Like the Campylobacter, Helicobacter is a deeply divergent genus which likely requires reclassiﬁcation into a number of distinct genera (indicated in Figure 2 by alphabetical suﬃxing). Sulfurovum and Nitratifractor are resolved as a robustly monophyletic group, independent of both the Helicobacteraceae and Nautiliaceae, for which we propose the name Sulfurovaceae (fam. nov., Figure 2). However, the concatenated rRNA gene tree does not support the monophyly of the Sulfurovaceae (Supplementary Figure S1). Inspection of individual protein trees reveals that the majority of markers (105/120) supports the association (Supplementary Figure S3), as well as the 16S rRNA gene by itself (Supplementary Figure S4). The revised Helicobacteraceae family comprises only species in the genera Helicobacter and Wolinella (Figure 2). Note that the genus Flexispira (Bryner et al., 1986) is not included in the Helicobacteraceae as it is a defunct basonym of Helicobacter (Dewhirst et al., 2000; Vandamme and On, 2001). There are three published genera presently classiﬁed as Epsilonproteobacteria for which genomic data are currently unavailable: Thiofractor, Candidatus Thioturbo, and Cetia. These genera are therefore provisionally placed within the Epsilonbacteraeota based on comparative analysis of 16S rRNA gene sequences alone (Supplementary Figure S4); however, their classiﬁcation may require future revision as genomic information becomes available. Thiofractor and Candidatus Thioturbo appear to be members of the order Campylobacterales consistent with previous ﬁndings (Muyzer et al., 2005; Makita et al., 2012), however, neither are clearly resolved into the family level groupings proposed for the Epsilonbacteraeota. Thiofractor may constitute its own family and Candidatus Thioturbo may be incorporated into the Arcobacteraceae, although this latter aﬃliation is only based on a partial 16S rRNA sequence (Supplementary Figure S4). Cetia paciﬁca clusters robustly within the family Nautiliaceae, which may or may not require reclassiﬁcation of the genus Caminibacter (Supplementary Figure S4; Grosche et al., 2015). Changes to the Helicobacteraceae Robustness of the placement of the Epsilonb FIGURE 1 \| Maximum likelihood phylogenetic inference (WAG + 0 model) of Epsilonproteobacteria and Desulfurellales in the context of 4,170 bacterial genomes and based on 120 concatenated protein sequences. Robustness of the placement of the Epsilonbacteraeota was assessed using bootstrap support and per-phylum jackkniﬁng. Symbols above each branch root (circles) represent bootstrap support of >90% (black) or ≥75% (hollow). Symbols below each root reﬂect jackknife reproducibility of a node, with black square representing nodes recovered with ≥90% support in ≥90% of tree topologies. Hollow squares indicate ≥75% support in ≥90% of topologies. Under every tree topology explored Epsilonproteobacteria and Desulfurellales were recovered as a strongly supported monophyletic group with no association to the remaining Proteobacteria. Key ﬁndings of tree topologies under alternate tree building models are summarized in Table 1 proteobacteria and Desulfurellales in the context of 4,170 April 2017 \| Volume 8 \| Article 682 Frontiers in Microbiology \| www.frontiersin.org 6 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. Functional Proﬁling of Epsilonbacteraeota Overlaying published phenotypic information of cultured Epsilonbacteraeota on the genome-based phylogeny suggests that the ancestor of this phylum was autotrophic and thermophilic (Figure 3). Mesophily arose later in the Campylobacterales, and heterotrophic growth appears to have arisen independently in the Campylobacterales and Desulfurellia (Figure 3). To quantify the extent to which taxonomy reﬂects functional variation amongst Epsilonbacteraeota, we performed PERMANOVA to quantify the contribution of predicted functional proﬁles using KO. The largest source of variation was taxonomy (family; R = 0.68, genus; R = 0.70, p = 0.001), followed by habitat (R = 0.28, p = 0.001), indicating that while vertical inheritance is a powerful predictor of functional capacity, a large portion of variation is not captured by this process and likely reﬂects habitat-speciﬁc adaptation. This variation was also reﬂected in PCA analysis of the functional proﬁles, where heterotrophic Campylobacter and Helicobacter genomes were clearly separate from other Epsilonbacteraeota (Figure 4). Furthermore, there was pronounced separation within these genera by functional PCA consistent with the suggestion to classify both Campylobacter and Helicobacter into multiple genera (Figure 2). To elucidate the metabolic components driving this separation, indicator analysis (De Cáceres and Legendre, 2009; De Cáceres et al., 2011) was applied to the gene annotation table to highlight key features responsible for functional divergence. TABLE 1 \| Support of lineage associations under different tree building methods. Tree method Model C-N C-N-D C-N-D-A C-N-P D-P ML (FastTree) WAG + Gamma ∗ ∗ ∗ JTT + Gamma ∗ ∗ ∗ LG + Gamma ∗ ∗ ∗ NJ (Clearcut) No correctiona ∗ Jukes-Cantor correction ∗ ∗ Kimura correction ∗ ∗ Columns refer to collections of orders or phyla which were robustly monophyletic, but not intermingled. Asterisk (∗) represents afﬁliations supported by both bootstrap resampling of the full tree (>90% support), and per-phylum jackknife analysis (>90% bootstrap support in >90% of jackknife trees). No instances were observed where an association was resolved under one criteria but not the other. Letters represent a robustly monophyletic group of genomes, taxonomically classiﬁed as C: Campylobacterales, N: Nautiliales, D: Desulfurellales, A: Aquiﬁcae, P: Proteobacteria. Between-group polyphyly was not observed for any permutation of groups. a The C-N-D afﬁliation is not supported, so C-N-D-A could not be tested. However, neither C-N-A or D-A afﬁliations were supported under this tree topology. TABLE 1 \| Support of lineage associations under different tree building methods. Frontiers in Microbiology \| www.frontiersin.org Functional Proﬁling of Epsilonbacteraeota Columns refer to collections of orders or phyla which were robustly monophyletic, but not intermingled. Asterisk (∗) represents afﬁliations supported by both bootstrap resampling of the full tree (>90% support), and per-phylum jackknife analysis (>90% bootstrap support in >90% of jackknife trees). No instances were observed where an association was resolved under one criteria but not the other. Letters represent a robustly monophyletic group of genomes, taxonomically classiﬁed as C: Campylobacterales, N: Nautiliales, D: Desulfurellales, A: Aquiﬁcae, P: Proteobacteria. Between-group polyphyly was not observed for any permutation of groups. a The C-N-D afﬁliation is not supported, so C-N-D-A could not be tested. However, neither C-N-A or D-A afﬁliations were supported under this tree topology. Columns refer to collections of orders or phyla which were robustly monophyletic, but not intermingled. Asterisk (∗) represents afﬁliations supported by both bootstrap resampling of the full tree (>90% support), and per-phylum jackknife analysis (>90% bootstrap support in >90% of jackknife trees). No instances were observed where an association was resolved under one criteria but not the other. Letters represent a robustly monophyletic group of genomes, taxonomically classiﬁed as C: Campylobacterales, N: Nautiliales, D: Desulfurellales, A: Aquiﬁcae, P: Proteobacteria. Between-group polyphyly was not observed for any permutation of groups. a The C-N-D afﬁliation is not supported, so C-N-D-A could not be tested. However, neither C-N-A or D-A afﬁliations were supported under this tree topology. Genes involved in carbon and nitrogen ﬁxation, assimilatory nitrate and nitrite reduction, thiosulfate oxidation and polysulﬁde reduction were signiﬁcantly associated with the environmental ecotype indicating that carbon, nitrogen, and sulfur cycling are primary drivers of functional divergence in environmental Epsilonbacteraeota. These results were visually apparent when aligning functional genes against the April 2017 \| Volume 8 \| Article 682 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 7 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. FIGURE 2 \| Phylogenetic analysis of the Epsilonbacteraeota. Maximum likelihood tree of the Epsilonbacteraeota based on 120 concatenated protein marker sequences using RAxML. Support of internal nodes was calculated using 100 bootstrap iterations, and junctions represent nodes with 100% bootstrap support (solid), and >75% support (hollow). All taxonomic groupings are identical to those in Figure 1 (calculated with FastTree), although the branching positions of Helicobacteraceae and Sulfurovaceae differs between the two topologies. [T]: denotes type species. FIGURE 2 \| Phylogenetic analysis of the Epsilonbacteraeota. April 2017 \| Volume 8 \| Article 682 Functional Proﬁling of Epsilonbacteraeota Maximum likelihood tree of the Epsilonbacteraeota based on 120 concatenated protein marker sequences using RAxML. Support of internal nodes was calculated using 100 bootstrap iterations, and junctions represent nodes with 100% bootstrap support (solid), and >75% support (hollow). All taxonomic groupings are identical to those in Figure 1 (calculated with FastTree), although the branching positions of Helicobacteraceae and Sulfurovaceae differs between the two topologies. [T]: denotes type species. April 2017 \| Volume 8 \| Article 682 Frontiers in Microbiology \| www.frontiersin.org 8 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. TABLE 2 \| Taxonomy of the Epsilonbacteraeota compared to existing Epsilonproteobacteria. Class/Order Family Genus Bergey’s NCBI SILVA LTP v123a Greengenes 13_5 LPSN Campylobacteria Epsilonproteobacteria – – – – Campylobacterales Arcobacteraceae Campylobacteraceae – – – – Arcobacter Campylobacteraceae Campylobacter Helicobacteraceae Helicobacter Wolinella Hydrogenimonaceae Hydrogenimonas Nitratiruptoraceae Epsilonproteobacteria Nautiliaceae Nautiliaceae Nitratiruptor Sulfurospirillaceae Campylobacteraceae – – – – Sulfurospirillum Sulfurovaceae Unclassiﬁed Helicobacteraceae/Nautiliaceaeb Helicobacteraceae/Nautiliaceaeb Sulfurovum Nitratifractor Thiovulaceae Helicobacteraceae – – – – Thiovulum Sulfuricurvum Sulfurimonas Family incertae sedis Thiofractorc Unclassiﬁed Campylobacteraceae Candidatus Thioturboc Nautiliales Campylobacterales Nautiliaceae Nautilia Caminibacter Lebetimonas Cetiac Thioreductoraceae Epsilonproteobacteria Nautiliaceae Nautiliales Thioreductord Desulfurellia Deltaproteobacteria – – – – Desulfurellales Desulfurellaceae Desulfurella Hippeaceae Desulfurellaceae Desulfurellaceae Desulfurellaceae Hippea Current classiﬁcation for each ranking is provided for commonly used taxonomic schemes comparing the proposed Epsilonbacteraeota (left) to currently accepted taxonomic schemes (right). Empty cells reﬂects an nchanged classiﬁcation for Epsilonbacteraeota. Dashed (–) cells represent a classiﬁcation consistent to Bergey’s in SILVA, Greengenes, or LPSN. Shaded cells represent an entry not recorded under the taxonomic cheme. Genera listed as ‘Unclassiﬁed’ in NCBI are recognized as a part of the Epsilonproteobacteria, but have no assignment below the class level. aThe genera Thiofractor and Sulfuricurvum are annotated in SILVA SSU taxonomy, but not recorded in LTP. bGenera of this family do not belong to a single family under this taxonomic scheme. cOnly 16S rRNA gene sequence data is available for this genus. dThioreductor placement s based on concatenated alignment of 14 protein markers. Novel families proposed in this study are bolded. Frontiers in Microbiology \| www.frontiersin.org 9 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. Campbell et al., 2006; Grote et al., 2012; Grosche et al., 2015; Florentino et al., 2016). Functional Proﬁling of Epsilonbacteraeota A recent biochemical analysis of hydrothermal vents demonstrated that abiotic processes sequester available carbon dioxide as formate (McDermott et al., 2015), and the presence of formate dehydrogenase may provide the ability to couple respiration with the creation of carbon dioxide to fuel rTCA. The Wood-Ljüngdahl (WL) pathway is more energetically favorable than the rTCA but requires strict anoxic conditions to operate (Berg, 2011), which may be incompatible with the microaerobic growth of many vent-associated Epsilonbacteraeota (Campbell et al., 2006). Monophyletic distribution of the WL-speciﬁc marker methylene-tetrahydrofolate reductase (MetF, Figure 3 and Supplementary Figure S7) amongst the Campylobacteria suggests that this pathway was an ancestral trait which has been supplanted by the rTCA, possibly in response to the adoption of (micro)aerobic habitats. Consistent with previous analyses (Hügler et al., 2005, 2011; Takai et al., 2005; Wright et al., 2013), RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) was not detected in any genome, and the rTCA remains the only known means for carbon ﬁxation amongst Epsilonbacteraeota. The pattern of auto- and heterotrophic lifestyles within the Epsilonbacteraeota suggests that autotrophy is an ancestral trait, punctuated by recent adaptations to heterotrophy mostly in host-associated habitats. Additionally, the Epsilonbacteraeota and Aquiﬁcae citrate lyase proteins are robustly monophyletic (Supplementary Figure S8), consistent with the inference that carbon ﬁxation in the Epsilonbacteraeota is descended from an ancestral Aquiﬁcae phenotype (Braakman and Smith, 2012). concatenated protein phylogeny (Figure 3). Host-associated Epsilonbacteraeota were deﬁned primarily by the absence of these functions, although consistent with their lifestyle the antimicrobial peptide resistance mechanism YejABEF (Wang et al., 2016) was a key signature of host-association. The separation of Helicobacter (containing the type species, H. pylori) from Helicobacter A to D was driven by the presence of genes involved in osmoprotectant and heme transport (Kappes et al., 1999; Létoﬀé et al., 2006), as well as the CAG pathogenicity island in H. pylori. Similarly, Campylobacter diﬀerentiation was in part due to the presence of additional transporters involved in lipopolysaccharide and capsular polysaccharide transport. These ﬁndings suggest that the transition toward a host-associated lifestyle in these lineages has occurred in a two-step manner. First, the loss of ‘environmental’ functions associated with the initial transition to host-association in Campylobacter and Helicobacter, followed by gene acquisition to enhance host adaptation. The distinct phylogenetic and functional diﬀerences within these genera suggest that future revision of their taxonomy may be warranted. Frontiers in Microbiology \| www.frontiersin.org Carbon Metabolism Analyzing the carbon metabolism of Epsilonbacteraeota revealed a complete tricarboxylic acid (TCA) cycle in all genomes with the exception of Helicobacter (Figure 3), which functions as a directed pathway producing succinate and α-ketoglutarate in this genus (Pitson et al., 1999). Phylogenetic analysis of the marker protein, 2-oxoglutarate oxidoreductase (KorA), support the TCA cycle being vertically inherited throughout the Epsilonbacteraeota (Supplementary Figure S6). Most genera also encode ATP citrate lyase (AclA and B, Figure 3) indicating the ability to perform autotrophic carbon ﬁxation via reductive TCA (rTCA; Buchanan and Arnon, 1990). The presence of this enzyme coincides with phenotypically established autotrophy (Hügler et al., 2005; Campbell et al., 2006), and genera which have lost the ability to ﬁx carbon have adopted an exclusively heterotrophic lifestyle in carbon rich habitats (Figure 4) with the exception of a number of autotrophic Arcobacter strains (Gevertz et al., 2000; Wirsen et al., 2002) for which genomes are not currently available. Phylogenetic inference of both citrate lyase subunits indicates a monophyletic history of carbon ﬁxation in the Epsilonbacteraeota with independent losses of this capability inferred in the Campylobacter, Helicobacteraceae, and Arcobacter (Figure 3). Citrate lyase was detected in a population genome basal to the Arcobacter genus (UBA6211, Figure 3) which, combined with previous observation of citrate lyase activity and the operation of the rTCA in Arcobacter (Hügler et al., 2005), suggests that carbon ﬁxation was present in the common ancestor of this genus. Vertical inheritance was also observed in Epsilonbacteraeota homologs of the cytosolic [NiFe] hydrogenase group 2D, which is hypothesized to provide reducing power for the rTCA (Brugna-Guiral et al., 2003; Greening et al., 2016). Nitrogen Metabolism FIGURE 3 \| Habitat and functional annotation of selected Epsilonbacteraeota pathways. Tree topology is that of Figure 2, collapsed at the genus level. Cell intensity reﬂects the proportion of genomes within a clade that possessed the column function. A: Trophism is inferred from genomic data. B: Although some members of Arcobacter are capable of autotrophic growth (discussed in text) all genomes in this study were obtained from heterotrophic species. Column descriptions are as follows: Carbon pathways: aconite hydratase (Acn), 2-oxoglutarate oxidoreductase alpha-, beta-, gamma-, and delta- subunits (KorABGD), succinyl-CoA synthetase alpha and beta subunits (SucCD), malate dehydrogenase (Mdh), ATP citrate lyase alpha subunit (AclA) and beta subunit (AclB), [NiFe] hydrogenase family 2D (NiFe 2D), formate dehydrogenase (Fdh), methylenetetrahydrofolate reductase (MetF) and carbon-monoxide dehydrogenase catalytic and iron sulfur subunits (CooS/CooF). Nitrogen pathways: periplasmic nitrate reductase components NapA, NapB, NapG, and NapH (NapABGH), periplasmic nitrate reductase c-type cytochrome (NapC), hydroxylamine oxidoreductase (HaoA), hydroxylamine reductase/hybrid cluster protein (Har/Hcp), predicted reductive Fe-S protein (FeS protein), ferredoxin-nitrate reductase (NarB), assimilatory nitrate reductase (catalytic subunit, NasA), ferredoxin-nitrite reductase (NirA), nitrogenase molybdenum-iron (alpha- and beta-chains) and iron protein (NifDKH), ammonium transporter (AmtB), nitrite reductase (NADH) large subunit (NirB), cytochrome c nitrite reductase (NrfA). Sulfur pathways: sulﬁde:quinone oxidoreductase (SQR), polysulﬁde reductase chain A (PsrA), thiosulfate oxidating Sox proteins (SoxXA, SoxY, SoxB, SoxCD). Motility: ﬂagellar hook protein (FlgE), ﬂagellin (FliC), methyl-accepting chemotaxis protein (Mcp), chemotaxis response regulator (CheB), chemotaxis methyltransferase (CheR). Phylogenetic inference of the HaoA and Har/Hcp proteins suggest that this more complex nitrite ammoniﬁcation pathway is monophyletic and ancestral to the Epsilonbacteraeota, despite widespread partial or complete loss of the pathway in the group, and putative lateral transfers of component genes from Epsilonbacteraeota donors to other bacteria (Supplementary Figure S9). Most Epsilonbacteraeota have multiple potential mechanisms for obtaining ﬁxed nitrogen and the acquisition of chemoorganotrophic catabolic machinery, which provide cells with higher and more reliable reducing power, likely facilitated the functional replacement of the HaoA-type with the NrfA-type nitrite reductase. The majority of extant autotrophic Epsilonbacteraeota possess one or more ammoniﬁcation pathways including dinitrogen reduction based on nitrogenase and/or a variety of pathways based on the reduction of nitrate the remaining Nautiliales genomes, as well as Campylobacter mucosalis, C. geochelonis, C. gracilis, C. hyointestinalis, and C. iguaniorum (Figure 3). Furthermore, H. thermophila and D. Nitrogen Metabolism Anaerobic respiration using nitrate as an electron acceptor is widespread throughout the Epsilonbacteraeota, as evidenced by the presence of soluble periplasmic nitrate reductase (NapA) in most genera (Figure 3), and membrane-bound nitrate reductase (NarG) in Desulfurella (Vetriani et al., 2014). The NAP system of many Campylobacteria lack the NapC protein (Simon et al., 2003; Sievert et al., 2008; Kern and Simon, 2009) and where a homolog is present, it is proposed to act as the quinol-oxidizing protein component of a reversely operating hydroxylamine:quinone redox module. This pathway operates as a quinone-reducing module in ammonia-oxidizing bacteria and Thaumarchaeota, and has recently been proposed in N. profundicola (Klotz and Stein, 2008; Campbell et al., 2009; Hanson et al., 2013; Simon and Klotz, 2013; Kozlowski et al., 2016). As a component of an ammoniﬁcation pathway, this module produces ammonium by reduction of nitrite either directly (analogous to the NrfHA ammoniﬁcation module) or via a hydroxylamine intermediate, which is thought to be transported to the cytoplasm and further reduced to ammonium by a putative hydroxylamine reductase (Har/Hcp), powered by an Fe-S ferredoxin (Hanson et al., 2013). While hydroxylamine reduction to ammonium and nitrate-dependent expression of genes encoding the entire pathway has, so far, only been demonstrated in N. profundicola, genes for this pathway are present in Campylobacter concisus, C. curvus, and C. fetus (Hanson et al., 2013). We further identiﬁed the complete inventory for this proposed pathway in No other complete carbon ﬁxation pathways were identiﬁed in the genomes. Formate dehydrogenase is widely distributed in many autotrophs (Schuchmann and Müller, 2014) and was common in hydrothermal vent-associated lineages (Figures 2, 3), many of which are known to oxidize formate (Macy et al., 1996; April 2017 \| Volume 8 \| Article 682 10 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. FIGURE 3 \| Habitat and functional annotation of selected Epsilonbacteraeota pathways. Tree topology is that of Figure 2, collapsed at the genus level. Cell intensity reﬂects the proportion of genomes within a clade that possessed the column function. A: Trophism is inferred from genomic data. B: Although some members of Arcobacter are capable of autotrophic growth (discussed in text) all genomes in this study were obtained from heterotrophic species. Frontiers in Microbiology \| www.frontiersin.org Nitrogen Metabolism Column descriptions are as follows: Carbon pathways: aconite hydratase (Acn), 2-oxoglutarate oxidoreductase alpha-, beta-, gamma-, and delta- subunits (KorABGD), succinyl-CoA synthetase alpha and beta subunits (SucCD), malate dehydrogenase (Mdh), ATP citrate lyase alpha subunit (AclA) and beta subunit (AclB), [NiFe] hydrogenase family 2D (NiFe 2D), formate dehydrogenase (Fdh), methylenetetrahydrofolate reductase (MetF) and carbon-monoxide dehydrogenase catalytic and iron sulfur subunits (CooS/CooF). Nitrogen pathways: periplasmic nitrate reductase components NapA, NapB, NapG, and NapH (NapABGH), periplasmic nitrate reductase c-type cytochrome (NapC), hydroxylamine oxidoreductase (HaoA), hydroxylamine reductase/hybrid cluster protein (Har/Hcp), predicted reductive Fe-S protein (FeS protein), ferredoxin-nitrate reductase (NarB), assimilatory nitrate reductase (catalytic subunit, NasA), ferredoxin-nitrite reductase (NirA), nitrogenase molybdenum-iron (alpha- and beta-chains) and iron protein (NifDKH), ammonium transporter (AmtB), nitrite reductase (NADH) large subunit (NirB), cytochrome c nitrite reductase (NrfA). Sulfur pathways: sulﬁde:quinone oxidoreductase (SQR), polysulﬁde reductase chain A (PsrA), thiosulfate oxidating Sox proteins (SoxXA, SoxY, SoxB, SoxCD). Motility: ﬂagellar hook protein (FlgE), ﬂagellin (FliC), methyl-accepting chemotaxis protein (Mcp), chemotaxis response regulator (CheB), chemotaxis methyltransferase (CheR). FIGURE 3 \| Habitat and functional annotation of selected Epsilonbacteraeota pathways. Tree topology is that of Figure 2, collapsed at the genus level. Cell intensity reﬂects the proportion of genomes within a clade that possessed the column function. A: Trophism is inferred from genomic data. B: Although some members of Arcobacter are capable of autotrophic growth (discussed in text) all genomes in this study were obtained from heterotrophic species. Column descriptions are as follows: Carbon pathways: aconite hydratase (Acn), 2-oxoglutarate oxidoreductase alpha-, beta-, gamma-, and delta- subunits (KorABGD), succinyl-CoA synthetase alpha and beta subunits (SucCD), malate dehydrogenase (Mdh), ATP citrate lyase alpha subunit (AclA) and beta subunit (AclB), [NiFe] hydrogenase family 2D (NiFe 2D), formate dehydrogenase (Fdh), methylenetetrahydrofolate reductase (MetF) and carbon-monoxide dehydrogenase catalytic and iron sulfur subunits (CooS/CooF). Nitrogen pathways: periplasmic nitrate reductase components NapA, NapB, NapG, and NapH (NapABGH), periplasmic nitrate reductase c-type cytochrome (NapC), hydroxylamine oxidoreductase (HaoA), hydroxylamine reductase/hybrid cluster protein (Har/Hcp), predicted reductive Fe-S protein (FeS protein), ferredoxin-nitrate reductase (NarB), assimilatory nitrate reductase (catalytic subunit, NasA), ferredoxin-nitrite reductase (NirA), nitrogenase molybdenum-iron (alpha- and beta-chains) and iron protein (NifDKH), ammonium transporter (AmtB), nitrite reductase (NADH) large subunit (NirB), cytochrome c nitrite reductase (NrfA). Sulfur pathways: sulﬁde:quinone oxidoreductase (SQR), polysulﬁde reductase chain A (PsrA), thiosulfate oxidating Sox proteins (SoxXA, SoxY, SoxB, SoxCD). Motility: ﬂagellar hook protein (FlgE), ﬂagellin (FliC), methyl-accepting chemotaxis protein (Mcp), chemotaxis response regulator (CheB), chemotaxis methyltransferase (CheR). Nitrogen Metabolism acetivorans lack only the predicted Fe-S ferredoxin of this pathway, but as hydroxylamine reductase is known to obtain reductant from other oxidoreductases (Pino et al., 2006), it is possible that these organisms are also capable of ammoniﬁcation using a hydroxylamine intermediate. The NapC homolog and HaoA proteins are homologous to NrfH and NrfA, respectively (Bergmann et al., 2005; Kern et al., 2011a; Simon and Klotz, 2013) and diﬀer mainly by the inﬁdelity of the HaoA oligomer as an ammonium-producing nitrite reductase. These homologs facilitate incomplete electron transfer and leak hydroxylamine due to looser redox coupling with the NapC homolog compared to that of the NrfA-NrfH module (Simon and Klotz, 2013). April 2017 \| Volume 8 \| Article 682 Frontiers in Microbiology \| www.frontiersin.org 11 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. FIGURE 4 \| Principal component analysis of Epsilonbacteraeota gene annotations. Analysis was performed using the KEGG Orthology (KO) annotation table. Circles denote host-associated individuals, and squares those from other environments. Genomes are colored by family, with exceptions of the Campylobacteraceae and Helicobacteraceae, which are separated based on sufﬁxing described in text. Vectors represent pathway level aggregations of indicator KOs ﬁtted to the ordination using the enﬁt function in vegan. Only vectors with R > 0.75 are displayed for clarity. Pathways are as follows: A, arginine biosynthesis; At, atrazine degradation; B, biotin metabolism; N, nitrogen metabolism; PTT, phenylalanine, tyrosine, and tryptophan biosynthesis; S, sulfur metabolism; Syn, synthesis and degradation of ketone bodies; T, two-component system; VLI, valine, leucine, and isoleucine degradation. FIGURE 4 \| Principal component analysis of Epsilonbacteraeota gene annotations. Analysis was performed using the KEGG Orthology (KO) annotation table. Circles denote host-associated individuals, and squares those from other environments. Genomes are colored by family, with exceptions of the Campylobacteraceae and Helicobacteraceae, which are separated based on sufﬁxing described in text. Vectors represent pathway level aggregations of indicator KOs ﬁtted to the ordination using the enﬁt function in vegan. Only vectors with R > 0.75 are displayed for clarity. Pathways are as follows: A, arginine biosynthesis; At, atrazine degradation; B, biotin metabolism; N, nitrogen metabolism; PTT, phenylalanine, tyrosine, and tryptophan biosynthesis; S, sulfur metabolism; Syn, synthesis and degradation of ketone bodies; T, two-component system; VLI, valine, leucine, and isoleucine degradation. Nitrogen Metabolism multiple lateral transfer events (Supplementary Figure S10), possibly the result of selective pressure exerted by the host immune system (Kern et al., 2011a; Kassem et al., 2012). (Figure 3). Annotation of the Thiovulum sp. ES genome did not reveal any ammoniﬁcation inventory; nevertheless and consistent with its original annotation (Marshall et al., 2012), we detected the gene encoding an ammonium transporter (AmtB) in this lineage, shared with most other Epsilonbacteraeota (Figure 3). Frontiers in Microbiology \| www.frontiersin.org Concluding Remarks y g ( ) Oxidation of sulfur is also a well known trait of Epsilonproteobacteria, although it is only reported in a handful of genera (Campbell et al., 2006). Sulfurimonas-like organisms have been suggested as the primary sulfur-oxidisers in deep-sea vent ecosystems (Akerman et al., 2013) and consistent with this observation, Sulfurimonas was one of the few Epsilonbacteraeota genera to possess the complete sulfur oxidation (sox) pathway (Figure 3). The absence of SoxC, and drastic reduction in electron liberation (Friedrich et al., 2005), in most genera suggests that sulfur oxidation is not the primary means of electron generation amongst Epsilonbacteraeota, with hydrogenases likely a more productive source of reducing power. The evolutionary history of sox in the Epsilonbacteraeota is convoluted. Consistent with previous analysis of sox genes (Meyer et al., 2007; Ghosh et al., 2009), SoxA and SoxB homologs in non-Arcobacter species shared a common ancestor with Aquiﬁcae, while Arcobacter acquired a complete sox cassette through lateral gene transfer with Gammaproteobacteria (Supplementary Figure S13). However, homologs of SoxC are monophyletic in the Epsilonbacteraeota, a ﬁnding also supported by previously literature (Ghosh et al., 2009). These data suggest that after acquiring the ability to oxidize thiosulfate independently of other Epsilonbacteraeota, an ancestor of Arcobacter introduced soxCD to Sulfurimonas and the Sulfurovaceae. g The rise of high-throughput DNA sequencing has fundamentally changed the landscape of microbial ecology and systematics. The 16S rRNA gene has led the way in providing an evolutionary backbone for microbial taxonomy over the past 30 years, but can now be complemented by whole genome methods which use multiple genetic markers to infer evolutionary relationships. More importantly, classiﬁcations are not static. As genomically described bacterial diversity expands, historic classiﬁcations require revision as new clades are discovered and tree topologies change. The class Epsilonproteobacteria and order Desulfurellales are an excellent case in point. Comparative analysis of these groups with a broad genomic representation of the bacterial domain indicates that they constitute a monophyletic unit not speciﬁcally related to the phylum Proteobacteria. We propose the reclassiﬁcation of this group as the phylum Epsilonbacteraeota, together with a small number of subordinate changes described in this manuscript. Additional changes may be required in future as, e.g., Campylobacter and Helicobacter appear to be phylogenetically and functionally diverse for genera. Concluding Remarks Metabolic reconstruction of the Epsilonbacteraeota suggests, perhaps unsurprisingly, that the ancestor to this phylum was a chemolithoautotrophic thermophile related to Aquiﬁcae, from which several heterotrophic and mesophilic lineages have evolved. Sulfur Metabolism It is possible that the acquisition of SQR represents a means of simultaneously detoxifying hydrogen sulﬁde, and utilizing it as an electron donor, as Epsilonproteobacteria have been seen to bloom in response to hydrogen sulﬁde releases on the African shelf (Lavik et al., 2009). Figure S12). These ﬁndings suggest that unlike nitrate reduction, sulfur reduction has been acquired in the Epsilonbacteraeota in response to immediate environmental needs. Polysulﬁde can be formed abiotically under hydrothermal vent conditions (Rushdi and Simoneit, 2005; Boyd and Druschel, 2013) and, similar to formate, presents a resource that early colonizing Epsilonbacteraeota may exploit. It is possible that the acquisition of SQR represents a means of simultaneously detoxifying hydrogen sulﬁde, and utilizing it as an electron donor, as Epsilonproteobacteria have been seen to bloom in response to hydrogen sulﬁde releases on the African shelf (Lavik et al., 2009). Description of Campylobacteria class. nov. Campylobacteria (Cam.py.lo.bac.te′ri.a. N.L. masc. n. Campylobacter type genus of the type order of the class; suﬀ. -ia, ending to denote a class; N.L. neut. pl. n. Campylobacteria the class of the order Campylobacterales). Description is the same as for the order Campylobacterales (Garrity et al., 2006b). Type order: Campylobacterales, phylum: Epsilonbacteraeota phyl. nov. Proposed Taxonomy Epsilonbacteraeota Description of Campylobacteria class. nov. Sulfur Metabolism It is possible that the acquisition of SQR represents a means of simultaneously detoxifying hydrogen sulﬁde, and utilizing it as an electron donor, as Epsilonproteobacteria have been seen to bloom in response to hydrogen sulﬁde releases on the African shelf (Lavik et al., 2009). Oxidation of sulfur is also a well known trait of Epsilonproteobacteria, although it is only reported in a handful of genera (Campbell et al., 2006). Sulfurimonas-like organisms have been suggested as the primary sulfur-oxidisers in deep-sea vent ecosystems (Akerman et al., 2013) and consistent with this observation, Sulfurimonas was one of the few Epsilonbacteraeota genera to possess the complete sulfur oxidation (sox) pathway (Figure 3). The absence of SoxC, and drastic reduction in electron liberation (Friedrich et al., 2005), in most genera suggests that sulfur oxidation is not the primary means of electron generation amongst Epsilonbacteraeota, with hydrogenases likely a more productive source of reducing power. The evolutionary history of sox in the Epsilonbacteraeota is convoluted. Consistent with previous analysis of sox genes (Meyer et al., 2007; Ghosh et al., 2009), SoxA and SoxB homologs in non-Arcobacter species shared a common ancestor with Aquiﬁcae, while Arcobacter acquired a complete sox cassette through lateral gene transfer with Gammaproteobacteria (Supplementary Figure S13). However, homologs of SoxC are monophyletic in the Epsilonbacteraeota, a ﬁnding also supported by previously literature (Ghosh et al., 2009). These data suggest that after acquiring the ability to oxidize thiosulfate independently of other Epsilonbacteraeota, an ancestor of Arcobacter introduced soxCD to Sulfurimonas and the Sulfurovaceae. Sulfurovaceae, suggesting a single loss of ﬂagella genes in the ancestor of this family. The presence of the chemotactic regulator Mcp and response regulating CheB and CheR within this family (Figure 3) provides additional evidence that the ancestor of the Sulfurovaceae possessed ﬂagella. Campylobacter hominis and C. gracilis are sister species in the Campylobacter B clade (Figure 2) suggesting a single loss of motility occurred in their common ancestor for as yet, unclear reasons given that the rest of the genus has retained motility. Figure S12). These ﬁndings suggest that unlike nitrate reduction, sulfur reduction has been acquired in the Epsilonbacteraeota in response to immediate environmental needs. Polysulﬁde can be formed abiotically under hydrothermal vent conditions (Rushdi and Simoneit, 2005; Boyd and Druschel, 2013) and, similar to formate, presents a resource that early colonizing Epsilonbacteraeota may exploit. Proposed Taxonomy of Epsilonbacteraeota The majority of Epsilonbacteraeota possess uni- or bipolar ﬂagella and are highly motile, with Thiovulum majus capable of achieving a speed of over 600 microns per second, or 2 m per hour (Garcia-Pichel, 1989). The ﬂagella of Epsilonbacteraeota diﬀer from that of other bacteria in several respects. For example, the C-ring of model Campylobacter jejuni and Helicobacter hepaticus diﬀers structurally from that of other bacteria (Chen et al., 2011), and recent knock-out experiments identiﬁed six novel genes exclusive to ﬂagella activity in Epsilonbacteraeota (Gao et al., 2014). Given the novelty of described Epsilonbacteraeota ﬂagella, we predicted that they should be an ancestral feature of this phylum. Indeed, phylogenetic analysis of the ﬂagellar hook protein (FlgE) and ﬂagellin (FliC, Figure 3) indicate a monophyletic origin of this trait in the Epsilonbacteraeota (Supplementary Figure S14). Flagella are known to be absent in several Epsilonbacteraeota, including Nitratifractor salsuginis, the genus Sulfurovum, and at least two Campylobacter species, C. hominis and C. gracilis (Vandamme et al., 1995; Lawson et al., 2001). The proposed Epsilonbacteraeota taxonomy groups the genera Nitratifractor and Sulfurovum into the novel family Sulfur Metabolism Anaerobic respiration of Epsilonbacteraeota frequently occurs through the reduction of sulfur compounds, and is a major source of primary production in some environments (Wright et al., 2013). Sulﬁde-oxidizing sulﬁde:quinone oxidoreductase (SQR) catalyzes the bidirectional oxidation of hydrogen sulﬁde to polysulﬁde, and this gene is inferred to be abundant in environments where Epsilonbacteraeota (Epsilonproteobacteria) are dominant members (Wright et al., 2013; Rossmassler et al., 2016). Consistent with this inference, we observed SQR genes in most Epsilonbacteraeota lineages (Figure 3). In contrast to the monophyletic origins of the carbon and most nitrogen cycling proteins described above, phylogenetic analysis of SQR reveals extensive polyphyly of Epsilonbacteraeota homologs, including a putative lateral transfer from a Bacteroidetes donor to the Nautilaceae ancestor (Supplementary Figure S11). An additional enzyme involved in polysulﬁde reduction, polysulﬁde reductase (PsrA) was also common in the Epsilonbacteraeota (Figure 3) and possessed polyphyletic origins (Supplementary Pentaheme cytochrome c nitrite reductase (NrfA) is found almost exclusively in heterotrophic Campylobacteria (Figure 3) and is known to provide protection against nitrosative stress to Campylobacter and Wolinella in their natural environments (Pittman et al., 2007; Kern et al., 2011b). This protein may also serve the same function in the majority of Helicobacter species, although this has not been well studied since the model species, H. pylori, lacks NrfA and has alternative mechanisms for coping with nitrosative stress (Flint et al., 2016). In contrast to the concatenated protein-based monophyly of the heterotrophs (Figure 3), phylogenetic inference of NrfA reveals at least two independent clades, the ﬁrst comprising Sulfurospirillum, Wolinella and some Arcobacter, and the second Campylobacter and Helicobacter (Supplementary Figure S10). It is likely at least one of these clusters is the result of lateral gene transfer, which we predict to be the Campylobacter and Helicobacter clade as members of these two genera are intermingled indicating April 2017 \| Volume 8 \| Article 682 Frontiers in Microbiology \| www.frontiersin.org 12 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. Figure S12). These ﬁndings suggest that unlike nitrate reduction, sulfur reduction has been acquired in the Epsilonbacteraeota in response to immediate environmental needs. Polysulﬁde can be formed abiotically under hydrothermal vent conditions (Rushdi and Simoneit, 2005; Boyd and Druschel, 2013) and, similar to formate, presents a resource that early colonizing Epsilonbacteraeota may exploit. Description of Nitratiruptoraceae fam. nov. Nitratiruptoraceae (Ni.tra.ti.rup.to.ra.ce′ae. N.L. masc. n. Nitratiruptor type genus of the family; suﬀ. -aceae, ending to denote a family; N.L. fem. pl. n. Nitratiruptoraceae the family of the genus Nitratiruptor). Emended Description of the Order Desulfurellales Kuever et al. (2006b) The order is as previously described (Kuever et al., 2006b) but is transferred from the class Deltaproteobacteria to Desulfurellia class. nov. The order consists of the families Desulfurellaceae and Hippeaceae fam. nov. Cells are Gram-negative, thermophilic, and motile. Type family: Desulfurellaceae, class: Desulfurellia class. nov., phylum: Epsilonbacteraeota phyl. nov. Described on the basis of divergent nature with all other members of Campylobacterales using both protein marker, as well as 16S and 23S rRNA gene sequence analysis. Description is the same as given by Nakagawa et al. (2005). Type genus: Nitratiruptor, order: Campylobacterales, class: Campylobacteria class. nov., phylum: Epsilonbacteraeota phyl. nov. Description of Sulfurospirillaceae fam. nov. Sulfurospirillaceae (Sul.fu.ro.spir.il.la.ce′ae. N.L. neut. n. Sulfurospirillum type genus of the family; suﬀ. -aceae, ending to denote a family; N.L. fem. pl. n. Sulfurospirillaceae the family of the genus Sulfurospirillum). Description is as previously (Kuever et al., 2006a), with the exception that the genus Hippea is moved to the family Hippeaceae fam. nov.. Cells are Gram-negative, obligately anaerobic, and motile. Uses short-chain fatty acids including acetate, fumarate, malate, as well as long-chain saturated fatty acids as growth substrates. Type species D. acetivorans is capable of dissimilatory sulfur reduction (Bonch-Osmolovskaya et al., 1990). Type genus: Desulfurella, order: Desulfurellales, class: Desulfurellia class. nov., phylum: Epsilonbacteraeota phyl. nov. The description of the family is drawn from the description of the type genus from Schumacher et al. (1992); cells are Gram-negative, motile, microaerobic, and mesophilic. Oxidize hydrogen or formate to reduce a range of electron acceptors. Type genus: Sulfurospirillum, order: Campylobacterales, class: Campylobacteria class. nov., phylum: Epsilonbacteraeota phyl. nov. Description of Desulfurellia class. nov. Desulfurellia (De.sul.fu.rel′li.a. N.L. fem. n. Desulfurella type genus of the type order of the class; suﬀ. -ia, ending to denote a class; N.L. neut. pl. n. Desulfurellia the class of the order Desulfurellales). Description is the same as for the order Desulfurellales (Kuever et al., 2006b). Type order: Desulfurellales, phylum: Epsilonbacteraeota phyl. nov. Description of Nitratiruptoraceae fam. nov. Emended Description of the Family Helicobacteraceae Garrity et al. (2006a) The description is the same as given by Garrity et al. (2006a) for the type genus with the following changes. Currently includes only the genera Helicobacter (type genus) and Wolinella. The genera Sulfurimonas, Sulfuricurvum, Sulfurovum, and Thiovulum are removed owing to a lack of monophyly with the type genus. Type genus: Helicobacter, order: Campylobacterales, class: Campylobacteria class. nov., phylum: Epsilonbacteraeota phyl. nov. Description of Desulfurellia class. nov. Description of Arcobacteraceae fam. nov. Arcobacteraceae (Ar.co.bac.ter.a.ce′ae. N.L. masc. n. Arcobacter type genus of the family; suﬀ. -aceae, ending to denote a family; N.L. fem. pl. n. Arcobacteraceae the family of the genus Arcobacter). Description is identical to that given by Vandamme et al. (1991), with the acknowledgment that some species are capable of autotrophic carbon dioxide ﬁxation via the reverse tricarboxylic acid cycle (Hügler et al., 2005). Type genus: Arcobacter, order: April 2017 \| Volume 8 \| Article 682 Frontiers in Microbiology \| www.frontiersin.org 13 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. Campylobacterales, class: Campylobacteria, class. nov., phylum: Epsilonbacteraeota phyl. nov. class: Campylobacteria class. nov., phylum: Epsilonbacteraeota phyl. nov. Campylobacterales, class: Campylobacteria, class. nov., phylum: Epsilonbacteraeota phyl. nov. Description of Hippeaceae fam. nov. Description of Hippeaceae fam. nov. Hippeaceae (Hippe.a.ce′ae. N.L. fem. n. Hippea type genus of the family; suﬀ. -aceae, ending to denote a family; N.L. fem. pl. n. Hippeaceae the family of the genus Hippea). The family is deﬁned based on phylogenetic analysis of concatenated marker proteins, and is separated from the Desulfurellaceae due to deep branching distance. Cells are Gram-negative, obligately anaerobic, thermophilic, and motile. Capable of heterotrophic growth using acetate and various other organic substrates by species. Type genus: Hippea, order: Desulfurellales, class: Desulfurellia class. nov., phylum: Epsilonbacteraeota phyl. nov. Hippeaceae (Hippe.a.ce′ae. N.L. fem. n. Hippea type genus of the family; suﬀ. -aceae, ending to denote a family; N.L. fem. pl. n. Hippeaceae the family of the genus Hippea). Emended Description of the Family Campylobacteraceae Vandamme and De Ley (1991) Thiovulaceae (Thio.vu.la.ce’ae. N.L. neut. dim. n. Thiovulum type genus of the family; suﬀ. -aceae, ending to denote a family; N.L. fem. pl. n. Thiovulaceae the family of the genus Thiovulum). Description is drawn from that of Vandamme and De Ley (1991), with the removal of the genera Arcobacter and Sulfurospirillum due to a lack of robust monophyly with the type genus. Type genus: Campylobacter, order: Campylobacterales, class: Campylobacteria class. nov., phylum: Epsilonbacteraeota phyl. nov. The family is deﬁned based on phylogenetic analysis of concatenated protein marker and ribosomal gene sequences. The family currently includes the genera Thiovulum (type genus), Sulfurimonas, and Sulfuricurvum. Cells are Gram- negative, motile, microaerobic, and mesophilic. Growth occurs in the mesophilic range. Type genus: Thiovulum, order: Campylobacterales, class: Campylobacteriaclass. nov., phylum: Epsilonbacteraeota phyl. nov. REFERENCES Bolger, A. M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a ﬂexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. doi: 10.1093/bioinformatics/btu170 Abby, S. S., Tannier, E., Gouy, M., and Daubin, V. (2012). Lateral gene transfer as a support for the tree of life. Proc. Natl. Acad. Sci. U.S.A. 109, 4962–4967. doi: 10.1073/pnas.1116871109 Bonch-Osmolovskaya, E. A., Sokolova, T. G., Kostrikina, N. A., and Zavarzin, G. A. (1990). Desulfurella acetivorans gen. nov. and sp. nov. A new thermophilic sulfur-reducing eubacterium. Arch. Microbiol. 153, 151–155. doi: 10.1007/ BF00247813 Akerman, N. H., Butterﬁeld, D. A., and Huber, J. A. (2013). Phylogenetic diversity and functional gene patterns of sulfur-oxidizing subseaﬂoor Epsilonproteobacteria in diﬀuse hydrothermal vent ﬂuids. Front. Microbiol. 4:185. doi: 10.3389/fmicb.2013.00185 Boyd, E. S., and Druschel, G. K. (2013). Involvement of intermediate sulfur species in biological reduction of elemental sulfur under acidic, hydrothermal conditions. Appl. Environ. Microbiol. 79, 2061–2068. doi: 10.1128/AEM. 03160-12 Alain, K., Zbinden, M., Le Bris, N., Lesongeur, F., Quérellou, J., Gaill, F., et al. (2004). Early steps in microbial colonization processes at deep-sea hydrothermal vents. Environ. Microbiol. 6, 227–241. doi: 10.1111/j.1462-2920. 2003.00557.x Braakman, R., and Smith, E. (2012). The emergence and early evolution of biological carbon-ﬁxation. PLoS Comput. Biol. 8:e1002455. doi: 10.1371/ journal.pcbi.1002455. Albertsen, M., Hugenholtz, P., Skarshewski, A., Nielsen, K. L., Tyson, G. W., and Nielsen, P. H. (2013). Genome sequences of rare, uncultured bacteria obtained by diﬀerential coverage binning of multiple metagenomes. Nat. Biotechnol. 31, 533–538. doi: 10.1038/nbt.2579 Brugna-Guiral, M., Tron, P., Nitschke, W., Stetter, K. O., Burlat, B., Guigliarelli, B., et al. (2003). [NiFe] hydrogenases from the hyperthermophilic bacterium Aquifex aeolicus: properties, function, and phylogenetics. Extremophiles 7, 145–157. doi: 10.1007/s00792-002-0306-3 Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410. doi: 10.1016/S0022- 2836(05)80360-2 Bryner, J. H., Littleton, J., Gates, C., Kirkbridge, C. A., and Richie, A. E. (1986). “Flexispira rappini gen. nov., sp. nov., a Gram-negative rod from mammalian fetus and feces,” in Proceedings of the XIV International Congress of Microbiology, Manchester. Anderson, I., Sikorski, J., Zeytun, A., Nolan, M., Lapidus, A., Lucas, S., et al. (2011). Complete genome sequence of Nitratifractor salsuginis type strain (E9I37-1T). Stand. Genomic Sci. 4, 322–330. doi: 10.4056/sigs.1844518 Buchanan, B. B., and Arnon, D. I. (1990). A reverse KREBS cycle in photosynthesis: consensus at last. Photosynth. Res. 24, 47–53. ACKNOWLEDGMENTS We are grateful to Maria Chuvochina, Aharon Oren, and Brian Tindall for advice on etymology. The genomes of Hydrogenimonas thermophila and Thioreductor were sequenced by the US Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/) in collaboration with the user community. SUPPLEMENTARY MATERIAL DW, DP, and PH performed the bioinformatic analysis, interpretation, and proposed taxonomic changes. KT, MK, SS, and TW sequenced additional genomes for analysis. BC, IV, JS, and MK proposed avenues for functional analysis and The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2017.00682/full#supplementary-material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2017.00682/full#supplementary-material Description of Epsilonbacteraeota phyl. nov. assisted with interpretation of data. DW and PH wrote the draft manuscript and all authors contributed to, and approved, the ﬁnal manuscript. Epsilonbacteraeota (Ep.si.lon.bac.ter.ae.o′ta. Gr. n. epsilon, name of the ﬁfth letter of Greek alphabet; Gr. n. baktêria, staﬀ, cane; suﬀ. -aeota, proposed ending to denote a phylum; N.L. neut. pl. n. Epsilonbacteraeota the phylum of bacteria sharing a common ancestry and possessing ribosomal gene sequence and protein marker similarity to those of the members of the classes Campylobacteria class. nov. and Desulfurellia class. nov) FUNDING The study was supported by a Discovery Outstanding Researcher Award (DP120103498) and an Australian Laureate Fellowship (FL150100038) from the Australian Research Council. The study was supported by a Discovery Outstanding Researcher Award (DP120103498) and an Australian Laureate Fellowship (FL150100038) from the Australian Research Council. The phylum is described based upon concatenated protein marker and ribosomal gene sequence phylogeny. Owing to the diverse ecological range of members of this phylum there is little unifying physiology. All members are Gram-negative, and motility as well as the reduction of nitrate and polysulﬁde are ancestral and common traits. The phylum Epsilonbacteraeota currently comprises two classes: Campylobacteria class. nov. and Desulfurellia class. nov.; and three orders: Campylobacterales, Nautiliales, and Desulfurellales. The type class of the phylum is Campylobacteria class. nov. Description of Sulfurovaceae fam. nov. Description of Sulfurovaceae fam. nov. Sulfurovaceae (Sul.fur.o.va.ce′ae. N.L. neut. n. Sulfurovum type genus of the family; suﬀ. -aceae, ending to denote a family; N.L. fem. pl. n. Sulfurovaceae the family of the genus Sulfurovum). The family is deﬁned based on phylogenetic analysis of concatenated marker proteins, and is separated from the Desulfurellaceae due to deep branching distance. Cells are Gram-negative, obligately anaerobic, thermophilic, and motile. Capable of heterotrophic growth using acetate and various other organic substrates by species. Type genus: Hippea, order: Desulfurellales, class: Desulfurellia class. nov., phylum: Epsilonbacteraeota phyl. nov. The family is deﬁned based on phylogenetic analysis of protein markers (concatenated and single) and the 16S ribosomal gene sequence. Includes the genera Sulfurovum (type genus) and Nitratifractor. Cells are Gram-negative, non-motile, and mesophilic. Growth occurs in the microaerobic range. Type genus: Sulfurovum, order: Campylobacterales, April 2017 \| Volume 8 \| Article 682 Frontiers in Microbiology \| www.frontiersin.org 14 Reclassiﬁcation of Epsilonproteobacteria to Epsilonbacteraeota (phyl. nov.) Waite et al. REFERENCES Bacterial diversity and successional patterns during bioﬁlm formation on freshly exposed basalt surfaces at diﬀuse-ﬂow deep-sea vents. Front. Microbiol. 6:901. doi: 10.3389/fmicb.2015.00901 Eisen, J. A. (1995). The RecA protein as a model molecule for molecular systematic studies of bacteria: comparison of trees of RecAs and 16S rRNAs from the same species. J. Mol. Evol. 41, 1105–1123. doi: 10.1007/BF00173192 vents. Front. 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https://openalex.org/W4229852867	https://www.researchsquare.com/article/rs-24291/latest.pdf	English	null	Influence of Participation in a Quality Improvement Collaborative on Staff Perceptions of Organizational Sustainability	Research Square (Research Square)	2,020	cc-by	8,611	Influence of Participation in a Quality Improvement Collaborative on Staff Perceptions of Organizational Sustainability James H Ford II (  jhfordii@wisc.edu ) Research article Keywords: Sustainability, Quality Improvement Collaborative, Participation, Staff perceptions, NIATx, Intervention DOI: https://doi.org/10.21203/rs.3.rs-24291/v5 ork is licensed under a Creative Commons Attribution 4.0 International License. Read Full License cense:   This work is licensed under a Creative Commons Attribution 4.0 International License Version of Record: A version of this preprint was published on January 7th, 2021. See the published version at https://doi.org/10.1186/s12913-020-06026-3. Page 1/16 Background An organization’s sustainability capacity (SC) represents its ability to implement and maintain the benefits of a systems change over time [1]. Substance use treatment clinics provide services to individuals with an opioid use disorder or alcohol use disorder, including clinical counseling and access to medications, and many of these individuals have co-occurring mental health disorders [2, 3]. These clinics face unique challenges when trying to implement and sustain changes, such as disengaged staff, lack of organizational capacity to sustain change, unease with making changes, or administrative process barriers such as multiple phone calls to schedule an appointment [4-7]. It is important, therefore, to understand how substance use clinic staff perceive the likelihood that changes within their organization will be sustained. Sustainability frameworks suggest that an organizations’ capacity to sustain change is influenced by multilevel factors or constructs related to organizational attributes, environmental contextual features, and intervention characteristics [1, 8-10]. Examples of organizational attributes include leadership support, champion roles, revised policies and procedures or expert coaching support; external contextual features relate to regulatory or financial changes; and innovation attributes focus on ease of use or understanding how likely the benefits of the change would be sustained. Multiple studies have shown that various organizational, external, and innovation attributes inform the likelihood of sustaining an evidence-based practice within an organization [11-19]. Efforts to sustain change within an organization are not only a function of its SC but also depends on staff involvement. Although leadership support and the role of a champion are factors or constructs within these frameworks, less emphasis is placed on the role of staff involvement in implementing and sustaining change. As such, little is known about how participation in change efforts influence staff perceptions of an organization’s SC changes over time. Abstract Background: Sustainability capacity (SC), which is an organization’s ability to implement and maintain change, is influenced by internal attributes, environmental contextual influencers, and intervention attributes. Temporal changes in staff SC perceptions, as well as the influence of quality improvement collaborative (QIC) participation, has generally not been explored. This project addresses this gap, measuring staff SC perceptions at four time points (baseline and every 9 months) for clinics participating in an intervention – the Network for the Improvement of Addiction Treatment QIC initiative (called NIATx200). Methods: A mixed linear model repeated measures analysis was applied to matched staff members (n=908, representing 2,329 total cases) across the evaluation timeframe. Three separate statistical models assessed potential predictors of SC perceptions: Time (Models I-III); NIATx200 intervention, staff job function, and tenure (Models II &III); and NIATx200 participation hours and four organizational variables (Model III). Results: For Model I, staff perceptions of total SC increased throughout most of the study (t1,4=-6.74, p<.0001; t2,4=-3.100, p<.036; t3,4=-0.23, p=ns). Model II did not change Model I’s overall Time effect, but combined NIATx200 services (t=-2.23, p=.026), staff job function (t=-3.27, p=.001), and organizational administrators (t=-3.50, p=.001) were also significantly associated with greater perceptions of total SC. Inclusion of additional variables in Model III demonstrated the importance of a higher participation level (t=-3.09, p<.002) and being in a free-standing clinic (t=-2.06, p<.04) on staff perceptions of total SC. Conclusion: Although staff exposure to sustainability principals was minimal in NIATx200, staff perceptions about their organization’s SC significantly differed over time. However, an organization’s participation level in a QIC became the principal predictor of staff SC perceptions, regardless of other factors’ influence. Given these findings, it is possible to develop and introduce specific sustainability content within the structure of a QIC to assess the impact on staff SC perceptions over time and the sustainment of organizational change. Trial Registration: ClinicalTrials.gov, NCT00934141 Registered July 6, 2009. Retrospectively registered. https://clinicaltrials.gov/ct2/show/NCT00934141 Staff Perceptions of Sustainability Capacity Staff Perceptions of Sustainability Capacity Conceptually, the ISF and CFSC frameworks treat sustainability capacity as a process by which certain organizational attributes such as leadership support or staff involvement influence how change is sustained in an organization. Despite extensive research on sustainability constructs and associated frameworks, few instruments have been developed and extensively utilized in research to quantitatively assess staff perceptions about organizational sustainability capacity associated with the constructs in the ISF or CFSC [22-24]. These constructs represent elements often included in the structure of a quality improvement collaborative (QIC), such as understanding how implementation support and improvement methods might interact with stakeholder participation in training and capacity building activities to influence staff perceptions about an organization’s ability to sustain change. Although the Normalization Process Theory established a framework for evaluating staff perceptions and participation [25], research has not, to date, explored staff perceptions about SC as an outcome measure and how participation in a QIC influences those changes. Further, sustainability instruments have not been utilized to prospectively assess how staff perceptions about sustainability change over time while participating in a QIC. Our objective in this manuscript was to explore temporal changes in staff SC perceptions for individuals working in substance use providers, as measured by the British National Health Services Sustainability Index (BNHS-SI), and how participation in a Network for the Improvement of Addiction Treatment (NIATx) quality improvement collaborative (QIC), called NIATx200, influenced those changes. Data collected during the NIATx200 initiative was utilized to begin addressing this important implementation research issue. This analysis builds on a prior analysis [26] and seeks to answer the research question, “What staff and organizational characteristics predict sustainability levels across the study timeframe?” guided study design and sample selection. The specific aims of the current paper were to: (1) explore temporal changes in staff perceptions about sustainability and (2) assess how staff and organizational characteristics as well as organizational participation in their assigned implementation strategy within a QIC influence changes in sustainability over time. Sustainability Capacity Recent efforts sought to define and refine the classification of sustainability factors or constructs. The Integrated Sustainability Framework (ISF) identified 36 factors across multiple settings (e.g., community, school, clinical/social services) as being associated with sustainability [20]. These factors were grouped into five contexts: (a) outer context, (b) inner context, (c) intervention characteristics, (d) processes, and (e) implementer and population characteristics. Example ISF factors within each context include sociopolitical context Page 2/16 and funding environment (outer context); funding/resources, staffing and turnover (inner context); adaptability, fit with population or context and benefits/need (intervention); partnership/engagement and program evaluation (process); and implementer motivation and attitudes (implementer/population) [20]. Alternatively, the Consolidated Framework for Sustainability Constructs (CFSC) conceptualized 40 constructs across six themes associated with sustainability of change in healthcare settings [21]. Themes include: (a) initiative design and delivery, (b) negotiations related to the initiative processes, (c) organizational setting, (d) people or individuals involved, (e) resources, and (f) external environment. The CFSC also explored approaches [retrospective (after the implementation has occurred) versus prospective (explored throughout implementation)] for assessing staff perceptions about sustainability and the level of focus [organizational (e.g., substance use provider) versus intervention (e.g., a single improvement project)] associated with the assessment of sustainability capacity [21]. The efforts resulted in the identification of the ten most prevalent sustainability constructs within each category (Additional File 1) [21]. Four constructs are common across both level of focus and assessment timing. These include: demonstrating effectiveness and monitoring progress over time (initiative design and delivery), leadership and champions (people involved), and general resources to support sustainability (resources) [21]. Other CFSC constructs varied according to whether they should be assessed at the organizational or intervention level. For example, training and capacity building, and integration with existing programs and policies were not typically assessed within an organizational level of focus; staff perceptions about the belief in the initiative is not assessed for the intervention level of focus and stakeholder participation in the retrospective approach [21]. Study Setting: NIATx200 The NIATx200 initiative built on prior successful NIATx research [4, 27-30]. NIATx200 evaluated the effectiveness of implementation strategies commonly used a QIC. To achieve this objective, NIATx200 recruited 201 addiction treatment clinics in five states (Massachusetts, Michigan, New York, Oregon, and Washington). Clinic eligibility criteria included: 60+ admissions per year, outpatient or intensive outpatient levels of care as defined by the American Society of Addiction Medicine (ASAM); and received some public funding in the past year [31]. Clinics, randomized within states, were stratiﬁed by size (number of patients per year) and management score [32] and assigned to one of four implementation strategies: (1) interest circle calls (n=49), (2) learning sessions (n=54), (3) coaching (n=50), or (4) a combination of all three implementation strategies (n=48). The NIATx200 initiative consisted an 18-month active implementation timeframe. During three distinct implementation periods lasting 6 months, participating clinics implemented organizational changes designed to improve wait time (mean days between first contact and first treatment), retention in treatment (percent of patients retained Page 3/16 Page 3/16 from first to fourth treatment session), and annual admissions. Data was also collected at the staff level about their perceptions associated with organizational readiness for change and sustainability propensity. The structure of the NIATx200 initiative and the description of the implementation strategies are described in more detail elsewhere [31, 33, 34]. from first to fourth treatment session), and annual admissions. Data was also collected at the staff level about their perceptions associated with organizational readiness for change and sustainability propensity. The structure of the NIATx200 initiative and the description of the implementation strategies are described in more detail elsewhere [31, 33, 34]. Mixed-effect regression models determined which implementation strategy was most effective in improving outcomes, as well as being most cost-effective [31]. Improvements in the wait time and admission outcomes for clinics assigned to the coaching and combination strategies significantly differed from clinics assigned to the interest circle strategy and the coaching strategy was the more cost-effective as compared to interest circles [34]. Although no NIATx implementation strategy significantly improved treatment retention (as defined), an exploratory analysis, accounted for early treatment drop-off (i.e., a client not making it to the first treatment session) when measuring retention, showed clinic-level improvements for providers assigned to the coaching, combination and learning session implementation strategies which suggest that how retention was defined impacted the findings [34]. Study Setting: NIATx200 Results from this exploratory analysis clearly indicated that clinic participation in the three intervention (i.e., learning sessions, coaching, and the combination arm) improved the outcomes, with coaching being the most cost-effective strategy. Although differences in clinic attributes did not affect improvements in the outcomes examined in other studies, organizational characteristics were included in these secondary data analyses. Organizational characteristics comprised: (1) non-profit status, (2) whether the clinic was free-standing Alcohol and Drug Abuse Treatment Program or part of a healthcare system, (3) whether the clinic had received accreditation from a national organization such as the Joint Commission on Accreditation of Healthcare Organizations or the Commission on Accreditation of Rehabilitation Facilities, and (4) the metropolitan statistical area (rural or urban status). Implementation Strategies Implementation Strategies The structure of the four NIATx200 implementation strategies represented clinic participation levels. Interest circles involved monthly multi-clinic teleconferences for a total of 18 direct contact hours (18 calls, each one hour in length), and allowed change teams from participating clinics to receive advice from peers and learn new skills. The learning session strategy consisted of three face-to-face multi- day sessions held approximately every six months, which were led by a core faculty team and utilized a common curriculum to offer didactic and experiential learning opportunities. The first learning session consisted of 8.5 hours of content delivered over a single day, while another 13 content hours were delivered over 1.5 days during each of the second and third learning sessions, resulting in a potential for 34.5 total direct contact hours. Clinics assigned to the coaching strategy received a one-day, 4-hour, site visit, as well as participated in monthly one-hour coaching calls; 22 direct contact hours were possible for the coaching strategy. On the calls, the coach and change leader, executive sponsor, and change team reviewed the impact of organizational changes to improve the study outcomes, discussed successes, and identified ideas for future change projects. The combination strategy involved the interest circle calls, coaching, and learning sessions, and consisted of a cumulative possibility of 74.5 direct contact hours. NIATx200 results indicated that clinics assigned to interventions with higher participation hours, where interest circles were the referenced intervention, showed greater improvements in wait time and admissions. As such, staff in the clinics assigned to the interventions with more opportunities to participate in the intervention and be exposed to sustainability concepts would have higher perceptions about the likelihood that changes would be sustained. Outcomes and Measurement Outcomes and Measurement Outcomes and Measurement The NIATx200 initiative utilized the British National Health Services Sustainability Index (BNHS-SI) to assess staff perceptions about the likelihood that a change will be sustained in the organization [23, 24]. The BNHS-SI has been utilized across multiple healthcare settings to assess staff perceptions about the sustainability of an organizational change [24, 35-45] and as a qualitative framework to qualitatively identify factors associated with the concept of sustainability [44, 46-48]. The tool (see Additional File 2 for questions) consists of 10 factors designed to assess overall staff perceptions about sustainability as well as their perceptions across three domains: The tool (see Additional File 2 for questions) consists of 10 factors designed to assess overall staff perceptions about sustainability as well as their perceptions across three domains: 1. Process– benefits beyond helping patients, credibility of the benefits, adaptability of the improved process, and effectiveness of systems to monitor progress. 2. Staff– staff involvement and training to sustain the process, staff attitudes toward sustaining the change, senior leadership engagement, and clinical leadership engagement. 3. Organization– fit with organization’s strategic aims and culture, and infrastructure for sustainability. Organization– fit with organization’s strategic aims and culture, and infrastructure for sustainabilit Page 4/16 Page 4/16 The BNHS-SI utilizes an additive, multi-attribute, utility model to summarize the scores across the three domains (see Additional File 3) which are then totaled to arrive at an overall organization sustainability propensity score [24]. The BNHS-SI utilizes an additive, multi-attribute, utility model to summarize the scores across the three domains (see Additional File 3) which are then totaled to arrive at an overall organization sustainability propensity score [24]. In the NIATx200 initiative, a staff sustainability survey [31] was developed and distributed at baseline and at every subsequent 9-month period (see Figure 1) to prospectively assess clinic staff perceptions about sustainability capacity. The BNHS-SI measures the likelihood that a change will be sustained; therefore, it does not rely on a set sustainability definition (e.g., clinic continued to maintain the intervention after funding ended) when asking staff to complete the instrument. Instead, survey instructions stated that the BNHS-SI was “designed to gauge your organization’s propensity for sustaining changes”. As such staff were asked to “think about one specific change implemented as part of the NIATx200 project”; and then select one of four options for each of the 10 factors that best describes sustainability in their organization. Data Collection Staff were invited to complete a paper survey or use a link in the invitation letter to complete the survey online. The survey also collected staff demographic information related to job function, employment status, and tenure within the organization. Two additional questions (i.e., “What is the first initial of your mother’s maiden name?” and “On what day of the month is your birthday?”) were combined with staff demographic characteristics and the clinic ID to create a unique identifier for individual staff members that allowed matching of individual survey responses to be tracked over time. Clinic participation (direct contact hours) in the assigned implementation strategy and the number of persons from the clinic participating in the assigned implementation strategy were recorded in real time by NIATx200 research staff and coaches. Design and Sample The unique identifier was utilized to match individual survey responses across the four different time points (Figure 1). As a result, all analyses are based on responses from the same staff members (n=908, representing 2,329 total cases) across the evaluation timeframe. An important variable for this analysis is each clinic’s cumulative level of staff participation in QIC activities throughout the 27-month intervention interval (at baseline and approximately every nine months) (called Total Participation). Participation in each of the four study interventions was measured separately but, for the purpose and this study, was aggregated into a Total Participation metric. This variable is used to determine the influence of the number of encounters with the implementation strategy during the 27-month period. Although this factor does not represent the total number of staff who took part in each activity, and therefore reflects a clinic-level influence, it remains dependent on overall staff involvement. As such, degree of staff participation is considered appropriate and relevant, and is retained for this sample. Outcomes and Measurement Our team utilized a similar approach when assessing sustainability capacity within the Veterans Administration [38-40]. For this analysis, the cumulative extent of staff beliefs that changes implemented as part of the NIATx200 initiative would be sustained (called the Total Sustainability Score) was the primary outcome. Three secondary outcomes also were evaluated – representing scores from the process, staff, and organization domains from the BNHS-SI tool (called Process, Staff, and Organization Domain Scores, respectively). Data Collection Data Collection Analysis IBM SPSSv26® was used to calculate all descriptive statistics and to estimate each model by calculating the parameter estimates for fixed effects at 95% confidence intervals. This study is reported in full accordance with the StaRI checklist [Additional File 4] [49]. Analysis Analysis comprised both simple descriptive statistics and multivariate model building. Descriptive statistics were calculated for all variables used for this study. The type of descriptive values depended on whether the variables were continuous or categorical. For continuous variables, the mean, standard deviation (SD), and min/max are reported, while the frequencies of each category are provided for the categorical variables. Bivariate analyses were conducted on the primary outcome measure (Total Sustainability Score) and each anticipated study variable before entry into model; all variables used in the model demonstrated a significant independent association with the sustainability total. The multivariate method was a linear mixed model repeated measures analysis that fit three separate statistical models to assess potential predictors of staff-level Total Sustainability Score, as well as on Process, Staff and Organization Domain Scores. For each model, a Repeated Covariance Type – A1(1): Heterogeneous – was used, which assumes different variances at each measurement time as well as correlations across time points that become weaker over those successive assessment times. All variables were entered into the models as fixed effects, and a maximum likelihood method was used to estimate the variable parameters. The statistical models are as follows: Page 5/16 Page 5/16 containing only the variable representing the four time points during the NIATx200 initiative (Time) 1. Model I – containing only the variable representing the four time points during the NIATx200 init 1. Model I – containing only the variable representing the four time points during the NIATx200 initiative (Time), 2. Model II – containing Time, plus NIATx200-provided Implementation Strategies (i.e., learning sessions, interest circle calls, coaching sessions, or service combinations) and Job Function (i.e., administrative vs. clinical), and 2. Model II – containing Time, plus NIATx200-provided Implementation Strategies (i.e., learning sessions, interest circle calls, coaching sessions, or service combinations) and Job Function (i.e., administrative vs. clinical), and 2. Model II – containing Time, plus NIATx200-provided Implementation Strategies (i.e., learning se sessions, or service combinations) and Job Function (i.e., administrative vs. clinical), and 3. Model III – containing the variables from Models I and II, plus organizational characteristics and the cumulative extent of participation in NIATx200-provided strategies (i.e., total number of hours). 3. Model III – containing the variables from Models I and II, plus organizational characteristics and the cumulative extent of participation in NIATx200-provided strategies (i.e., total number of hours). Descriptive variables The final sample size represents responses from the same staff members (n=908, representing 2,329 total cases) across the evaluation timeframe. The Total Participation in the NIATx200 implementation strategies represented the only continuous independent variable used in the models and ranged from no time to 64 hours (mean = 26.03, SD = 18.23). Table 1 lists the category frequencies for the remaining model variables, as well as the mean Total Sustainability Scores associated with each variable category. Repeated measures Model I Repeated measures Model I Page 6/16 Page 6/16 Page 6/16 Primary Outcome: Model I (see Table 2) yielded strong overall predictive significance for Time (F=7.270, p<.0001), with staff perceptions about overall sustainability capacity increasing throughout most of the study. Primary Outcome: Model I (see Table 2) yielded strong overall predictive significance for Time (F=7.270, p<.0001), with staff perceptions about overall sustainability capacity increasing throughout most of the study. Secondary Outcomes: The time effect pattern for the primary outcome was also identified for the process and organizational domains. However, Staff Domain Scores evidenced a statistically significant increase only when comparing the study endpoints. Secondary Outcomes: The time effect pattern for the primary outcome was also identified for the process and organizational domains. However, Staff Domain Scores evidenced a statistically significant increase only when comparing the study endpoints. Repeated measures Model II Repeated measures Model II Primary Outcome: Table 3 shows that Model II did not change the overall Time effect profile identified in Model I for overall staff perceptions about sustainability capacity. However, the assigned NIATx200 strategy and staff job function were significant – participation in combined services (compared to learning sessions only) and organization administrators were associated with greater perceptions about sustainability propensity. Secondary Outcomes: While the overall Time effect did not differ between Model II and Model I for the Staff Domain Score, the assigned NIATx200 strategy comparing combined services to learning sessions only and staff job function were significantly associated with perceptions about staff involvement. The Time effect for the Organization Domain Score did not change from Model I to Model II, and only the assigned NIATx200 strategy was significantly associated with perceptions related to organizational capacity to support sustainability. For the Process Domain Score, the pattern of effect for time changed somewhat between Model II and Model I, and only staff job function was significant. Page 7/16 Page 7/16 Repeated measures Model III Primary Outcome: The addition of Total Participation levels and organizational characteristics in Model III demonstrated the importance of being in a free-standing agency and having a higher participation, as measured by direct exposure hours, on overall beliefs about sustainability, but changed the statistical profiles for other model variables (see Table 4). For example, including Total Participation resulted in staff perceptions about sustainability being statistically significant only when comparing the two study endpoints. In addition, higher Total Sustainability Scores shifted to other implementation strategies (i.e.., coaching). Finally, administrators continued to report a greater sustainability propensity than clinicians. Secondary Outcomes: The findings for the Staff Domain Score were similar to the Model III results for overall sustainability capacity. Total Participation hours strongly influenced staff perceptions about the Process and Organization Domain Scores. Participation in the coaching implementation strategy, staff who were administrators, and working in a for-profit, free-standing, facility was associated with greater sustainability propensity for the process domain. For the Organizational Domain Score, the time effect increased throughout most of the study and assignment to the coaching strategy was significant, as was being involved in a free-standing facility or an agency located in a rural setting. Page 8/16 Discussion Such efforts could be support through sustainability specific modules (introduced early in the collaborative) followed by a re-enforcement of the sustainability concepts. In addition, the QIC could be structured to incorporate sustainability learning sessions or a coach-led sustainability site visit to re-enforce sustainability concepts or help the organization develop a sustainability plan. In addition to staff-level factors, organizational characteristics had some notable associations with sustainability levels. Interestingly, affiliation with a free-standing Alcohol and Drug Treatment Program had the most prevalent effect, being associated with higher total sustainability, as well as with the process and organization sustainability sub-domains. For-profit facilities demonstrated higher levels of process sustainability, while rural facilities were associated with greater organizational sustainability. Perhaps not surprisingly, none of the organizational factors were statistically related to the staff sub-domain of sustainability. Study results suggest, as outlined in the Normalization Process Theory, that (1) staff involvement (working individually and collectively) to implement the change and (2) social processes (coherence, cognitive participation, collection action and reflexive monitoring) are associated with how an innovation is embedded, integrated and sustained within the organization [25, 50, 51]. Specifically, our results demonstrated that increased exposure promotes greater belief in sustainability, which was supported both through combinations of implementation strategies and extent of participation. When added to the multivariate statistical model, an organization’s cumulative participation level in a QIC became a principal predictor of staff SC perceptions, over-riding the effects of the other factors. This finding is consistent with prior research showing that free-standing clinics participated more in NIATx200 [33]. Indeed, the pattern of effects found for the combined implementation strategies seems to be more a function of the hours of participation; when controlling for Total Participation hours, combined strategies are not as predictive of Total Sustainability as individual strategies. Process sustainability focuses on staff beliefs in the benefits of and the credibility of the evidence for the change; how easy it is to adapt the change to the organization; and the presence of systems to monitor change. Higher levels of process sustainability in for-profit facilities may suggest that the infrastructure (e.g., training or culture) is better suited to support the constructs associated with cognitive participation (e.g., legitimation and buy-in) as well as reflexive monitoring (e.g., monitoring implementation impact) within the Normalization Process Theory [51]. Discussion Our study explored how the extent of participation in a QIC, based on implementation strategy assignment, was associated with staff perceptions about sustainability. The study was framed in the context of two conceptual sustainability frameworks (i.e., ISF and CFSC) with clear operational definitions using a rigorous outcome measure of sustainability capacity – the BNHS-SI. To the best of our knowledge, this study was the first to track how staff perceptions about sustainability changed longitudinally. We accomplished this by using data collected over a 27-month period from a convenience sample of responses from the same provider staff members participating in a QIC (NIATx200). In addition, the study is the first to measure the number of hours of provider participation in the assigned implementation strategy of the QIC and utilize the level of participation as a predictor of how staff perceptions about sustainability change over time. Although NIATx200 offered minimal exposure to sustainability principals, staff perceptions about their organization’s overall SC continued throughout the QIC implementation period, with the most significant improvement occurring over the first 9-month period. However, there was a clear indication of a saturation effect for perceptions of improved sustainability, with subsequent changes in overall sustainability scores eventually showing no noticeable differences as time progressed. Similar patterns occurred for the process, staff, and organizational domains. These findings support a need to continue reinforcing the importance of sustainability within organizations, especially later during the implementation process. An evaluation of long-term sustainment in the NIATx200 initiative found that between 27% to 40% of participating clinics sustained changes for one of the three study outcomes but only 12% of the clinics sustained changes Page 9/16 Page 9/16 for two of the three study outcomes [45]. In some instances, improvement and subsequent sustainment occurred after the end of the active NIATx200 implementation period. It was additionally noteworthy that there were noticeable staff differences in perceptions about whether change will be sustained within organizations. Administrators and managers were much more likely to anticipate a propensity for enhanced sustainability than were clinicians within the organization. These differences are more prominent for attributes associated with the process and staffing domains of the BNHS-SI. As a result, the implementation process requires further effort to engage clinicians to convince them that effective change is beneficial and can be both attained and sustained. Discussion Collective Action emphasizes the organizational resources needed to support change as well as the workability of the change in the organization [25, 50, 51]. In the BNHS-SI, the idea of workability may be associated with adaptability (i.e., ease of adapting the change to fit the organization) with organizational resources being associated with the infrastructure (i.e., policies and procedures and resources) to support the sustainability of change [23, 24]. Rural treatment facilities most likely do not have the resources to invest in changes that will not be sustained and therefore, take steps to establish an infrastructure needed to support and sustain change. The results suggest that repeat exposure in different implementation strategies to sustainability concepts may help to ingrain within staff the importance of sustaining organizational change. Given these findings, it seems possible to develop and introduce specific sustainability content (e.g., how to develop and implement a sustainability plan) within the structure of a QIC as a means to assess the impact on staff SC perceptions over time and the sustainment of organizational change. The effectiveness of a QIC with a sustainability component, compared to one without, could be evaluated within a randomized control trial that assigns organizations to the implementation strategy using baseline staff perceptions about organizational readiness or sustainability capacity The BNHS-SI assesses the six sustainability constructs from the CFSC that consistently have been the most commonly found in at least 75% of both the sustainability approaches (retrospective versus prospective) and level of focus (organizational versus intervention) within the CFSC framework [21]. Table 5 outlines the conceptual relationships between CFSC, and ISF constructs, the prominent attributes associated with these constructs and how these attributes are measured within the BNHS-SI. For example, staff involvement and training emphasize the need for orienting and training staff to be able to deliver the initiative successfully in the CFCS and aligns with two constructs in the ISF Process domain (i.e., partnership/engagement and training/support/supervision). However, the idea measured in the BNHS is that staff have been involved from the beginning of the change and are adequately trained to sustain the improved process. Study results indicated that each hour of total participation in the NIATx QIC increased staff perceptions, as measured by the BNHS-SI, Page 10/16 Page 10/16 related to these six common sustainability constructs within the CFCS. Strengths and Limitations Strengths and Limitations Several strengths characterize this study. First, the study design involved data collection at four points in time, allowing prospective analyses. Second, the use of a unique identifier algorithm permitted a repeated measures statistical approach. The same cases could be tracked for changes across time, allowing for a profile of the evaluation of individuals’ perceptions about sustainability. Finally, the structure of the implementation strategies within the NIATx200 initiative allowed for clinic-level participation to be tracked within the assigned implementation strategy. Although this study had strengths, certain limitations also need to be considered. First, as mentioned previously, the data collected do not directly represent staff-level participation in QIC activities. Participation was calculated for each clinic, but still ultimately depended on staff involvement. Second, although repeated-measures analysis allowed for profiles of unique, individual, cases across time, case variation between the time phases might have existed. However, the repeated covariance model likely lessened the impact of such variation. Third, while the study comprised a 27-month intervention timeframe, consisting of baseline measurement and three 9-month intervals, there was some variation in the 9-month periods when staff surveys were completed (Figure 1). Fourth, the sample included only responses from linked staff surveys over the four data collection time periods. Although we had staff responses from 83.6% of the participating clinics, the exclusion of certain clinics precluded controlling for successful improvement in the outcomes (wait time, retention, or admission) or exploring how success could influence staff attitudes towards sustainability. Finally, the data represented limited staff demographic information, related to employment only. Further application of the NIATx framework, which utilizes a broader variety of staff characteristics, could provide additional insights into their influences on sustainability propensity. Conclusion Sustainability of organizational change represents an increasingly important focus of implementation research. Research has shown that the scientific evidence for how staff perceptions about organizational sustainability capacity, as well as what influences changes over time, represent a gap in dissemination and implementation research. These findings addressed this recognized gap in the literature. Although staff perceptions about sustainability capacity changed over time, this analysis determined that staff participation, representing the level of involvement in the assigned implementation strategy, is the most significant contributor that influenced changes in staff perception about sustainability propensity over time. The impact of participation “dose exposure” on sustainability perceptions highlight the need for dissemination and implementation strategies to re-enforce concepts associated with sustainability to improve staff perceptions about the sustainability capacity of the organization Since a QIC is often utilized in implementation efforts, a recognition that staff participation influences staff perceptions about sustainability can inform its design and structure and could provide a foundational step toward determining how change is sustained in substance abuse clinics. The unique perspectives from this study address recognized gaps in the literature, including when and how to incorporate sustainability concepts in a QIC to increase the likelihood that changes will be sustained. Knowing that participation in a QIC influences staff perceptions, researchers and practitioners can work to improve the structure of a QIC by developing activities or interventions (e.g., develop and introduce specific sustainability content) or modify implementation strategies (e.g., coaching for sustainability). Such efforts might promote greater staff involvement or participation in the QIC. Future research could assess the impact of these changes on the relationship between how staff perceptions of sustainability capacity change over time and whether a change is successfully sustained in the organization. Discussion Future research can attempt to directly replicate these findings by integrating the BNHS-SI as a measurement tool within the CFCS. Further research should explore exactly how these organizational characteristics independently and within the Normalization Process Theory impact participation in a QIC and staff perceptions that changes will be sustained. Evidence suggests that factors associated with sustainability include the fit of the innovation or change within the organizational culture or a culture that encourages flexibility and adaptability [52, 53], and one items in the organizational sub-process in the BNHS-SI assesses the degree that staff perceives the innovation as fitting within the organizations’ strategic aims and culture. Our study found that staff perceptions in this sub-process changed over time and that participation was associated with the improvement. Furthermore, another study identified six guiding principles associated with the sustainability of a culture change [54]. Several enabling factors associated with these principles are assessed, in part, by the BNHS-SI, such as: the perceived value of organizational data (Principle: Continuously assess and learn from cultural change and assessed within the Process sub-domain in the BNHS-SI); the perceived value of organizational data (Principle: Continuously assess and learn from cultural change and assessed within the Process sub-domain in the BNHS-SI); willingness to relinquish control (Principle: Promote staff engagement and assessed within the and willingness to relinquish control (Principle: Promote staff engagement and assessed within the Staff sub-domain in the BNHS-SI); and perception that the change is legitimate and credible (Principle: Align vision and action and assessed within the Process sub-domain in the BNHS-SI). Page 11/16 Page 11/16 The association of these factors with sustained cultural change would suggest that staff perceptions of organizational sustainability might be associated with the organizational culture that drives those perceptions. However, we did not assess either the changes in organizational culture or a relationship between culture and staff perceptions about sustainability. Future research should explore the role of organizational culture on staff perceptions about sustainability. References 1. Schell S, Luke D, Schooley M, Elliott M, Herbers S, Mueller N, Bunger A: Public health program capacity for sustainability: a new framework. Implementation Science 2013, 8:15. 1. Schell S, Luke D, Schooley M, Elliott M, Herbers S, Mueller N, Bunger A: Public health program capacity for sustainability: a new framework. Implementation Science 2013, 8:15. 2. SAMHSA: Substance Abuse Mental Health Services Administration, Center for Behavioral Health Statistics and Quality. Behavioral Health Services Information Series: National Directory of Drug and Alcohol Abuse Treatment Facilities 2015, NSDUH Series H-48, HHS Publication No.(SMA) 16-4940. In Substance Abuse and Mental Health Services Administration, Rockville, MD; 2015. 2. SAMHSA: Substance Abuse Mental Health Services Administration, Center for Behavioral Health Statistics and Quality. Behavioral Health Services Information Series: National Directory of Drug and Alcohol Abuse Treatment Facilities 2015, NSDUH Series H-48, HHS Publication No.(SMA) 16-4940. In Substance Abuse and Mental Health Services Administration, Rockville, MD; 2015. 3. Substance Abuse and Mental Health Services Administration: Key substance use and mental health indicators in the United States: Results from the 2018 National Survey on Drug Use and Health. (Center for Behavioral Health Statistics and Quality SAaMHSA ed. Rockville, MD; 2019. 3. Substance Abuse and Mental Health Services Administration: Key substance use and mental health indicators in the United States: Results from the 2018 National Survey on Drug Use and Health. (Center for Behavioral Health Statistics and Quality SAaMHSA ed. Rockville, MD; 2019. 4. Ford II JH, Green CA, Hoffman KA, Wisdom JP, Riley KJ, Bergmann L, Molfenter T: Process improvement needs in substance abuse treatment: Admissions walk-through results. Journal of substance abuse treatment 2007, 33:379-389. 4. Ford II JH, Green CA, Hoffman KA, Wisdom JP, Riley KJ, Bergmann L, Molfenter T: Process improvement needs in substance abuse treatment: Admissions walk-through results. Journal of substance abuse treatment 2007, 33:379-389. 5. Stumbo SP, Ford JH, Green CA: Factors influencing the long-term sustainment of quality improvements made in addiction treatment facilities: a qualitative study. Addiction science & clinical practice 2017, 12:26. 5. Stumbo SP, Ford JH, Green CA: Factors influencing the long-term sustainment of quality improvements made in addiction treatment facilities: a qualitative study. Addiction science & clinical practice 2017, 12:26. 6. Alanis-Hirsch K, Croff R, Ford JH, 2nd, Johnson K, Chalk M, Schmidt L, McCarty D: Extended-Release Naltrexone: A Qualitative Analysis of Barriers to Routine Use. J Subst Abuse Treat 2016, 62:68-73. 7. Declarations Ethics approval and consent to participate. This study has been reviewed and was approved by was approved by the University of Wisconsin Social and Behavioral Sciences Institutional Review Board (SE-2006-0521) and Health Sciences minimal risk IRB (2014-1048). Consent for publication. Not applicable Consent for publication. Not applicable Availability of data and materials. Datasets generated and/or analyzed during the current study are not publicly available because the data contains potentially identifying information but are available from the corresponding author on reasonable request. ompeting interests. The authors declare that they have no competing interests. Competing interests. The authors declare that they have no competing interests. Funding. This study was funded by NIDA (R01 DA020832, R21 DA36700). NIDA was not involved in data collection, data analysis or writing of this paper. The statements made here are those of the authors. Authors’ contributions. All authors made significant contributions to this manuscript. JHF planned and implemented the study design, was involved in the data collection, and led the development and writing of the manuscript. AMG conducted the data analysis and was a major contributor in writing the manuscript. All authors read and approved the final manuscript. Portions of the research was presented at the 11th Annual Dissemination and Implementation conference. Acknowledgements. The authors would like thank Ka Xiong for her review of the manuscript. We would also like to acknowledge the thousands of very dedicated, very busy staff from the substance abuse clinics in Massachusetts, Michigan, New York, Oregon, and Washington who participated in the NIATx200 study as well as members of the original NIATx200 research team. An earlier version of this research was presented at the 11th Annual Conference on the Science of Dissemination and Implementation. 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Higuchi KS, Davies B, Ploeg J: Sustaining guideline implementation: A multisite perspective on activities, challenges and supports. Journal of clinical nursing 2017, 26:4413-4424. 49. Pinnock H, Barwick M, Carpenter CR, Eldridge S, Grandes G, Griffiths CJ, Rycroft-Malone J, Meissner P, Murray E, Patel A: Standards for reporting implementation studies (StaRI) statement. Bmj 2017, 356:i6795. 50. May C, Finch T: Implementing, embedding, and integrating practices: an outline of normalization process theory. Sociology 2009, 43:535-554. 51. Figure 1 NIATx 200 Study Timeline References McEvoy R, Ballini L, Maltoni S, O’Donnell CA, Mair FS, MacFarlane A: A qualitative systematic review of studies using the normalization process theory to research implementation processes. Implementation Science 2014, 9:2. 52. Palinkas LA, Spear SE, Mendon SJ, Villamar J, Reynolds C, Green CD, Olson C, Adade A, Brown CH: Conceptualizing and measuring sustainability of prevention programs, policies, and practices. Transl Behav Med 2020, 10:136-145. Page 15/16 Page 15/16 53. Xiang X, Robinson-Lane SG, Rosenberg W, Alvarez R: Implementing and sustaining evidence-based practice in health care: The Bridge Model experience. J Gerontol Soc Work 2018, 61:280-294. 54. Willis CD, Saul J, Bevan H, Scheirer MA, Best A, Greenhalgh T, Mannion R, Cornelissen E, Howland D, Jenkins E, Bitz J: Sustaining organizational culture change in health systems. J Health Organ Manag 2016, 30:2-30. Fi 53. Xiang X, Robinson-Lane SG, Rosenberg W, Alvarez R: Implementing and sustaining evidence-based practice in health care: The Bridge Model experience. J Gerontol Soc Work 2018, 61:280-294. 54. Willis CD, Saul J, Bevan H, Scheirer MA, Best A, Greenhalgh T, Mannion R, Cornelissen E, Howland D, Jenkins E, Bitz J: Sustaining organizational culture change in health systems. J Health Organ Manag 2016, 30:2-30. 53. Xiang X, Robinson-Lane SG, Rosenberg W, Alvarez R: Implementing and sustaining evidence-based practice in health care: The Bridge Model experience. J Gerontol Soc Work 2018, 61:280-294. 54. Willis CD, Saul J, Bevan H, Scheirer MA, Best A, Greenhalgh T, Mannion R, Cornelissen E, Howland D, Jenkins E, Bitz J: Sustaining organizational culture change in health systems. J Health Organ Manag 2016, 30:2-30. Figures Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. AdditionalFile1Lennox.pdf AdditionalFile2SustainabilityModelScoresheetNoscores.pdf AdditionalFile3SustainabilityModelScoresheetwscores.pdf AdditionalFile4StaRIchecklist.pdf AdditionalFile4StaRIchecklist.pdf Page 16/16 Page 16/16
https://openalex.org/W2775103183	https://europepmc.org/articles/pmc5749501?pdf=render	English	null	STAT5BN642H is a driver mutation for T cell neoplasia	The Journal of clinical investigation/The journal of clinical investigation	2,017	cc-by	14,043	Conflict of interest: The authors have declared that no conflict of interest exists. Submitted: April 12, 2017; Accepted: October 5, 2017. License: This work is licensed under the Creative Commons Attribution 4.0 Inter- national License. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Reference information: J Clin Invest. 2018;128(1):387–401. https://doi.org/10.1172/JCI94509. The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E Introduction due to differences in the level of STAT5 proteins and possible dis- tinct chromatin-remodeling capabilities as a result of interactions with other transcriptional regulatory proteins (10–12). Recently, the hSTAT5BN642H mutation was described as a gain-of-function (GOF) mutation in leukemic patients that causes enhanced and prolonged tyrosine phosphorylation (13–27). This mutation is associated with a poor prognosis and an increased risk of relapse, despite the use of combination chemotherapy (13). The N642H mutation is located in the center of the STAT5B SH2 domain, the phosphotyrosine-binding domain that is essential for the forma- tion of parallel STAT5 dimers and efficient nuclear transloca- tion (1). STAT5BN642H has been found in more than 90 patients with 7 types of aggressive leukemia or lymphoma including γδ T cell–derived lymphoma, hepatosplenic T cell lymphomas, large granular lymphocytic (LGL) leukemia, T cell acute lymphoblas- tic leukemia (T-ALL), T cell prolymphocytic leukemia, NK/T cell lymphoma, and enteropathy-associated T cell lymphoma (13–25). The 2 signal transducer and activator of transcription 5 proteins, STAT5A and STAT5B, encoded by 2 different genes with 89% DNA sequence homology, are downstream targets of cytokines and growth factors (1, 2). STATs are highly expressed and/or hyperactivated by tyrosine and serine phosphorylation in numer- ous hematopoietic cancers (3–6). The 2 STAT5 proteins have been reported to have redundant functions largely due to overlapping genome binding sites (7–9). There are different phenotypes upon genetic loss or somatic point mutation resulting in hyperactivation of STAT5A or STAT5B. STAT5B has a dominant role in immune cells, as suggested by its higher expression levels compared with STAT5A in NK or T cell subsets (7–9). Interestingly, mutations in cancer patients have predominantly been found in the Src homology 2 (SH2) domain of human STAT5B (hSTAT5B). This suggests that differences in hematopoietic transformation are Studies in mice have implicated STAT5 signaling in the expan- sion of CD8+ T cells as well as the development of lymphoblastic lymphoma (28). Nevertheless, there is no evidence of whether the hSTAT5BN642H mutation is capable of driving the development and progression of leukemia. Drug-sensitivity tests on hSTAT5BN642H- expressing leukemic blasts from T-ALL patients indicated that the mutation provides a survival advantage in leukemic cells (17). STAT5BN642H is a driver mutation for T cell neoplasia Ha Thi Thanh Pham,1,2 Barbara Maurer,1,2 Michaela Prchal-Murphy,3 Reinhard Grausenburger,3 Eva Grundschober,3 Tahereh Javaheri,1,2 Harini Nivarthi,4 Auke Boersma,5 Thomas Kolbe,6,7 Mohamed Elabd,1 Florian Halbritter,4 Jan Pencik,1 Zahra Kazemi,8,9 Florian Grebien,1 Markus Hengstschläger,10 Lukas Kenner,1,11,12 Stefan Kubicek,4 Matthias Farlik,4 Christoph Bock,4,8,13 Peter Valent,14,15 Mathias Müller,2 Thomas Rülicke,5 Veronika Sexl,3 and Richard Moriggl1,2,8 1Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria. 2Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria. 3Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Vienna, Austria. 4CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria. 5Institute of Laboratory Animal Science, and 6Biomodels Austria (Biat), University of Veterinary Medicine Vienna, Vienna, Austria. 7IFA-Tulln, University of Natural Resources and Life Sciences, Tulln, Austria. 8Medical University of Vienna, Vienna, Austria. 9Center of Physiology and Pharmacology, Vienna, Austria. 10Center of Pathobiochemistry and Genetics, Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria. 11Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria. 12Unit of Pathology of Laboratory Animals, University of Veterinary Medicine Vienna, Vienna, Austria. 13Max Planck Institute for Informatics, Saarbrücken, Germany. 14Department of Internal Medicine I, Division of Hematology and Hemostaseology, and 15Ludwig Boltzmann-Cluster Oncology, Medical University of Vienna, Vienna, Austria. STAT5B is often mutated in hematopoietic malignancies. The most frequent STAT5B mutation, Asp642His (N642H), has been found in over 90 leukemia and lymphoma patients. Here, we used the Vav1 promoter to generate transgenic mouse models that expressed either human STAT5B or STAT5BN642H in the hematopoietic compartment. While STAT5B- expressing mice lacked a hematopoietic phenotype, the STAT5BN642H-expressing mice rapidly developed T cell neoplasms. Neoplasia manifested as transplantable CD8+ lymphoma or leukemia, indicating that the STAT5BN642H mutation drives cancer development. Persistent and enhanced levels of STAT5BN642H tyrosine phosphorylation in transformed CD8+ T cells led to profound changes in gene expression that were accompanied by alterations in DNA methylation at potential histone methyltransferase EZH2-binding sites. Aurora kinase genes were enriched in STAT5BN642H-expressing CD8+ T cells, which were exquisitely sensitive to JAK and Aurora kinase inhibitors. Together, our data suggest that JAK and Aurora kinase inhibitors should be further explored as potential therapeutics for lymphoma and leukemia patients with the STAT5BN642H mutation who respond poorly to conventional chemotherapy. jci.org Volume 128 Number 1 January 2018 Results (TET2) and DNA methyltransferase 3α (DNMT3A) affecting DNA methylation are frequently found in T cell lymphoma (29). TET1/2 was also shown to interact with STAT5 in Tregs that are strictly dependent on STAT5 because of direct regulation of the STAT5 target genes FOXP3 and CD25 (30). Interestingly, the DNMT3A gene was shown to be controlled by STAT5 in AML cells (31). Drugs interfering with epigenetic changes are powerful tools in cancer drug development and have found entry into therapeu- tic strategies (29). A key role of STAT5 is to support the process of histone acetylation and methylation in T cells, which was shown for the TCR locus (32, 33). Furthermore, the histone methyltrans- ferase EZH2 and histone deacetylase 1 (HDAC1) were shown to be recruited via STAT5 binding (34, 35). hSTAT5BN642H is an activating mutation in hematopoietic cells. Somatic mutations of STAT5B, many of which are located in the SH2 domain, have been found in patients with lymphoid neoplasia (Figure 1A) (13–26, 36). To investigate the impact of these somatic mutations on hSTAT5B activity, we analyzed different potential GOF mutations in 293T cells using tyrosine phosphorylation of STAT5 (pY-STAT5) as a correlation for activity. We observed high pY-STAT5 levels under steady-state conditions in cells expressing the N642H muta- tion, the most frequent STAT5 mutation in patients with leukemia or lymphoma. The 2 SH2 domain variants Y665H and Y665F also showed enhanced activity in the absence of cytokine stimulation (Figure 1B). We observed a similar pattern of pY-STAT5B upon expression of the N642H mutant in the murine pro–B cell line Ba/ F3 and the murine myeloid cell line 32D (Figure 1C). In contrast, expression of hSTAT5B at comparable levels failed to induce signif- icant pY-STAT5 in the absence of IL-3 stimulation (Figure 1C and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI94509DS1). Impor- tantly, hSTAT5BN642H rendered Ba/F3 and 32D cells cytokine inde- pendent, supporting the finding of a proto-oncogenic potential of hSTAT5BN642H (15) (Supplemental Figure 1B). Here, we investigated the oncogenic potential of the hSTAT5BN642H mutation compared with the nonmutated hSTAT5B using Vav1-driven transgenic mouse models. In contrast to WT hSTAT5B, moderate hSTAT5BN642H expression levels triggered leu- kemia or lymphoma development, which manifested as a transplant- able CD8+ T cell disease. Related Commentary: p. 113 Conflict of interest: The authors have declared that no conflict of interest exists. Submitted: April 12, 2017; Accepted: October 5, 2017. License: This work is licensed under the Creative Commons Attribution 4.0 Inter- national License. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Reference information: J Clin Invest. 2018;128(1):387–401. https://doi.org/10.1172/JCI94509. Epigenetic abnormalities are major drivers of hematopoiet- ic malignancies. Mutations in Tet methylcytosine dioxygenase 2 jci.org Volume 128 Number 1 January 2018 3 8 7 The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 1. hSTAT5BN642H is an activating mutation. (A) Schematic of STAT5B mutations identified in leukemia and lymphoma patients. Each dot represents 1 patient. (B) WB analysis of pY-STAT5, total STAT5 protein, and HSC70 in 293T cells that were transiently transfected with different hSTAT5B (hS5B) variants using a pMSCV-IRES-GFP vector, with or without growth hormone (GH) stimulation. (C) WB analysis of pY-STAT5, STAT5, FLAG, and HSC70 in hSTAT5B- or hSTAT5BN642H-expressing (N642H) Ba/F3 cells with or without IL-3 stimulation. (B and C) Nontransfected and pMSCV-transfected cells are shown as controls. Data presented in B and C are representative of 3 independent experiments. Samples were run on parallel gels for B and C, and a load- ing control is provided for each gel. Figure 1. hSTAT5BN642H is an activating mutation. (A) Schematic of STAT5B mutations identified in leukemia and lymphoma patients. Each dot represents 1 patient. (B) WB analysis of pY-STAT5, total STAT5 protein, and HSC70 in 293T cells that were transiently transfected with different hSTAT5B (hS5B) variants using a pMSCV-IRES-GFP vector, with or without growth hormone (GH) stimulation. (C) WB analysis of pY-STAT5, STAT5, FLAG, and HSC70 in hSTAT5B- or hSTAT5BN642H-expressing (N642H) Ba/F3 cells with or without IL-3 stimulation. (B and C) Nontransfected and pMSCV-transfected cells are shown as controls. Data presented in B and C are representative of 3 independent experiments. Samples were run on parallel gels for B and C, and a load- ing control is provided for each gel. R E S E A R C H A R T I C L E Quantification of the WB was performed using ImageJ. Data are representative of 3 independent experiments. (C) Flow cytometric analysis of the percentage of LSKs, LT-HSCs (CD150+CD48–), ST-HSCs (CD150+CD48+), MPPs (CD150–CD48+), (D and E) common lymphoid progenitors (lineage−Sca1+IL-7R+AA4+), MPCs (lineage−Sca1–IL-7R–c-Kit+), and CD3+ cells in the BM of WT, hSTAT5B, and hSTAT5BN642H mice. Analyses in C–E included 7-week-old WT (n = 7), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. Data represent the mean ± SD. P < 0.05, P < 0.01, and *P < 0.001, by 1-way ANOVA with Bonferroni’s correction. Analysis of WBC counts in hSTAT5BN642H mice revealed an increase of approximately 20-fold compared with that detected in hSTAT5B and WT mice (Figure 3C). The WBC count in hSTAT5B mice only increased slightly with age but remained within a phys- iological range (Supplemental Figure 3B). The drastic increase in the WBC count in STAT5BN642H mice was correlated with an expan- sion of CD8+ T cells (Figure 3C). Similarly, CD8+ T cells increased by 3-fold in the lymph nodes (LNs) of hSTAT5BN642H mice (Fig- ure 3D), which was confirmed by immunohistochemical staining (Supplemental Figure 3C). The numbers of CD4+ T cells were also moderately increased, whereas the percentage, but not the total number, of CD19+ B cells was reduced in the LNs of hSTAT5BN642H mice compared with controls (Figure 3E and Supplemental Fig- ure 3D). Hematocrit levels were comparable in all mouse models (Supplemental Figure 3E). We also observed a mild expansion of other hematopoietic cell types such as CD19+ B cells, CD4+ T cells, and CD11b+Gr1+ myeloid cells in the spleen (Figure 3E and Supple- mental Figure 3F). tem, including hematopoietic stem cells (HSCs) (37) (Supplemen- tal Figure 2, A and B). Transgenic mice expressing hSTAT5BN642H rapidly developed malignant disease leading to death between 40 and 100 days of age. hSTAT5B-transgenic mice showed no signs of disease when sacrificed at the age of 12 months or older (Figure 2A). Despite expressing comparable levels of total STAT5, only hSTAT5BN642H-transgenic mice showed elevated pY-STAT5 signals, indicating strong and persistent tyrosine phosphorylation (Figure 2B). In line with this observation, Vav1-driven expression of hSTAT5BN642H, but not hSTAT5B, led to increased numbers of progenitor cells throughout all early hematopoietic compart- ments, including lineage–Sca1+c-Kit+ cells (LSKs), long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), and multipotent progen- itors (MPPs) (CD150+CD48–, CD150+CD48+, CD150–CD48+ frac- tions) (Figure 2C). R E S E A R C H A R T I C L E Figure 2. Moderate Vav1-driven expression of hSTAT5BN642H in mice leads to HSC expansion. (A) Survival curve shows the percentages of disease-free survival of hSTAT5BN642H (N642H) mice (n = 21) compared with that of hSTAT5B (hS5B) (n = 20) and WT (n = 10) mice. (B) WB analysis of pY-STAT5, total STAT5, and HSC70 in the LNs and spleens of WT mice and hSTAT5BN642H- and hSTAT5B-transgenic mice. Quantification of the WB was performed using ImageJ. Data are representative of 3 independent experiments. (C) Flow cytometric analysis of the percentage of LSKs, LT-HSCs (CD150+CD48–), ST-HSCs (CD150+CD48+), MPPs (CD150–CD48+), (D and E) common lymphoid progenitors (lineage−Sca1+IL-7R+AA4+), MPCs (lineage−Sca1–IL-7R–c-Kit+), and CD3+ cells in the BM of WT, hSTAT5B, and hSTAT5BN642H mice. Analyses in C–E included 7-week-old WT (n = 7), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. Data represent the mean ± SD. P < 0.05, P < 0.01, and P < 0.001, by 1-way ANOVA with Bonferroni’s correction. Figure 2. Moderate Vav1-driven expression of hSTAT5BN642H in mice leads to HSC expansion. (A) Survival curve shows Figure 2. Moderate Vav1-driven expression of hSTAT5BN642H in mice leads to HSC expansion. (A) Survival curve shows the percentages of disease-free survival of hSTAT5BN642H (N642H) mice (n = 21) compared with that of hSTAT5B (hS5B) (n = 20) and WT (n = 10) mice. (B) WB analysis of pY-STAT5, total STAT5, and HSC70 in the LNs and spleens of WT mice and hSTAT5BN642H- and hSTAT5B-transgenic mice. Quantification of the WB was performed using ImageJ. Data are representative of 3 independent experiments. (C) Flow cytometric analysis of the percentage of LSKs, LT-HSCs (CD150+CD48–), ST-HSCs (CD150+CD48+), MPPs (CD150–CD48+), (D and E) common lymphoid progenitors (lineage−Sca1+IL-7R+AA4+), MPCs (lineage−Sca1–IL-7R–c-Kit+), and CD3+ cells in the BM of WT, hSTAT5B, and hSTAT5BN642H mice. Analyses in C–E included 7-week-old WT (n = 7), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. Data represent the mean ± SD. P < 0.05, P < 0.01, and P < 0.001, by 1-way ANOVA with Bonferroni’s correction. Figure 2. Moderate Vav1-driven expression of hSTAT5BN642H in mice leads to HSC expansion. (A) Survival curve shows the percentages of disease-free survival of hSTAT5BN642H (N642H) mice (n = 21) compared with that of hSTAT5B (hS5B) (n = 20) and WT (n = 10) mice. (B) WB analysis of pY-STAT5, total STAT5, and HSC70 in the LNs and spleens of WT mice and hSTAT5BN642H- and hSTAT5B-transgenic mice. Results Transcriptome and DNA methylome anal- yses illustrated profound changes in gene expression and reduced DNA methylation of potential EZH2-binding sites, with Aurora kinas- es being one of the most altered genes in hSTAT5BN642H-transgenic animals. In line with this, we found that Aurora kinase and JAK inhib- itors were effective in blocking neoplastic T cell expansion and organ infiltration driven by hSTAT5BN642H. This suggested that inhibitors of Aurora kinases and JAK have potential as a treatment for patients suf- fering from hSTAT5N642H-driven lymphoma or leukemia. Vav1-driven expression of hSTAT5BN642H induces cancer. Given that hSTAT5BN642H was found in different hematopoietic cancers, we expressed hSTAT5B or hSTAT5BN642H in mice during early hematopoiesis using the Vav1 oncogene promoter. This led to transgene expression primarily in cells of the hematopoietic sys- 3 8 8 jci.org Volume 128 Number 1 January 2018 The Journal of Clinical Investigation The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Likewise, the numbers of common lymphoid progenitors (CLPs) and myeloid progenitor cells (MPCs) were sig- nificantly elevated in mice expressing hSTAT5BN642H (Figure 2D). hSTAT5BN642H mice had 3 times more CLPs than did WT mice, which translated into expansion of CD3+ T cells, but not CD19+, B cells in their BM (Figure 2E and Supplemental Figure 2C). The elevated number of MPCs was also accompanied by a signifi- cant increase in the percentage of CD11b+Gr1+ cells in the BM of hSTAT5BN642H mice (Supplemental Figure 2C). We used flow cytometry to analyze markers for T cell acti- vation (CD25) and surface markers for T cell subpopulations, including naive CD8+ T cells (CD62LhiCD44lo), central memory CD8+ T cells (CD62LhiCD44hi), and effector memory CD8+ T cells (CD62LloCD44hi) (38–40). This analysis showed that the leukemic jci.org Volume 128 Number 1 January 2018 3 8 9 The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 3. hSTAT5BN642H mice suffer from an aggressive CD8+ T cell lymphoma. (A) Macroscopic comparison of hSTAT5BN642H and hSTAT5B mouse spleens and LNs with those from WT mice. Scale bars: 1 cm. (B) Modified Wright staining of blood smears from hSTAT5BN642H (N642H), hSTAT5B (hS5B), and WT mice (original magnification, ×100). (C) WBC count using an animal blood counter (scil Vet ABC). CD8/CD4 ratios in the peripheral blood were determined using flow cytometry. Analysis included 7- to 10-week-old WT (n = 20), hSTAT5B (n = 15), and hSTAT5BN642H (n = 20) mice. (D) CD8/CD4 T cell ratios in LNs were determined using flow cytometry. Analyses included 7-week-old WT (n = 5), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. (E) Quantification of the absolute number of CD4+ and CD8+ T cells, myeloid cells (CD11b+Gr1+), and B cells (CD19+) in spleens from hSTAT5BN642H- and hSTAT5B-transgenic mice and WT mice. Analyses included 7-week-old WT (n = 13), hSTAT5B (n = 6), and hSTAT5BN642H (n = 6 and 11) mice. (F) CD3+CD8+ splenic cells were analyzed by flow cytometry for their expression of CD25. Analyses included 8-week-old WT (n = 8), hSTAT5B (n = 9), and (n = 6) hSTAT5BN642H mice. (G) CD3+CD8+ splenic cells were further analyzed for CD62L and CD44 expression. Analyses included WT (n = 8), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice at 8 weeks of age. Data represent the mean ± SD. R E S E A R C H A R T I C L E n ≥ 6. P < 0.01 and P < 0.001, by 1-way ANOVA with Bonferroni’s correction. Figure 3. hSTAT5BN642H mice suffer from an aggressive CD8+ T cell lymphoma. (A) Macroscopic comparison of hS Figure 3. hSTAT5BN642H mice suffer from an aggressive CD8+ T cell lymphoma. (A) Macroscopic comparison of hSTAT5BN642H and hSTAT5B mouse spleens and LNs with those from WT mice. Scale bars: 1 cm. (B) Modified Wright staining of blood smears from hSTAT5BN642H (N642H), hSTAT5B (hS5B), and WT mice (original magnification, ×100). (C) WBC count using an animal blood counter (scil Vet ABC). CD8/CD4 ratios in the peripheral blood were determined using flow cytometry. Analysis included 7- to 10-week-old WT (n = 20), hSTAT5B (n = 15), and hSTAT5BN642H (n = 20) mice. (D) CD8/CD4 T cell ratios in LNs were determined using flow cytometry. Analyses included 7-week-old WT (n = 5), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. (E) Quantification of the absolute number of CD4+ and CD8+ T cells, myeloid cells (CD11b+Gr1+), and B cells (CD19+) in spleens from hSTAT5BN642H- and hSTAT5B-transgenic mice and WT mice. Analyses included 7-week-old WT (n = 13), hSTAT5B (n = 6), and hSTAT5BN642H (n = 6 and 11) mice. (F) CD3+CD8+ splenic cells were analyzed by flow cytometry for their expression of CD25. Analyses included 8-week-old WT (n = 8), hSTAT5B (n = 9), and (n = 6) hSTAT5BN642H mice. (G) CD3+CD8+ splenic cells were further analyzed for CD62L and CD44 expression. Analyses included WT (n = 8), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice at 8 weeks of age. Data represent the mean ± SD. n ≥ 6. P < 0.01 and **P < 0.001, by 1-way ANOVA with Bonferroni’s correction. cells expressed surface markers indicative of mature T cells with an activated phenotype and high expression of IL-2Rα (CD25), a direct target gene of STAT5 (41) (Figure 3F). Fifty percent of the diseased CD8+ T cells also expressed markers reminiscent of cen- tral memory T cells. Moreover, we found that the percentage of cells expressing markers for effector memory T cells was elevated in the diseased mice compared with that observed in WT controls (Figure 3G). High numbers of proliferating T cells were associated with splenomegaly and lymphoma formation, and proliferating T cells were found to heavily infiltrate peripheral organs, leading to fatal pulmonary obstruction (Figure 4 and Supplemental Figure 4). jci.org Volume 128 Number 1 January 2018 R E S E A R C H A R T I C L E Figure 4. Highly proliferative T cells infiltrate into the peripheral organs of hSTAT5BN642H mice. Histological anal- ysis using H&E, CD3, and Ki67 staining of the lungs of 8- to 10-week-old hSTAT5B, hSTAT5BN642H, and WT mice. Data are a representative of 3 independent experiments. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). Figure 4. Highly proliferative T cells infiltrate in peripheral organs of hSTAT5BN642H mice. Histolo ysis using H&E, CD3, and Ki67 staining of the lu 10-week-old hSTAT5B, hSTAT5BN642H, and WT m are a representative of 3 independent experime bar: 100 μm. Original magnification, ×20 and ×4 Figure 4. Highly proliferative T cells infiltrate into the peripheral organs of hSTAT5BN642H mice. Histological anal- ysis using H&E, CD3, and Ki67 staining of the lungs of 8- to 10-week-old hSTAT5B, hSTAT5BN642H, and WT mice. Data are a representative of 3 independent experiments. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). Figure 4. Highly proliferative T cells infiltrate into the peripheral organs of hSTAT5BN642H mice. Histological anal- ysis using H&E, CD3, and Ki67 staining of the lungs of 8- to 10-week-old hSTAT5B, hSTAT5BN642H, and WT mice. Data are a representative of 3 independent experiments. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). lation of 371 genes in T cells derived from hSTAT5BN642H compared with that observed in WT T cells (FDR-adjusted P < 0.05) (Sup- plemental Figure 7A). As expected, known STAT5 targets such as Ccl5, Ccr5, Pim1, Bcl2, and Il2r were among the top upregulated genes, confirming hSTAT5BN642H transgene specificity (Supple- mental Figure 7, B and C and Supplemental Tables 2 and 3) (7–9). Importantly, gene set enrichment analysis (GSEA) confirmed that genes upregulated in CD8+ T cell lymphoma patients were high- ly enriched, which emphasized the validity of our model (Figure 7B) (43, 44). Additional pathway analysis showed that E2F targets, the G2M checkpoint, and MYC targets were the most upregulated pathways, underlining the high proliferation rate of leukemic cells and indicating hSTAT5BN642H as a driver for cell-cycle progres- sion (Figure 7C and Supplemental Figure 7, D and E). In contrast, inflammatory gene pathways or developmental core cancer path- ways were significantly downregulated (P < 0.05), as analyzed by pathway analysis (Supplemental Figure 7E). whether cells carrying the hSTAT5BN642H mutation are sensitive to JAK inhibition. R E S E A R C H A R T I C L E As expected, the FDA-approved JAK inhibitors rux- olitinib and tofacitinib reduced the activation of STAT5 and cell viability, with an IC50 of 0.11 μM (ruxolitinib) and 0.12 μM (tofaci- tinib) and comparable IC50 values for all genotypes (Figure 6, B and C, and Supplemental Figure 6A). Moreover, other FDA-approved drugs such as HDAC inhibitors for the treatment of T cell lympho- ma were tested (42). Entinostat and several other drugs were also found to be effective in inducing apoptosis in T cells, with an IC50 in the nanomolar range, but did not exert differential effects between hSTAT5B- and hSTAT5N642H-expressing cells (Supplemental Figure 6A and Supplemental Table 1). Following the in vitro data, we investigated the effect of rux- olitinib in vivo by treating hSTAT5BN642H CD8+ T cell recipient Ly5.1+CD45.1 mice, 60 days after transplantation, with ruxolitinib (45 mg/kg) for a period of 30 days. The treatment significantly reduced the size of LNs and spleens (Figure 6, D and E). The WBC count as well as CD25 expression on donor hSTAT5BN642H CD8+ T cells were also reduced upon ruxolitinib treatment (Figure 6F and Supplemental Figure 6B). Furthermore, ruxolitinib decreased the degree of T cell infiltration into the lungs, skin, BM, LNs, and spleens of treated mice, leading to a substantial reduction in dis- ease burden (Figure 6G and Supplemental Figure 6C). The treat- ment did not significantly affect the myeloid cell population in the hematopoietic organs (Supplemental Figure 6D). Besides its function as a transcription factor, STAT5 can shape chromatin by interacting with other chromatin-remodeling enzymes such as EZH2 (35, 45). As changes in DNA methylation patterns have recently been associated with malignant disease and particularly with leukemia (46, 47), we questioned wheth- er the dramatic changes observed in the expression profiles of hSTAT5BN642H CD8+ T cells would also be reflected by alterations in the DNA methylome. Using reduced representation bisul- fite sequencing (RRBS), we found that overall DNA methylation across CpG islands (CGIs) among hSTAT5BN642H and WT T cells was highly consistent (Pearson’s r = 0.98), with only 1,380 CGIs being substantially different (absolute difference ≥5 percentage points) (Figure 7D) (48). When comparing WT and hSTAT5B CD8+ T cells, we found weaker differences and overlaps (Supplemental Figure 8A). R E S E A R C H A R T I C L E tion of CD8+ T cells (Figure 5, C and D). Of note, the i.v. injection of CD8+ T cells from diseased mice into nonirradiated Ly5.1+CD45.1+ recipient mice was sufficient to phenotypically recapitulate the pri- mary disease, identifying the CD8+ T cells as the malignant cell pool (Figure 5, E–G, and Supplemental Figure 5, C and D). JAK inhibitors suppress disease progression in the hSTAT5BN642H- driven disease model. A number of treatment regimens have been suggested for leukemia and lymphoma patients carrying the hSTAT5BN642H mutation. However, there is limited knowledge about the effectiveness of these treatments, partially because of the lack of a suitable preclinical model (17, 18). Typically, STAT5 is activated in response to cytokine signaling, and cells harboring the hSTAT5BN642H mutant show prolonged pY-STAT5 levels upon stimulation rather than being constitutively active (18). When we analyzed the level of pY-STAT5 in primary T cells derived from the LNs of WT and hSTAT5B- and hSTAT5BN642H-transgenic mice, we detected dras- tically reduced levels of pY-STAT5 one hour after IL-2 deprivation in WT and hSTAT5B-expressing T cells. In contrast, low levels of pY-STAT5 remained detectable up to 4 hours after IL-2 removal in hSTAT5BN642H-expressing T cells (Figure 6A). The finding that cytokines efficiently activated hSTAT5BN642H prompted us to test To test whether the T cell disease in hSTAT5BN642H-transgenic mice was transplantable, we transferred BM cells from mutant or WT control mice i.v. into nonirradiated, immunocompromised NSG recipient mice. The recipients of mutant cells became termi- nally sick approximately 3 months after injection (Figure 5A). Bone marrow transplantations (BMTs) induced disease, with a phenotype comparable to that of hSTAT5BN642H-transgenic mice. The disease was characterized by enlarged spleens and lymphoma formation, with T cell infiltration into peripheral organs (Figure 5B and Supple- mental Figure 5, A and B) caused by excessive expansion and infiltra- 3 9 0 jci.org Volume 128 Number 1 January 2018 The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 4. Highly proliferative T cells infiltrate into the peripheral organs of hSTAT5BN642H mice. Histological anal- ysis using H&E, CD3, and Ki67 staining of the lungs of 8- to 10-week-old hSTAT5B, hSTAT5BN642H, and WT mice. Data are a representative of 3 independent experiments. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). The Journal of Clinical Investigation The Journal of Clinical Investigation The Journal of Clinical Investigation The Journal of Clinical Investigation Data represent the mean ± SD. P < 0.05, P < 0.01, and P < 0.001, by unpaired, 2-tailed Student’s t test. pared with that detected in WT murine STAT5B and hSTAT5B. The mutated STAT5 increased its binding to the Cis promoter and was also found at the promoter regions of the EZH2 known targets Cdkn2a and Ccnd2. In addition, it bound to the less methylated CGI in association with Aurkb (Figure 8C). Although EZH2 bind- ing was found to be reduced in hSTAT5BN642H CD8+ T cells, EZH2 retained its binding at the same CGI (Figure 8D and Supplemental Figure 9C). However, hSTAT5BN642H was not shown to have direct interactions with EZH2 (Figure 8E). of DNA methylation at nearby CGIs included the mitotic check- point protein KNTC1 (49) and the oncogene topoisomerase type IIα (TOP2A) (36, 50), which is known to regulate DNA topologi- cal structure and cell-cycle progression (Supplemental Figure 8B, right, sector II). None of these genes was substantially affected in hSTAT5B CD8+ T cells (Supplemental Figure 8B). Specific DNA methylation changes in hSTAT5BN642H reveal targets for therapy. Location overlap analysis (LOLA) (51) of regions that lost methylation in T cells expressing hSTAT5BN642H compared with WT cells revealed significant enrichment for sites known to bind EZH2 and/or SUZ12 proteins. These are components of poly- comb repressor complex 2 (PCR2), which promotes methylation of histone 3 at lysine 27 (FDR-adjusted P ≤ 0.05, Fisher’s exact test) (Figure 8A, top, Supplemental Figure 9A, and Supplemental Table 4). STAT5 has recently been reported to oppose a network of transcription factors such as NF-κB and IKAROS in B cell acute lymphoblastic leukemia (52) and to interact with EZH2 (35). Fur- thermore, TOP2A expression has been previously linked to EZH2 expression in aggressive prostate cancer (53). Consistently, target genes of EZH2 and SUZ12 were found to be enriched in CD8+ T cells derived from hSTAT5BN642H mice (Figure 8B, Supplemental Figure 9B, and Supplemental Table 5). STAT5BN642H-expressing T cells are sensitive to AURKB inhibition. Among EZH2 target genes, the genes encoding Aurora kinase B (Aurkb) and DNA topoisomerase 2α (Top2a) were significantly upreg- ulated, and AURKB targets were highly enriched in hSTAT5BN642H- expressing CD8+ T cells (Figure 8B and Supplemental Figure 10A). The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 5. hSTAT5BN642H CD8+ T cells are the cancer-initiating cells. (A) Percentage of disease-free survival following hSTAT5BN642H whole BMT into 8-week- old NSG recipient mice compared with WT BMT. (B) Macroscopic view of LNs and spleen from a hSTAT5BN642H BMT recipient mouse compared with those from a WT BMT recipient mouse. Scale bar: 1 cm. (C) Flow cytometric analysis shows the quantity of CD3+ cells and CD8/CD4 T cell ratio in the spleens of BMT recipient mice. (D) Histological analysis of CD3+ cells from the lungs of NSG recipient mice after hSTAT5BN642H or WT BMT. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). (E) Percentage of disease-free survival after hSTAT5BN642H or WT CD8+ T cell transplantation into nonirradiated 8-week-old Ly5.1/CD45.1 recipient mice. (F) Flow cytometric analysis shows the quantity of splenic CD3+CD8+ cells in CD8+ T cell–transplanted mice. (G) Spleen versus BW ratios of WT and hSTAT5BN642H CD8+ T cell–transplanted Ly5.1/CD45.1 mice. (A–C) n = 4 WT mice and n = 5 hSTAT5BN642H mice; (E–G) n = 6 WT mice and n = 5 hSTAT5BN642H mice. Data represent the mean ± SD. P < 0.05, P < 0.01, and P < 0.001, by unpaired, 2-tailed Student’s t test. Figure 5. hSTAT5BN642H CD8+ T cells are the cancer-initiating cells. (A) Percentage of disease-free survival following hSTAT5BN642H whole BMT into 8-week- old NSG recipient mice compared with WT BMT. (B) Macroscopic view of LNs and spleen from a hSTAT5BN642H BMT recipient mouse compared with those from a WT BMT recipient mouse. Scale bar: 1 cm. (C) Flow cytometric analysis shows the quantity of CD3+ cells and CD8/CD4 T cell ratio in the spleens of BMT recipient mice. (D) Histological analysis of CD3+ cells from the lungs of NSG recipient mice after hSTAT5BN642H or WT BMT. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). (E) Percentage of disease-free survival after hSTAT5BN642H or WT CD8+ T cell transplantation into nonirradiated 8-week-old Ly5.1/CD45.1 recipient mice. (F) Flow cytometric analysis shows the quantity of splenic CD3+CD8+ cells in CD8+ T cell–transplanted mice. (G) Spleen versus BW ratios of WT and hSTAT5BN642H CD8+ T cell–transplanted Ly5.1/CD45.1 mice. (A–C) n = 4 WT mice and n = 5 hSTAT5BN642H mice; (E–G) n = 6 WT mice and n = 5 hSTAT5BN642H mice. jci.org Volume 128 Number 1 January 2018 R E S E A R C H A R T I C L E Combining DNA methylation analysis with mRNA expression data, we identified a small set of genes with substan- tial and concordant changes in DNA methylation and expression of genes within the proximity of differentially methylated CGIs (Supplemental Figure 8, B and C). Interestingly, the genes with higher expression in hSTAT5BN642H T cells and concordant loss hSTAT5BN642H CD8+ T cells exhibit substantial changes in gene expression profile, accompanied by specific changes in DNA methyla- tion. Given the leukemogenic effect of hSTAT5BN642H, which is not shared by WT hSTAT5B, we next investigated alterations in gene expression and epigenetic modifications in T cells derived from both mouse models. CD8+ T cells were isolated from the LNs of 13-week-old WT and hSTAT5B and hSTAT5BN642H diseased mice, and mRNA sequencing analysis was performed. While the glob- al expression patterns of WT and hSTAT5B CD8+ T cells were comparable, the gene expression signature of cells expressing hSTAT5BN642H showed a distinct pattern (Figure 7A). We found a significant upregulation of 564 genes and a significant downregu- 3 9 1 jci.org Volume 128 Number 1 January 2018 The Journal of Clinical Investigation The Journal of Clinical Investigation Western blot analysis showed that hSTAT5BN642H mice had higher AURKB activity, and quantitative PCR (qPCR) analysis validat- ed the hSTAT5BN642H-dependent upregulation of Aurkb levels in CD8+ T cells (Figure 9A and Supplemental Figure 10B). This led us to test the dual-specific JAK and Aurora kinase inhibitor AT9283 as a potential therapeutic in hSTAT5BN642H-expressing cells. We found that hSTAT5BN642H-expressing T cells were exquisitely more sensitive to AT9382, with a 10-fold lower IC50 compared with that of hSTAT5B-expressing T cells (Figure 9B), but not to etoposide, a TOP2A inhibitor (Supplemental Figure 10C). AT9283 was not effective in reducing STAT5 activation compared with ruxolitinib but efficiently reduced AURKB activity (Figure 9C). The high sen- To investigate whether STAT5BN642H has a role in the upregula- tion of these genes, we performed ChIP with isolated CD8+ T cells from WT, hSTAT5B, and hSTAT5BN642H mice. Given its hyperac- tivation status, binding of hSTAT5BN642H to DNA increased com- 3 9 2 jci.org Volume 128 Number 1 January 2018 The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L Figure 6. hSTAT5BN642H-driven diseased T cells can be treated with JAK inhibitors. (A) WB analysis of pY-STAT5 levels in isolated and cultivated LN T cells from hSTAT5BN642H, hSTAT5B, and WT mice after IL-2 removal. (B) Dose-response curve of WT, hSTAT5BN642H, and hSTAT5B T cells 72 hours after ruxolitinib treatment, analyzed using CellTiter-Glo (CTG) assay. IC50 values were determined using GraphPad Prism. Error bars indicate the mean ± SEM. DMSO (100% viability) and 10 μM bortezomib (0% viability) on each plate served as controls. (C) WB of hSTAT5BN642H, hSTAT5B, and WT T cell cultures after 5 hours of treatment with ruxolitinib or tofacitinib, analyzed for pY-STAT5. (D) Macroscopic view of LNs and spleens from CD8+ T cell–transplanted mice treated with ruxolitinib compared with vehicle controls. CD8+ T cell–recipient mice were treated with ruxolitinib at the dosage of 45 mg/kg twice a day for 30 days. (E) Quantification of spleen versus BW ratio of vehicle- and ruxolitinib-treated hSTAT5BN642H CD8+ cell–transplanted mice. (F) WBC counts of vehicle- and ruxolitinib-treated hSTAT5BN642H CD8+ cell–transplanted mice, measured using a scil Vet ABC animal blood counter. Flow cytometric analysis of CD25 expression in peripheral blood CD8+ T cells. MFI, mean fluorescence intensity. The Journal of Clinical Investigation (G) Histological analysis of CD3+ cells in the lungs of recipient mice after treatment with ruxolitinib compared with the vehicle-treated group. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). n = 5 vehicle-treated mice and n = 4 ruxolitinib-treated mice. Data represent the mean ± SD. n ≥ 6. P < 0.05, by unpaired, 2-tailed Student’s t test. Data presented in A–C are representative of 3 independent experiments. The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 6. hSTAT5BN642H-driven diseased T cells can be treated with JAK inhibitors. (A) WB analysis of pY-STAT5 levels in isolated and cultivated LN T cells from hSTAT5BN642H, hSTAT5B, and WT mice after IL-2 removal. (B) Dose-response curve of WT, hSTAT5BN642H, and hSTAT5B T cells 72 hours after ruxolitinib treatment, analyzed using CellTiter-Glo (CTG) assay. IC50 values were determined using GraphPad Prism. Error bars indicate the mean ± SEM. DMSO (100% viability) and 10 μM bortezomib (0% viability) on each plate served as controls. (C) WB of hSTAT5BN642H, hSTAT5B, and WT T cell cultures after 5 hours of treatment with ruxolitinib or tofacitinib, analyzed for pY-STAT5. (D) Macroscopic view of LNs and spleens from CD8+ T cell–transplanted mice treated with ruxolitinib compared with vehicle controls. CD8+ T cell–recipient mice were treated with ruxolitinib at the dosage of 45 mg/kg twice a day for 30 days. (E) Quantification of spleen versus BW ratio of vehicle- and ruxolitinib-treated hSTAT5BN642H CD8+ cell–transplanted mice. (F) WBC counts of vehicle- and ruxolitinib-treated hSTAT5BN642H CD8+ cell–transplanted mice, measured using a scil Vet ABC animal blood counter. Flow cytometric analysis of CD25 expression in peripheral blood CD8+ T cells. MFI, mean fluorescence intensity. (G) Histological analysis of CD3+ cells in the lungs of recipient mice after treatment with ruxolitinib compared with the vehicle-treated group. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). n = 5 vehicle-treated mice and n = 4 ruxolitinib-treated mice. Data represent the mean ± SD. n ≥ 6. *P < 0.05, by unpaired, 2-tailed Student’s t test. Data presented in A–C are representative of 3 independent experiments. The Journal of Clinical Investigation Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive and negative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from published gene signature cytotoxic T cells (43, 44). (C) Top enriched gene sets are the results of GSEA including E2F target, G2M checkpoint, MYC target, and cell-cycle progression in hSTAT5BN642H CD8+ T cells. P values in B and C were determined by Kolmogorov-Smirnov test. (D) Scatterplot contrasting the mean DNA methylation levels in WT and hSTAT5BN642H-mutant T cells in all CGIs covered in at least 1 sample per genotype (n = 15,209). The density of data points in each plot region is indicated by color intensity, and CGIs with lower DNA methylation in WT (n = 770) or hSTAT5BN642H (n = 610) cells are indicated by black and red crosses, respectively (absolute difference ≥5 percentage points, n = 2 per genotype). Analyses included 13-week-old mice. NES, normalized enrichment score. BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T c Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap stantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA me Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap showing Z scores of rlog-transformed and library size–normalized counts of genes upregulated (red) or downregulated (blue) in hSTAT5B or hSTAT5BN642H and WT CD8+ T cells (FDR-adjusted P < 0.05). Analyses included 13-week-old WT (n = 5), hSTAT5B (n = 4), and hSTAT5BN642H (n = 5) mice. Each column in the heatmap represents data from CD8+ T cells from 1 mouse of a given genotype, and each row represents data for a given gene. (B) Enrichment blot of the CD8+ T cell lymphoma expression signature. Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive and negative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from published gene signature cytotoxic T cells (43, 44). Discussion sitivity of AT9283 could be an attribute of Aurora serine/threonine and JAK tyrosine/serine kinase combinatorial inhibition, as IC50 values of ruxolitinib and tofacitinib were similar in all genotypes (Figure 6 and Supplemental Figure 6). Combinatory treatment with ruxolitinib and AZD1152, an AURKB-specific inhibitor, showed an additive effect, which further supported our hypothesis (Supple- mental Figure 10D). Although AZD1152 treatment did not affect STAT5 phosphorylation in all genotypes, it efficiently inhibited AURKB activity in hSTAT5BN642H-expressing T cells (Figure 9D). Here, we provide evidence that the STAT5BN642H mutation is a direct driver and not a bystander mutation for lymphoid malig- nancy. Expression of hSTAT5BN642H triggers the development of leukemia or lymphoma characterized by highly proliferative and invasive CD8+ T cells. hSTAT5BN642H activation remains large- ly cytokine dependent, which renders the diseased cells sensi- tive to JAK inhibition. When comparing T cells from transgenic hSTAT5BN642H mice with those from their hSTAT5B counterparts, jci.org Volume 128 Number 1 January 2018 3 9 3 The Journal of Clinical Investigation The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap showing Z scores of rlog-transformed and library size–normalized counts of genes upregulated (red) or downregulated (blue) in hSTAT5B or hSTAT5BN642H and WT CD8+ T cells (FDR-adjusted P < 0.05). Analyses included 13-week-old WT (n = 5), hSTAT5B (n = 4), and hSTAT5BN642H (n = 5) mice. Each column in the heatmap represents data from CD8+ T cells from 1 mouse of a given genotype, and each row represents data for a given gene. (B) Enrichment blot of the CD8+ T cell lymphoma expression signature. Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive and negative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from published gene signature cytotoxic T cells (43, 44). (C) Top enriched gene sets are the results of GSEA including E2F target, G2M checkpoint, MYC target, and cell-cycle progression in hSTAT5BN642H CD8+ T cells. P values in B and C were determined by Kolmogorov-Smirnov test. (D) Scatterplot contrasting the mean DNA methylation levels in WT and hSTAT5BN642H-mutant T cells in all CGIs covered in at least 1 sample per genotype (n = 15,209). The density of data points in each plot region is indicated by color intensity, and CGIs with lower DNA methylation in WT (n = 770) or hSTAT5BN642H (n = 610) cells are indicated by black and red crosses, respectively (absolute difference ≥5 percentage points, n = 2 per genotype). Analyses included 13-week-old mice. NES, normalized enrichment score. Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap showing Z scores of rlog-transformed and library size–normalized counts of genes upregulated (red) or downregulated (blue) in hSTAT5B or hSTAT5BN642H and WT CD8+ T cells (FDR-adjusted P < 0.05). Analyses included 13-week-old WT (n = 5), hSTAT5B (n = 4), and hSTAT5BN642H (n = 5) mice. Each column in the heatmap represents data from CD8+ T cells from 1 mouse of a given genotype, and each row represents data for a given gene. (B) Enrichment blot of the CD8+ T cell lymphoma expression signature. 3 9 4 jci.org Volume 128 Number 1 January 2018 The Journal of Clinical Investigation R E S E A R C H A R T I C L E ure 8. hSTAT5BN642H-driven DNA methylation changes accompanied by enhanced DNA-binding activity of STAT5 result in the induction of Aurora ase B. (A) Region set enrichment analysis testing CGIs with lower DNA methylation in hSTAT5BN642H cells than in WT cells (top) or lower DNA methyla- n in WT cells than in hSTAT5BN642H cells (bottom). Enrichment was determined using LOLA (51). Each dot represents 1 ChIP-seq experiment for a given nscription factor from the CODEX database. The vertical dashed line represents the significance threshold (FDR-adjusted P ≤ 0.05). (B) Enrichment t of EZH2 target genes in HSCs, together with their methylation states of EZH2-bound and EZH2-unbound CGIs 100 kb up- and downstream of the nscriptional start sites (TSSs). Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive and gative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from the MSigDB (72). Black circles indicate CGIs overlapping h EZH2-binding sites. p.p., percentage points. n = 2 per genotype. ChIP with anti-STAT5 (C) or anti-EZH2 (D) in CD8+ T cells isolated from WT (n = 7), TAT5B (n = 7), or hSTAT5BN642H (n = 4) mice. Binding of STAT5 to the Cis and Ccnd2 promoters or binding of EZH2 to the promoter regions of Cdkn2A and nd2 served as positive controls. Horizontal dotted line indicates the threshold for nonspecific binding. (E) ChIP with anti-STAT5, anti-EZH2, or IgG in AT5BN642H-expressing CD8+ T cells, followed by WB analysis. IB, immunoblot. Data presented in C–E are representative of 2 independent experiments. or bars indicate the mean ± SD. Figure 8. hSTAT5BN642H-driven DNA methylation changes accompanied by enhanced DNA-binding activity of STAT5 re Figure 8. hSTAT5BN642H-driven DNA methylation changes accompanied by enhanced DNA-binding activity of STAT5 result in the induction of Aurora kinase B. (A) Region set enrichment analysis testing CGIs with lower DNA methylation in hSTAT5BN642H cells than in WT cells (top) or lower DNA methyla- tion in WT cells than in hSTAT5BN642H cells (bottom). Enrichment was determined using LOLA (51). Each dot represents 1 ChIP-seq experiment for a given transcription factor from the CODEX database. The vertical dashed line represents the significance threshold (FDR-adjusted P ≤ 0.05). The Journal of Clinical Investigation (B) Enrichment blot of EZH2 target genes in HSCs, together with their methylation states of EZH2-bound and EZH2-unbound CGIs 100 kb up- and downstream of the transcriptional start sites (TSSs). Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive and negative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from the MSigDB (72). Black circles indicate CGIs overlapping with EZH2-binding sites. p.p., percentage points. n = 2 per genotype. ChIP with anti-STAT5 (C) or anti-EZH2 (D) in CD8+ T cells isolated from WT (n = 7), hSTAT5B (n = 7), or hSTAT5BN642H (n = 4) mice. Binding of STAT5 to the Cis and Ccnd2 promoters or binding of EZH2 to the promoter regions of Cdkn2A and Ccnd2 served as positive controls. Horizontal dotted line indicates the threshold for nonspecific binding. (E) ChIP with anti-STAT5, anti-EZH2, or IgG in STAT5BN642H-expressing CD8+ T cells, followed by WB analysis. IB, immunoblot. Data presented in C–E are representative of 2 independent experiments. Error bars indicate the mean ± SD. STAT5BN642H has been shown previously to render Ba/F3 cells cytokine independent and to be constitutively active in HeLa cells (13, 15, 17). Ba/F3 cells have been used to determine the oncogenic potential of many leukemogenic drivers, however, the expression level of the oncogene is often very high, and the cells might have acquired additional mutations as a result of long-term cultivation. In cytokine-independent cell lines such as HeLa or HEK293T, STAT5 might be activated by other available growth stimuli. Cells expressing low levels of STAT5BN642H, however, remain dependent on cytokine stimulation, as shown in our diseased T cell model. This was also observed in NK cells by Küçük and colleagues (18). development could be a result of the CD8+ T cell sensitivity to the Stat5a/b gene dosage that was described previously in mice (62). Moreover, it has been reported that CD8+ T cells are more susceptible to oncogenic drivers, especially when these drivers are activated by cytokines or triggered via T cell receptors (62, 63). Similarly, hSTAT5BN642H activation remains cytokine depen- dent, and the upregulation of IL-2Rα, a direct target of STAT5, resulted in CD8+ T cells becoming more sensitive to low doses of cytokine stimulation. jci.org Volume 128 Number 1 January 2018 The Journal of Clinical Investigation (C) Top enriched gene sets are the results of GSEA including E2F target, G2M checkpoint, MYC target, and cell-cycle progression in hSTAT5BN642H CD8+ T cells. P values in B and C were determined by Kolmogorov-Smirnov test. (D) Scatterplot contrasting the mean DNA methylation levels in WT and hSTAT5BN642H-mutant T cells in all CGIs covered in at least 1 sample per genotype (n = 15,209). The density of data points in each plot region is indicated by color intensity, and CGIs with lower DNA methylation in WT (n = 770) or hSTAT5BN642H (n = 610) cells are indicated by black and red crosses, respectively (absolute difference ≥5 percentage points, n = 2 per genotype). Analyses included 13-week-old mice. NES, normalized enrichment score. 59, 60). Transgenic mouse models expressing high levels of murine Stat5a or Stat5b developed lymphoblastic lymphoma at low pene- trance (5%–25%) and with a late onset (up to 456 days) (28, 61). we found reduced DNA methylation of EZH2-binding sites. This correlated with an increase in the transcription of STAT5B and EZH2 target genes including the cell-cycle regulators Top2A and Aurkb, for which AURKB represents a potential therapeutic target. We now show that moderate expression of hSTAT5BN642H, but not hSTAT5B, is sufficient to trigger an aggressive disease that causes rapid lethality at a young age, with full penetrance irrespective of gender, demonstrating the potent oncogenic role of the hSTAT5BN642H mutation. Despite the Vav1 promoter– dependent expression of hSTAT5BN642H throughout the entire hematopoietic system, malignancy evolved in CD8+ T cells. This T cells express considerably more STAT5B than do other cell types of the hematopoietic system (54–57), suggesting a privileged role for STAT5B in the T cell compartment (58). Moreover, STAT5B is the dominant STAT5 protein in effector and regulatory T cells, and the differences in STAT5A and STAT5B governing T cell func- tion are largely associated with paralog expression differences (7, 3 9 4 jci.org Volume 128 Number 1 January 2018 3 9 4 jci.org Volume 128 Number 1 January 2018 The Journal of Clinical Investigation The Journal of Clinical Investigation The Journal of Clinical Investigation (C and D) WB of hSTAT5BN642H, hSTAT5B, and WT T cell cultures after 5 hours of treatment with AT9283 or AZD1152, for determination of pY-STAT5, total STAT5, p-AURKB, total AURKB, p-H3 (Ser10), and total H3 levels. Data presented in A–D are representative of 3 independent experiments. gested that STAT5 and EZH2 compete for binding to regulatory sites, as shown in B cells and mammary epithelial cells (52, 75). We observed that, as a consequence of STAT5B hyperactivation, STAT5BN642H bound more to DNA and subsequently upregulated many cell-cycle–regulating genes including Top2a and Aurkb. The fact that the cells were particularly sensitive to Aurora kinase inhi- bition underlines this observation. tantly, the most upregulated genes were E2F and MYC targets, which highlights the proliferative nature of the diseased T cells and explains the upregulation of numerous genes (65). STAT5BN642H is hyperphosphorylated, and it would be interesting to study its potential different interactions with CD8+ T cell–specific activators or repressors compared with the less active WT STAT5B. Recent work suggested that altered DNA methylation patterns in T cells are indicative or even causative for T cell transformation and that methylation of gene bodies was correlated with active transcription contributing to carcinogenesis (66, 67). Epigenetic regulators such as EZH2, TET1/2, and HDAC play important roles in leukemogenesis (68–71) and have been shown to interact with STAT5 (30, 31, 34, 35). EZH2 has been linked to the long-term repopulating capability, proliferation, and inhibition of apoptosis of HSCs (72, 73), all of which are important for transformed cells as well as for governing peripheral T cell fates (74). We demonstrate here that the expression of hSTAT5BN642H not only led to transcrip- tional changes but also changed DNA methylation. Decreased methylation at EZH2- and SUZ12-binding sites in hSTAT5BN642H T cells resulted in the upregulation of EZH2 target genes. There are conflicting reports regarding the interaction between EZH2 and STAT5. In 2011, Mandal and colleagues reported that STAT5 plays an essential part in the recruitment of EZH2 to repress Ig κ-chain (Igk) transcription in progenitor B cells (35). Others sug- Work by many groups identified STAT5 as an important tar- get for therapy, since it is essential for JAK2V617F-, Flt3-ITD-, and BCR/ABL-driven diseases (76–78). Currently, intensive efforts are being made to inhibit STAT5 by blocking its SH2 domain (79). However, effective targeting of STAT5 remains challenging. The Journal of Clinical Investigation So far, hSTAT5BN642H mutations have primarily been found in patients with T cell or NK cell malignan- cies, pointing toward the sensitivity of these patients to aberrant STAT5 activation. When STAT5BN642H was identified in CD8+ T cells in patients, such as those with T cell LGL (T-LGL) or epi- theliotropic intestinal T cell lymphoma (13, 23, 64), it gave rise to more aggressive disease (26). The malignant transformation and expansion of CD8+ T cells in transgenic mice correlated with the upregulation of direct STAT5 target genes such as D-type cyclins, Bcl2 family members, and Pim kinases, which promote cell-cycle progression and survival. Impor- 3 9 5 jci.org Volume 128 Number 1 January 2018 The Journal of Clinical Investigation R E S E A R C H A R T I C L E The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 9. hSTAT5BN642H-driven diseased T cells are sensitive to Aurora kinase B inhibition. (A) WB analysis of p-AURKB, total AURKB, and HSC70 in LNs from WT and hSTAT5BN642H- and hSTAT5B-transgenic mice. WB quantification (bar graph) was performed using ImageJ. (B) Dose-response curves of WT, hSTAT5BN642H, or hSTAT5B T cells in response to AT9283 after 72 hours of treatment, analyzed using a CTG assay. IC50 values were determined using GraphPad Prism. Error bars indicate the mean ± SEM. DMSO (100% viability) and 10 μM bortezomib (0% viability) on each plate served as controls. n = 6 per genotype. (C and D) WB of hSTAT5BN642H, hSTAT5B, and WT T cell cultures after 5 hours of treatment with AT9283 or AZD1152, for determination of pY-STAT5, total STAT5, p-AURKB, total AURKB, p-H3 (Ser10), and total H3 levels. Data presented in A–D are representative of 3 independent experiments. Figure 9. hSTAT5BN642H-driven diseased T cells are sensitive to Aurora kinase B inhibition. (A) WB analysis of p-AURKB, total AURKB, and HSC70 in LNs from WT and hSTAT5BN642H- and hSTAT5B-transgenic mice. WB quantification (bar graph) was performed using ImageJ. (B) Dose-response curves of WT, hSTAT5BN642H, or hSTAT5B T cells in response to AT9283 after 72 hours of treatment, analyzed using a CTG assay. IC50 values were determined using GraphPad Prism. Error bars indicate the mean ± SEM. DMSO (100% viability) and 10 μM bortezomib (0% viability) on each plate served as controls. n = 6 per genotype. jci.org Volume 128 Number 1 January 2018 Methods Plasmid construction/mutagenesis and transfection. hSTAT5B variants were generated using site-directed mutagenesis (80). Mutagenic PCR was performed using KOD Polymerase (Novagen). PCR products were subsequently digested with DpnI enzyme (New England BioLabs) to remove the methylated template according to the manufacturer’s pro- tocol. E. coli was transformed with the digested reaction, and positive clones were selected by Sanger sequencing (81). Plasmid transfection was performed using Lipofectamine 2000 Reagent (Invitrogen, Ther- mo Fisher Scientific). Cytokine stimulation of T cells was performed with human IL-2 (100 U/ml; ProleukinÒ; Novartis), murine IL-4 (100 ng/ml; R&D Sys- tems), or murine IL-7 (10 ng/ml; R&D Systems). The 293T and 32D cell lines were gifts of M. Hengstschläger (Center of Pathobiochemistry and Genetics, Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria) and F. Grebien (Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria), respectively. The Ba/F3 cell line was provided by A. D’An- drea (Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA). The cases of patients harboring the STAT5BN642H mutation were assembled from previously published work (13–26, 36). Animals and generation of transgenic mice. Transgenic mice were generated and bred on a C57BL/6NCrl background and maintained in a specific pathogen–free environment in the experimental mouse facility at the University of Veterinary Medicine (Vienna, Austria). We used the Vav1-hematopoietic vector Vav1-hCD4 (HS21/45) (37) to generate several transgenic mouse lines expressing hSTAT5B and hSTAT5BN642H in the hematopoietic system and selected the lines B6N-Tg(STAT5B)731Biat and B6N-Tg(STAT5BN/H)726Biat, respec- tively, for further experiments. The hSTAT5BN642H construct was gen- erated using overlapping PCR technology as previously described (80) (forward primer: GAAAGAATGTTTTGGCATCTGATGCCTTTTAC; reverse primer: GTAAAAGGCATCAGATGCCAAAACATTCTTTC). The construct was digested with the HindIII restriction enzyme and gel purified for pronuclear injection (82). The transgenic mice were identified by genotyping PCR (forward primer: ACGCAGGACACA- GAGAATGAG; reverse primer: GTGATGGTGGCGTTGACCTC). WT (C57BL/6NCrl) mice and B6-Ly5.1/Cr (B6.SJL-PtprcaPepcb/BoyCrCrl) mice were purchased from Charles River Laboratories, and NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were purchased from The Jack- son Laboratory. Given the rapid development and strong phenotype of the hSTAT5BN642H-transgenic mice, the colony was propagated via in vitro fertilization with archived sperm cells (83). Transplantation experiments. BM cells (1 × 106) from hSTAT5BN642H or WT mice were transplanted by lateral tail vein injection into nonirra- diated NSG mice. Mice were monitored daily and evaluated at the first sign of disease onset. Methods CD8+ T cells were isolated using a CD8+ Magni Sort Mouse T Cell Enrichment Kit (eBioscience), and sorted cells were checked with flow cytometry for their purity. Cells (1 × 106) were inject- ed i.v. into nonirradiated Ly5.1/CD45.1 mice. IHC. Mouse organs were incubated overnight in 4% phos- phate-buffered formaldehyde solution (Roti-Histofix; Carl Roth), dehydrated, embedded, and cut (4-μm-thick sections). For immu- nohistochemical staining, heat-mediated antigen retrieval was per- formed in citrate buffer at pH 6.0 (Dako) and stained with antibod- ies against CD3 (Thermo Fisher Scientific; RM-9107-S0; dilution 1:300); Ki67 (Novocastra, Leica Biosystem; NCL-Ki67p; dilution 1:1,000); and STAT5B (Santa Cruz Biotechnology; sc-1656; dilution 1:200) using standard protocols. Images were taken using a Zeiss Imager Z.1 microscope. Western blot analysis. Western blotting (WB) was performed using standard protocols. The antibodies used were: monoclonal rabbit anti- mouse phosphorylated STAT5 (p-STAT5) (Invitrogen, Thermo Fisher Scientific; 716900; dilution 1:1,000); purified mouse anti-STAT5 (BD; 610191; dilution 1:2,000); monoclonal mouse anti-mouse HSC70 (Santa Cruz Biotechnology; sc-7298; dilution 1:10,000); monoclonal mouse anti-Flag M2 ( MilliporeSigma; F3156; dilution 1:1,000); mono- clonal rabbit anti–p–Aurora A (Thr288), p–Aurora B (Thr232), and p–Aurora C (Thr198) (Cell Signaling Technology; 2914; 1:1,000); monoclonal rabbit anti-Aurora B/AIM1 (Cell Signaling Technology; 3094; 1:1,000); monoclonal rabbit anti–histone H3 (anti-H3) (Cell Signaling Technology; 4499; 1:1,000); monoclonal rabbit anti–p-H3 (Ser10) (Cell Signaling Technology; 53348; 1:1,000); ECL anti-mouse IgG; HRP-linked whole antibody from sheep (GE Healthcare; NA931V; dilution 1:10,000); ECL anti-rabbit IgG; and HRP-linked whole anti- body from sheep (GE Healthcare; NA934V; dilution 1:10,000). WB quantification was performed using ImageJ software (NIH). (See the complete unedited blots in the supplemental material.) Hematocytometry and flow cytometry. Mouse blood was collect- ed into EDTA tubes (Greiner Bio-One Mini-Collect K3EDTA Tubes; Thermo Fisher Scientific) from the facial vein or from euthanized mice via cardiac puncture, and blood smears were stained using mod- ified Wright staining. WBC counts were measured using an animal blood counter (scil Vet ABC). For flow cytometry, erythrocytes were lysed using Gay’s solution (10 mM KHCO3 and 75 mM NH4Cl, pH 7.4). Single-cell suspensions were prepared by mincing organs through a 70-μm cell strainer (BD Biosciences). HSC staining was performed as previously described (84). All antibodies used for flow cytometry were purchased from eBioscience and BD (see Supplemental Table 6 for the list of the anti- bodies). All analyses were performed on the BD FACSCanto II using FACSDiva software (BD). Further analysis was performed using FlowJo software. The Journal of Clinical Investigation upstream inhibition of JAK activation or the chromatin-remodel- ing partners of STAT5 could be an alternative targeting strategy for enhanced STAT5 activation. mycin) (all from Gibco, Thermo Fisher Scientific) supplemented with IL-3 (1 ng/ml; ImmunoTools). IL-3 stimulation was performed with 10 ng/ml IL-3 for 20 minutes. The hSTAT5BN642H, hSTAT5B, and B6N WT T cells were isolated from LNs and spleens from 8- to 12-week-old mice. Following T cell activation by anti-CD3 (BD), T cells were grown in complete RPMI 1640 medium containing 10 mM HEPES, 1× MEM nonessential ami- no acids, 50 μM β-mercaptoethanol (all from Gibco, Thermo Fisher Scientific), 1 mM sodium pyruvate (MilliporeSigma), and 100 U/ml human IL-2 (ProleukinÒ; Novartis). The Journal of Clinical Investigation Sev- eral different strategies have been suggested for the treatment of hSTAT5BN642H-expressing cells including the use of BCL2, MEK1/2, and JAK inhibitors (17, 18). Although some patients respond to JAK inhibitors, the lack of sensitivity in other patients requires broader therapeutic targets (16, 17). We believe that the hSTAT5BN642H-transgenic mouse model will serve as a valuable preclinical model. Using this model, we showed that the com- bined use of Aurora kinase and JAK inhibitors is a potential thera- peutic strategy to treat lymphoma and leukemia patients with the STAT5BN642H mutation. We show here that hSTAT5BN642H acts as a driver mutation in the development of leukemia and lymphoma and propose that 3 9 6 jci.org Volume 128 Number 1 January 2018 The Journal of Clinical Investigation er). Sequences were trimmed for adapters using Trimmomatic (86) with the ILLUMINACLIP settings “:2:40:7 SLIDINGWINDOW:4:15 MAXINFO:20:0.50 MINLEN:18.” All reads were aligned to the GRCm38 (mm10) assembly of the mouse genome using BSMAP in its RRBS mapping mode (87, 88). DNA methylation levels for individual CpGs were calculated using custom Python scripts and loaded into RnBeads (89) for exploratory analysis and to aggregate DNA meth- ylation estimates per CGI. The aggregated values were loaded into R for further analysis. Differentially methylated regions (absolute dif- ference ≥5 percentage points) were compared with ChIP-seq peaks from the CODEX database (90) using LOLA (51) to find significant overlaps (FDR-adjusted P ≤ 0.05) with potential regulators and effec- tors of DNA methylation differences. To compare DNA methylation at CGIs with genes, each CGI was associated with all genes within a 10-kb window after conversion of the gene coordinates to the mm10 reference genome using the UCSC LiftOver tool (https://genome. ucsc.edu/cgi-bin/hgLiftOver). RRBS sequencing data were deposited in the NCBI’s GEO database (GEO GSE104557). were enriched using a CD8+ MagniSort Enrichment Kit, and mRNA was isolated using TRIzol (MilliporeSigma) in combination with an RNeasy Mini Kit (QIAGEN). mRNA library preparation (SENSE mRNA-Seq Library preparation) and RNA sequencing (RNA-seq) was performed with an Illumina HiSeq 2500 at the Vienna Biocenter Core Facility (VBCF) Next-Generation Sequencing (NGS) Unit (www. vbcf.ac.at). Adapter trimming and removal of low-quality bases were performed using cutadapt. After alignment of reads against contam- inating sequences (mitochondrial and ribosomal DNA), the remain- ing reads were aligned against GRCm37 using transcriptome-guided alignment with TopHat, version 1.4.1 (http://ccb.jhu.edu/software/ tophat/index.shtml). Next, the htseq-count (http://htseq.readthedocs. io/en/master/count.html) with mode union was used to obtain gene counts for union gene models. Then, differentially expressed genes (log2 fold change >2 and FDR-adjusted q < 0.1) were determined using DESeq2, version 1.12.4 (Bioconductor). For heatmaps, centered and scaled rlog-transformed library size– normalized counts were visualized using the heatmap.2 function of R package gplots, version 3.0.1 (https://www.rdocumentation.org/ packages/gplots/versions/3.0.1). Viability assay. Murine T cells (5 × 104) from hSTAT5BN642H and WT mice were seeded in triplicate in 96-well plates. JQ1, 5-azacyti- dine, entinostat, etoposide, AT9283, tofacitinib, and ruxolitinib (all from Selleckchem) were added and incubated for 72 hours. All com- pounds were solubilized in DMSO (MilliporeSigma). DMSO and bor- tezomib (Selleckchem) were used as a negative and positive control, respectively. CellTiter-Glo reagent (Promega) was used to determine viability, measured on an EnSpire plate reader (PerkinElmer). The Journal of Clinical Investigation IC50 val- ues were determined by nonlinear regression using GraphPad Prism 6 (GraphPad Software). Gene lists from differential expression analyses were ranked for the log2 fold changes between hSTAT5BN642H and WT or hSTAT5BN642H and hSTAT5B CD8+ T cells. Ranking lists were subsequently used for GSEA via the Broad Institute’s GSEAPreranked tool at the standard setting. Gene sets were obtained from current publications or from the Broad Institute’s Molecular Signatures Database (MSigDB). RNA-seq data and a description of the experimental design are available in the NCBI’s Gene Expression Omnibus (GEO) database (GEO GSE104557). RRBS and analysis. Genomic DNA from purified CD8+ T cells was isolated using an AllPrep DNA/RNA Mini Kit (QIAGEN) and subsequently subjected to RRBS and analysis. RRBS was carried out as described earlier (85). In brief, 100 ng genomic DNA was digest- ed for 12 hours at 37°C with 20 units of MspI (New England Bio- Labs; R0106L) in 30 μl of 1× NEB Buffer 2. Fill-in and A-tailing were performed by the addition of Klenow Fragment 3′→ 5′ exo- (New England BioLabs; M0212L) and dNTP mix (10 mM dATP, 1 mM dCTP, 1 mM dGTP). After ligation to methylated Illumina TruSeq LT v2 adaptors using Quick Ligase (New England BioLabs; M2200L), the libraries were size selected by performing a 0.75× clean-up with AMPure XP beads (Beckman Coulter; A63881). Up to 12 libraries were pooled in equal amounts on the basis of qPCR data and bisulfite converted using the EZ DNA Methylation Direct Kit (Zymo Research; D5020) with the following changes to the manufacturer’s protocol: the conversion reagent was used at ×0.9 concentration; incubation was performed for 20 cycles of 1 minute each at 95°C, followed by 10 minutes at 60°C; and the desulfonation time was extended to 30 minutes. Bisulfite-converted libraries were enriched for up to 17 cycles using PfuTurbo Cx Hotstart DNA Polymerase (Agilent Tech- nologies; 600412). After a 2× AMPure XP clean-up, quality control was performed using a Qubit dsDNA HS Assay Kit (Life Technolo- gies, Thermo Fisher Scientific; Q32854) and an Experion DNA 1K Analysis Kit (Bio-Rad; 700-7107). Sequencing was performed on an Illumina HiSeq 3000/4000 System using the 5-bp single-end mode. Initial data processing was carried out at the Biomedical Sequencing Facility of the Medical University of Vienna (http://www.biomed- ical-sequencing.at) using an in-house pipeline based on Pypiper (http://databio.org/pypiper) and Looper (http://databio.org/loop- In vivo ruxolitinib treatment. Methods Cell culture. 293T cells were cultivated with complete DMEM medium (10% FCS, 2 mM L-glutamine, 10 U/ml penicillin-streptomy- cin). Ba/F3 and 32D cells were cultivated with complete RPMI 1640 medium (10% FCS, 2 mM L-glutamine, 10 U/ml penicillin-strepto- RNA sequencing and analysis. mRNA was isolated from CD8+ T cells harvested from LNs from mice of all 3 genotypes. CD8+ T cells 3 9 7 jci.org Volume 128 Number 1 January 2018 2015;208(4):115–128. 10. Bamford S, et al. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and web- site. Br J Cancer. 2004;91(2):355–358. 20. Ma X, et al. Rare occurrence of a STAT5B N642H mutation in adult T-cell acute lymphoblastic leu- kemia. Cancer Genet. 2015;208(1–2):52–53. 2. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7(1–2):203–214. 11. Imada K, et al. Stat5b is essential for natural killer cell-mediated proliferation and cytolytic activity. J Exp Med. 1998;188(11):2067–2074. 21. Jiang L, et al. Exome sequencing identifies somat- ic mutations of DDX3X in natural killer/T-cell lymphoma. Nat Genet. 2015;47(9):1061–1066. 3. Nivarthi H, Friedbichler K, Moriggl R. Stat5 as a Hematopoietic Master Regulator for Differenti- ation and Neoplasia Development. In: Decker T, Müller M, eds. Jak-Stat Signaling: From Basics to Disease. Vienna: Springer;2012:153–167. 12. Lin JX, Leonard WJ. The role of Stat5a and Stat5b in signaling by IL-2 family cytokines. Oncogene. 2000;19(21):2566–2576. 22. López C, et al. Genes encoding members of the JAK-STAT pathway or epigenetic regulators are recurrently mutated in T-cell prolymphocytic leukaemia. Br J Haematol. 2016;173(2):265–273. 13. Rajala HL, et al. Discovery of somatic STAT5b mutations in large granular lymphocytic leuke- mia. Blood. 2013;121(22):4541–4550. 4. Bunting KD. STAT5 signaling in normal and pathologic hematopoiesis. Front Biosci. 2007;12:2807–2820. 23. Nairismägi ML, et al. JAK-STAT and G-protein- coupled receptor signaling pathways are fre- quently altered in epitheliotropic intestinal T-cell lymphoma. Leukemia. 2016;30(6):1311–1319. 14. Nicolae A, et al. Frequent STAT5B mutations in γδ hepatosplenic T-cell lymphomas. Leukemia. 2014;28(11):2244–2248. 5. Heltemes-Harris LM, Farrar MA. The role of STAT5 in lymphocyte development and transfor- mation. Curr Opin Immunol. 2012;24(2):146–152. 15. Bandapalli OR, et al. The activating STAT5B N642H mutation is a common abnormality in pediatric T-cell acute lymphoblastic leukemia and confers a higher risk of relapse. Haematologi- ca. 2014;99(10):e188–e192. 24. Lavallée VP, et al. Chemo-genomic interrogation of CEBPA mutated AML reveals recurrent CSF3R mutations and subgroup sensitivity to JAK inhibi- tors. Blood. 2016;127(24):3054–3061. 6. Ferbeyre G, Moriggl R. The role of Stat5 transcrip- tion factors as tumor suppressors or oncogenes. Biochim Biophys Acta. 2011;1815(1):104–114. 7. Villarino A, et al. Signal transducer and activator of transcription 5 (STAT5) paralog dose governs T cell effector and regulatory functions. Elife. 2016;5:e08384. 25. Simpson HM, et al. Concurrent mutations in ATM and genes associated with common γ chain signaling in peripheral T cell lymphoma. PLoS One. 2015;10(11):e0141906. 16. Kiel MJ, et al. Author contributions ium chloride wash buffer (0.5 M LiCl2, 50 mM HEPES, 1 mM EDTA, 0.7% sodium deoxycholate, 1% NP-40) and then once in Tris-EDTA (TE) buffer containing 50 mM NaCl. Chromatin was eluted in 2× 100 μl elution buffer (1% SDS, 50 mM Tris, 10 mM EDTA). Eluted chro- matin (20 μl) was used for WB analysis. Samples and inputs were incubated with 8 μl of 5 M NaCl at 65°C overnight and subsequently incubated with 0.5 M EDTA, 1 M Tris (pH 6.5), and proteinase K (10 mg/ml) for 2 hours at 55°C. RNA was lysed for 1 hour at 37°C using 0.2 mg/ml RNase-A (MilliporeSigma). Chromatin clean-up was per- formed using a PCR purification kit (QIAGEN). DNA was subjected to qPCR using GoTaq Real-Time qPCR (Promega), and the amount of amplification was quantified using standard curves. Primers are listed in Supplemental Table 7. RM designed and supervised the study. HTTP, BM, MPM, EG, TJ, HN, ZK, TK, AB, SK, MF, MM, TR, VS, and RM designed and/or performed experiments. HTTP, RG, FH, and MPM analyzed data. JP and FG contributed to the interpretation of the data. LK inter- preted IHC results. ME designed and performed experiments. PV, MH, and CB revised the manuscript with regard to critical intellec- tual content. HTTP, BM, VS, and RM wrote the manuscript. The Journal of Clinical Investigation hSTAT5BN642H CD8+ T cell transplant recipients were treated with ruxolitinib (Chemietek) twice a day by oral gavage at a dosage of 45 mg/kg. Ruxolitinib was dissolved in DMSO (MilliporeSigma) and subsequently diluted in 0.5% methylcel- lulose (w/v) (MilliporeSigma). ChIP. CD8+ T cells (107 cells) from WT, hSTAT5B, and hSTAT5BN642H mice were isolated using a CD8+ MagniSort Enrichment Kit. Isolat- ed cells were washed twice with ice-cold PBS supplemented with inhibitors (1 mM Na3VO4, 1 mM NaF, 1× cOmplete Protease Inhibitor Cocktail [PIC], Roche) and fixed with DSG (2 mM, 30 min; Thermo Fisher Scientific). Cells were washed twice with cold PBS supple- mented with inhibitor and fixed with formaldehyde (1%, 10 min; MilliporeSigma). Fixation was quenched by incubation with glycine (125 mM, 5 min; MilliporeSigma). T cells were subsequently harvest- ed by centrifugation (350 g, 5 min). Cell lysis was performed with 1% SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris [pH 8.1], 1 mM Na3VO4, 1 mM phenylmethylsulphonyl) at 4°C for 30 minutes and sonicated using a Diagenode Bioruptor (20 cycles with 30 sec- onds on, 30 seconds off, high magnitude). Sonication was followed by chromatin dilution (1:10) in dilution buffer (167 mM NaCl, 16.7 mM Tris [pH 8.1], 1.2 mM EDTA, 1.1% Triton-X, 0.01% SDS). Clear chromatin was harvested by centrifugation (10,000 g, 10 min, 4°C). Cleared chromatin was incubated with rolling at 4°C with 5 μg STAT5 (C-17) (Santa Cruz Biotechnology; sc-835 X), EZH2 (Diagenode; pAb-039-050), or IgG (Santa Cruz Biotechnology; sc-2027 X) over- night at 4°C. Diluted chromatin (1%) was kept as the input. Blocked Dynal Magnetic Beads (65 μl; Life Technologies, Thermo Fisher Sci- entific) were added per IP the following day and incubated for an additional 4 hours at 4°C. IP samples were washed 5 times with lith- 3 9 8 jci.org Volume 128 Number 1 January 2018 2. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7(1–2):203–214. 1. Stark GR, Darnell JE. The JAK-STAT pathway at twenty. Immunity. 2012;36(4):503–514. Acknowledgments We would like to thank Gregor Hörmann, Safia Zahma, Graham Tebb, Michaela Schlederer, Katrin Meissl, Johannes Schmöllerl, Patricia Stiedl, Helmut Dolznig, Margit Rosner, Thomas Weich- hart, Claus Vogl, and all members of Ludwig Boltzmann Institute for Cancer Research (LBI-CR) for their help and support. This work and RM, HTTP, BM, MM, VS, SK, CB, MF, and PV are sup- ported by the Austrian Science Fund (FWF) SFB grant F47 and F61 subprojects F4701-B20, F4704-B20, F4706-B20, F4707-B20, F6101, F6102, F6105, F6106, and F6107. FG is supported by the European Research Council (ERC) Starting Grant ONCOME- CHAML. FH is supported by a postdoctoral fellowship from the German Research Council (DFG) (HA 7723/1-1). Statistics. Flow cytometric data are reported as the mean ± SD and were analyzed using GraphPad Prism 6 (GraphPad Software). Differ- ences were assessed for statistical significance by an unpaired, 2-tailed Student’s t test and 1-way ANOVA with Bonferroni’s correction. Kaplan-Meier plots were analyzed using a log-rank (Mantel-Cox) test. P values for GSEA were determined using the Kolmogorov-Smirnov test. A P value of less than 0.05 was accepted as statistically significant. Study approval. All animal experiments were approved by the institutional ethics committee and the Austrian Ministry BMWFW authorities under the animal license protocols BMWFW-68.205/ 0166-WF/V/3b/2015), BMWFW-68.205/0117-WF/V/3b/2016, and BMWFW-68.205/0103-WF/V/3b/2015. All mice were bred and main- tained under standardized conditions at the University of Veterinary Medicine Vienna. 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https://openalex.org/W2038902706	http://hrcak.srce.hr/file/237068	English	null	Recipe Development Process Re-Design with ANSI/ISA-88 Batch Control Standard in the Pharmaceutical Industry	International journal of engineering business management	2,014	cc-by	10,707	ARTICLE ARTICLE International Journal of Engineering Business Management Special Issue: Innovations in Pharmaceutical Industry International Journal of Engineering Business Management Special Issue: Innovations in Pharmaceutical Industry DOI: 10.5772/59025 © 2014 The Author(s). Licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Reducing time-to-market is one of the most challenging tasks that pharmaceutical companies deal with. In this sense, the recipe development process represents one of the most critical phases. Multi-site production companies require an efficient recipe development model, with a robust modular structure, which must be appropriately shared among local laboratories and plants. To this extent, the ANSI/ISA-88 batch manufacturing standard, rising in the context of process control and automation, is rapidly becoming widely used in pharmaceutical companies. This paper presents a step-by-step approach to assessing the compliance to the ANSI/ISA-88 standard along with a BPM-oriented methodology applicable to the re-design of any generic recipe development process. Redesigning a recipe development process is a complex activity and can mask several pitfalls and criticalities. Thus, along with the methodology, some general evidence and suggestions are provided based on the experience of a project carried out in a large multinational pharmaceutical company. Margherita De Minicis1, Francesco Giordano1, Federico Poli1 and Massimiliano M. Schiraldi2,* 1 Operations Management Team srl, Roma, Italy 2 University of Rome “Tor Vergata”, Department of Enterprise Engineering, Roma, Italy * Corresponding author E-mail: schiraldi@uniroma2.it 1 Operations Management Team srl, Roma, Italy 1 Operations Management Team srl, Roma, Italy 1 Operations Management Team srl, Roma, Italy 2 University of Rome “Tor Vergata”, Department of Enterprise Engineering, Roma, Italy * Corresponding author E-mail: schiraldi@uniroma2.it y g p p g g * Corresponding author E-mail: schiraldi@uniroma2.it Received 03 Jun 2014; Accepted 26 Aug 2014 Received 03 Jun 2014; Accepted 26 Aug 2014 www.intechopen.com www.intechopen.com 1. Introduction Brief comments on the ANSI/ISA-88 standard (paragraph 2) and the BPM approach (paragraph 3) are also included, in order to introduce these concepts. Finally, the results and the conclusion (paragraph 6) are presented to highlight the benefits of this methodology and to discuss some common misalignments that may arise while assessing their compliance to the ANSI/ISA-88 standard. ANSI/ISA-88 is rapidly becoming a widely used standard in batch manufacturing, especially in pharmaceutical companies [6,7,8,9,10]. The standard, first approved in 1995 by the International Society of Automation (ISA) and then updated at a later stage in 2010, arises in the context of batch process control and automation. However, the recent growth of interest in this standard on the part of multinational firms can be attributed to the well- established set of models and terminology [11,12,13,14,15] provided, which proved to be useful for managing the end-to-end recipe development processes (from R&D to commercial production). Adopting a new product development model, according to ANSI/ISA-88 guidelines, affects both the conceptual and documental level. The American standard defines the recipe contents, differentiating between equipment independent recipes, which are managed by scientists at an enterprise/laboratory level, and equipment dependent recipes, which are enriched with specific information about plant and process cells required for commercial production [16,17,18]. Moreover, when dealing with numerous/different manufacturing sites, most difficulties in implementing the commercial production of drugs arise during the transformation of the generic recipe (which specifies product characteristics and overlooks processing equipment parameters) into a “master recipe”, calibrated to the specific machinery of a given plant. The reasonable need to differentiate the form and content of the documentation concerning different phases of the development process is clear, especially when we consider the scale-up required while passing from a pilot plant to a production plant: the parameters of materials and equipment usually change when processing a 10-litre batch or a 20,000 one. Despite this, all the information must be efficiently communicated between the main actors in the recipe development-chain. The transition from one recipe to another needs to be rationalized, and the knowledge acquired in every transformation activity must be accurately managed in order to reduce the overall time spent before commercial production, thus facilitating further development processes [19,20]. 1. Introduction As most recent pharmaceutical companies’ trends attests, the time needed for the development process of a new drug is continuously speeding up, especially concerning the post-approval of clinical phases [1,2,3]. Interest in process improvement and optimization has been a partial response to the observed trend of stagnant profit margins and the current growth of competition in this sector, regarding both general and patented drugs. Thus, reducing time-to-market and recipe development intervals are some of the principal features and challenges that pharmaceutical companies are dealing with [4,5]. Multinational corporations in particular require an efficient recipe development model, with a robust modular structure, which must be appropriately shared among local laboratories and plants. ANSI/ISA-88, in this sense, can be considered a valid contribution to reaching the so-called “golden batch”, providing a mature framework where information-streams properly integrate all the key actors involved. However, re- designing such a complex process, which is cross- functional and affects the main end-to-end development phases, can mask several pitfalls and difficulties. Keywords ANSI/ISA-88 Standard, Recipe Development Process, Business Process Management, Process Mapping co Giordano, Federico Poli and Massimiliano M. Schiraldi: 88 Batch Control Standard in the Pharmaceutical Industry Int J Eng Bus Manag, 2014, 6:16 \| doi: 10.5772/59025 1 ANSI/ISA-88 is rapidly becoming a widely used standard in batch manufacturing, especially in pharmaceutical companies [6,7,8,9,10]. The standard, first approved in 1995 by the International Society of Automation (ISA) and then updated at a later stage in 2010, arises in the context of batch process control and automation. However, the recent growth of interest in this standard on the part of multinational firms can be attributed to the well- established set of models and terminology [11,12,13,14,15] provided, which proved to be useful for managing the end-to-end recipe development processes (from R&D to commercial production). Business Process Management approach and the Business Process Model Notation 2.0 (BPMN 2.0) [21,22]. Any effective process improvement initiative requires a clear definition of the as-is and to-be scenario, in order to analyse the gaps and identify a proper road map to reach the expected results. In addition, a standard and appropriate notation is required to physically represent the flows of activities and documentation. Hereafter, paragraph 4 and 5 will describe in detail the path of the proposed method and a case study of the implementation of the method at a large pharmaceutical multinational company. 2. ANSI/ISA-88 standard ANSI/ISA-88 is an international standard, which widely addresses all the main features related to batch manufacturing processes, describing procedures and equipment requirements during all the development phases, from the enterprise/laboratory level to commercial production [23,24]. It is important to point out that reference models and guidelines provided by the ISA standard are not to be considered strictly normative (some clauses are informative as well). This ensures a good level of flexibility in representing the current structure in an “ANSI/ISA-88 way”, giving also the opportunity to collapse and expand parts of the reference model to better suit peculiar batch manufacturing cases. Hence, considering that the models provided are sufficiently abstract that they may be applied to a wide variety of batch manufacturing implementations, and that compliance certifications are usually performed by independent organizations, the assessment of the degree of ANSI/ISA-88 compliance is essentially to be meant as an alignment on the use of the terminology and models definition. Whereby a partial compliance exists, specifications and implementations must be described, and areas of non- compliance are to be identified. Since a full description of the standard is out of the scope of this article, a brief description is provided only for those elements that are significant for the described case, ensuring a clear understanding of the contents and omitting the non-essential information. Thus, in order to fully achieve the benefits of ANSI/ISA- 88 implementation, this paper presents an integrative methodology, applicable to the re-design of any generic recipe development process in compliance with the ANSI/ISA-88 standard. The aim is to provide a step-by- step approach to assess the standard compliance and perform a recipe development process re-design, by leveraging process oriented techniques, such as a 1. Introduction While the recipes and the process model are well defined, the main area lacking in the current ANSI/ISA-88 standard is the extent and content of the guidelines provided for the implementation of the general-to-specific recipe transformation. www.intechopen.com Int J Eng Bus Manag, 2014, 6:16 \| doi: 10.5772/59025 2 2.2. Reference model, Recipes and Libraries Fina proc com are the ally, the physic cess cells and t mposed of equip the list of de control activit cal model logic then into unit pment modules evices that car ties. ally groups eq ts. Farther on, s and control m rry out the pr quipment into , units can be modules, which rocessing and o e h d The landmar between the model, whi importance o physical enti forming the process. Eac structure th visualization equipment, Hence, starti overall proce separates pa phases. Proce a clear divisi physical cha processed. Fa of subdivisio of the same p rk of the enti e process, pro ch is depic of the model is ities, such as conceptual h model has hat allows it n of processe to a deeper ing from the A ss can be div arts of the p ess stages are on of processi anges follow arther on, pro on, identifying process operat ire standard i ocedural cont ted in Figu s the logical se process cells steps of a a multi-hier t to pass fr s, procedures description ANSI/ISA-88 P vided into pro process into then made of ing tasks whe one another ocess actions ar g the minor pr tion. is the relation trol and phy ure 1. The eparation betw s and equipm batch produc rarchy breakd rom a high- s and produc of each elem Process Mode ocess stages, w main concep f process operat ere the chemic r in the mat re the lowest rocessing activ nship ysical very ween ment, ction down level ction ment. el, an which ptual tions, cal or terial level vities The beco stru abov gene of th one 600- spra of a are phy or th linkages be ome clearer o ucture are und vementioned eric process ac he process oper or more proc -litre bin, asse aying paramet specific plant then linked t ysical model, s he specific 600 etween differe once the elem derstood. For drug oral ctions, charge ration wet gra cedural phases embling a sp ter, consistent t’s production to one or mor such as the ut 0-litre bin. ent levels of ments of each r example, co production and spray, w anulation, can s, such as the ray, or insert t with the wor n. 2.1 ANSI/ISA-88 structure The ANSI/ISA-88 standard is divided into five parts that respectively deal with different features of the batch manufacturing process and control. Part 1 (Models and Terminology) [25] defines the reference models (physical, Int J Eng Bus Manag, 2014, 6:16 \| doi: 10.5772/59025 2 process an relationship process step perform a b different hier that is comm also defined, Part 2 (Data defines the implementat Part 1. This graphical n represent t dependent re Site Recipe M recipe-indepe contents of how a stand created. In t represent an perform the t of the standa detailed defi providing a r of batch pro At the end, for Modular defines imp modular eq equipment co d procedura between the ps and the batch produc rarchical leve mon and consis , and generic a Structures an data mode ion, addressin s part as we notation (pro he execution ecipe procedu odels and Repre endent object equipment-in dard library o this part, we n equipment-in transformatio ard (Batch Pro nition of batc reference mod oduction recor Part 5 (Implem Equipment Co plementation quipment co ontrol concept al model), equipment, procedural t ction, subdiv ls. 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Schiraldi: Recipe Development Process Re-Design with ANSI/ISA-88 Batch Control Standard in the Pharmaceutical Industry 3 2.1 ANSI/ISA-88 structure ology draft, y for 1’s proc disp For proc cess stage is m pensing, blend the wet gran cess actions are made of severa ding, wet gra nulation proc charge, mix, s al process opera anulation and cess operation spray and dry ations, such as film coating. n some basic y. s . c Figuure 1. ANSI/ISAA-88 reference m model Figuure 1. ANSI/ISAA-88 reference m model The elem are phas proc rela prod spec oper analogue br ments of the p divided into u ses. The subst cedures is th tion with th duce a comm cific plant rational work reakdown ap procedural contr unit procedure, tantial differe hat procedure he equipmen mercial batch, conditions a instructions. pproach is us rol model. 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Reference model, Recipes and Libraries Finally, proc re equipment m tilized fluid be f the models h breakdown nsidering the process, the which are part n be linked to charging of a ting an initial rk instructions cedural phases modules of the ed granulator s n e e t o a l s s e r AN “rec info requ be SI/ISA-88 ha cipe”, descri ormation tha uirements for used from d s a wide in ibing it as at uniquely r a specific pr different par nterpretation the necess defines the roduct. Since rts of an en of the term sary set of e production e recipes may terprise, this m f n y s Considering production p could be man for example process, typical nufacturing an the pharmac l process stages d packaging. 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Int J Eng Bus Manag, 2014, 6:16 \| doi: 10.5772/59025 4 www.intechopen.com 2.2. Reference model, Recipes and Libraries Consi ur different mentation (fr ecipes are rela al evolution o production d vary in con nt requiremen a common s eaders, form s, and other the ANSI/ISA iefly mentione ring the var idering the r recipes exis rom enterpris ated to each o of documenta etails are ad ntents, espec nt to produ structure mad mulas, equipm information. A-88 standard ed below: rying recipe t at se to other ation dded. cially ce a de of ment The d are shown in Figure 2 and briiefly mentioned below: • General of infor will be u raw m process regard t at that s Recipe: is crea rmation about used to manu materials, the sing requirem to a particular site. ated without s the process ce ufacture the pr eir relative ments, but r site or the eq pecific knowl ell equipment roduct; it ident quantities, without spe quipment avai edge t that tifies and ecific lable Figuure 2. ANSI/ISAA-88 Recipe Moddel 3. A com A BPM approac mpliance projec ch to managin cts ng ANSI/ISA-888 To this extent, the proposed method was applied and validated on the case of a multinational pharmaceutical enterprise. but implementing an effective transformation from the as-is process model to a compliant to-be one can be quite challenging for a company. The introduction of a BPM method at this stage can help identify gaps and define the road map, while BPM notation is a practical tool which can effectively provide clear visibility of the process and documental flows, enabling wiser decisions. To this extent, the proposed method was applied and validated on the case of a multinational pharmaceutical enterprise. We identify four main sequential phases to perform the activities in a structured way: • Phase 1: initial scope and input definition. In each case, this cycle of improvement activities must be supported by a consistent graphical notation, capable of correctly representing the main subjects, events, activities and process rooting. Business Process Model Notation (BPMN 2.0) is considered one of the most efficient standards [33, 39] to represent in a graphical way a varied range of business and service processes. BPMN is an intuitive notation, suitable also for non-technicians figures, but capable of either representing complex process semantics or easing B2B and internal coordination. The activities of the flow and process representations are simple to understand, and clear communication is enhanced between who develops a new model and the final users who are affected by the analysed process. Furthermore, BPMN models consists of basic diagrams (BPD, Business Process Diagrams) which are built from a limited set of graphical elements, that are flow objects, connection objects, swim lanes and artefacts [40]. The following paragraphs explain why such a process oriented approach and flow-chart technique were chosen for the proposed methodology, highlighting the advantages obtained in the case study while assessing the ANSI/ISA-88 compliance. • Phase 2: identification of ANSI/ISA-88 applicable areas. • Phase 3: process analysis & mapping. • Phase 4: gap analysis and road map definition. Moreover, two additional cross-activities must be run to Moreover, two additional cross-activities must be run to support the implementation of the method itself: • Core team members’ education in ANSI/ISA-88 standard. • Convergence meetings, following a project • Convergence meetings, following a project management methodology. management methodology. 3. A com A BPM approac mpliance projec ch to managin cts ng ANSI/ISA-888 Proc [29,3 oper rega cess modelli 30,31,32,33], i rational efficie ardless of the ing, as man s currently be ency and proc e industry sec ny publicati ecoming a co cess improvem ctor to which ons confirm ornerstone for ment projects, it is applied. m r , . Int J Eng Bus Manag, 2014, 6:16 \| doi: 10.5772/59025 4 www.intechopen.com Adopting a process oriented approach and using a visual representation ensures a better control of performance and enables the detection of those existing inefficiencies that are hardly recognizable. Corporate process flows are often made of many figures at different stages, and the aggregation of several complex phases in few synthetic diagrams should be avoided. Indeed, a clear visibility of the entire stream of events is required in order to properly understand the linkages among activities, identify the strengths and weakness of a process and, eventually, facilitate interaction and integration with outsourcers [34,35] or with suppliers [36]. Additionally, process mapping and modelling can be considered one of the first steps for almost every improvement methodology that take place when analysing processes [37,38]. Both to identify opportunities and constraints, or even to handle a correct change management process, an as-is analysis and mapping are required. Having a clear understanding of the current stream of activities leads to a correct definition of an optimal to-be scenario. Once the desired future state is also clear, it is finally possible to perform a gap analysis and define an improvement implementation road map in order to achieve the expected results. Management approach. The aim of the method is to provide a reference framework which allows practitioners to perform an ANSI/ISA-88 compliance assessment in different contexts. The motivations for such an effort lie in the need to support the implementation of ANSI/ISA-88 guidelines with a methodological and structured approach. Proven experiences have evidenced that implementing a new product development process in pharmaceutical companies is a difficult task, hiding several inefficiencies or inconsistencies concerning the flow of activities. ANSI/ISA-88 certainly provides a strong reference model, but implementing an effective transformation from the as-is process model to a compliant to-be one can be quite challenging for a company. The introduction of a BPM method at this stage can help identify gaps and define the road map, while BPM notation is a practical tool which can effectively provide clear visibility of the process and documental flows, enabling wiser decisions. Margherita De Minicis, Francesco Giordano, Federico Poli and Massimiliano M. Schiraldi: Recipe Development Process Re-Design with ANSI/ISA-88 Batch Control Standard in the Pharmaceutical Industry 5 www.intechopen.com 3. A com A BPM approac mpliance projec ch to managin cts ng ANSI/ISA-888 Phase 1: this phase covers the initial scope and input definition, where project boundaries are defined and preliminary data gathering is performed, in order to discuss the project main objectives, together with the identification of expected benefits from applying the standard. Phase 2: the results of phase 1 are used to identify ANSI/ISA-88 applicable / not applicable areas and define specific process requirements. Indeed, as already mentioned, the ANSI/ISA-88 standard is not strictly normative and some areas of the reference model can be collapsed or expanded due to peculiarities of the recipe development process. 4. Proposal for an ANSI/ISA-88 compliance assessment and roadmap identification 4. Proposal for an ANSI/ISA-88 compliance assessment and roadmap identification This paragraph illustrates the proposed methodological approach to carryng out an ANSI/ISA-88 assessment and provides a roadmap for full compliance, leveraging the above-mentioned tools of the Business Process In order to point out the key information of the as-is process, accountable personnel, with deep knowledge of the company’s process architecture and documentation, need to be involved. These core team members should belong to the corporate functions affecting the recipe development process, such as the introduction of new products, the technical integration of drug product development, process validation, manufacturing execution systems, operation quality, quality assurance, and IT/automation. Phase 4: At the end of the previously described phases, it is possible to design the processes in a to-be version, highlighting similarities with the current model and detecting gaps to be covered. During this phase, actions to bridge the gaps and highlight priorities must be identified and formalized into a specific road map. In more detail, the main topics to be addressed are summarized as follows: Data gathered within phase 2 will feed the subsequent steps, whereby all the fragmented information must be consolidated to provide clear visibility of the end-to-end stream process and to identify the requirements of the specific case, thus restricting the focus only to applicable areas of the standard reference model. Examples of collapsible and extendable areas are: recipe types, recipe information structure, recipe graphical representation, the transformation process of equipment-independent to master recipes, and contents of the equipment- independent recipe object model. • Alignment on ANSI/ISA-88 terminology (process model, physical model and procedural model). • Process elements (process stage, operation and action) and process element standard libraries definition, following ANSI/ISA-88 data architecture. • General, site, master and control recipe contents and structure. • Graphical representation of equipment independent and dependent processes. • General-to-master recipe transformation process • General-to-master recipe transformation proce activities and document definition. Moreover, as mentioned above, during this phase and the subsequent ones, training on ANSI/ISA-88 to all main figures interviewed is required, in order to ease the as- is/to-be transformation process model. activities and document definition. The identified road map represents the main deliverable of the proposed method. Phase 3: once the main data are collected and ANSI/ISA- 88 applicable/ not applicable areas are defined, the process can be properly analysed and mapped in phase three. 4. Proposal for an ANSI/ISA-88 compliance assessment and roadmap identification First, an as-is recipe development process analysis is required. In order to depict this information in diagrams and have a visual representation of the process, the proposed method takes advantage of the BPMN 2.0, which provides an effective process modelling tool. Considering the complexity of the activities performed by different actors, operating both at a corporate, R&D and plant level, process end-to-end visibility is essential to defining the degree of similarity to the ANSI/ISA-88 standard’s expectations. The reason why BPMN notation was chosen rather than other process flow methods is that it belongs to both the specific and adaptive set of graphical elements, which can be used to represent the process. Moreover, this tool gives us the possibility of using diagrams to create simulations and automated workflows in further phases. Another important reason for this choice is the increasing adoption of BPMN as a common standard in most companies for process modelling, workflow automation and direct execution. These considerations become more valuable considering that ANSI/ISA-88 initiatives often take place in large multinational multi-location companies, where the use of standards among different production plants enhance knowledge sharing [41,42,43] and information exchanges. Together with the explanation of the method, our proposal also provides a list of recommendations derived from the experience obtained from a real case implementation: redesigning a recipe development process is a complex activity, and can mask several pitfalls and criticalities. In order to facilitate the adoption of the proposed method and correctly implement the different phases, some general evidence and suggestions are provided. Below the most relevant considerations supporting phase 3 and phase 4 of the proposed reference model are indicated: • Generate templates for ANSI/ISA-88 compliant documents from company existing ones (Phase 3): considering that in pharmaceutical companies recipe development processes are well established by standard operative procedures, one of the most critical tasks is the effective representation of the future recipe development process, together with the transition itself. The proposed method suggests starting the redesign process by transforming existing documents related to a new product development (NPD) process of a specific pharmaceutical product, in order to generate standard ANSI/ISA-88 documents, such as a general and master recipe, to be used as a reference. By creating these new documents, a direct link between the main information of the current and future documentation is possible and the degree of similarity to the ANSI/ISA-88 standard is verified. Int J Eng Bus Manag, 2014, 6:16 \| doi: 10.5772/59025 6 4. Proposal for an ANSI/ISA-88 compliance assessment and roadmap identification Identifying all the elements and the perfect linkages between them is a significant strategy for improving the general recipe standardization. As discussed above, a great amount of effort is made to transform the general recipe into a master recipe. This difficulty is generally due to the absence of standard contents and graphical representation used in all the documents involved in the recipe development process. Knowledge and information sharing is essential to reduce the time-to-market of new products. To overcome this problem, the proposed method suggests creating a set of stencils (e.g., in Microsoft Visio), according to the notation suggested by the ANSI/ISA-88 standard. p p Standardize process elements and share process element libraries (Phase 3/4): the definition of standard elements to describe the recipe development process is only a starting point. These elements, used to represent the recipe flow, should be exclusively selected from a shared library with a strong modular structure. Process actions represent the main building block to start from, in order to define the to-be standard process operations and stages. Identifying all the elements and the perfect linkages between them is a significant strategy for improving the general recipe standardization. As discussed above, a great amount of effort is made to transform the general recipe into a master recipe. This difficulty is generally due to the absence of standard contents and graphical representation used in all the documents involved in the recipe development process. Knowledge and information sharing is essential to reduce the time-to-market of new products. To overcome this problem, the proposed method suggests creating a set of stencils (e.g., in Microsoft Visio), according to the notation suggested by the ANSI/ISA-88 standard. Enterprise Recipe Management solution (Phase 4): in order to reduce process variability and batch recipe development cycle time, the increased repeatability of production processes and the reverse flow of information for recipe changes, facilitate introduction of new product/machinery, the adoption of a dedicated content management system or a product life-cycle management system (PLMS) is suggested. These issues are in fact the most relevant for the adoption of ANSI/ISA-88. A PLM enterprise platform is usually able to integrate people, data, processes and business systems allowing the proper management of the entire lifecycle of a product. According to the ANSI/ISA-88 principles, recipe documents and process elements, libraries should be shared among the organization and continuously updated, following the new product introduction process. 4. Proposal for an ANSI/ISA-88 compliance assessment and roadmap identification Once the gaps between processes and documentation are clear, it is possible to outline short term and long term opportunities for improvement • Generate templates for ANSI/ISA-88 compliant documents from company existing ones (Phase 3): considering that in pharmaceutical companies recipe development processes are well established by standard operative procedures, one of the most critical tasks is the effective representation of the future recipe development process, together with the transition itself. The proposed method suggests starting the redesign process by transforming existing documents related to a new product development (NPD) process of a specific pharmaceutical product, in order to generate standard ANSI/ISA-88 documents, such as a general and master recipe, to be used as a reference. By creating these new documents, a direct link between the main information of the current and future documentation is possible and the degree of similarity to the ANSI/ISA-88 standard is verified. Once the gaps between processes and documentation are clear, it is possible to outline short term and long term opportunities for improvement. • Generate templates for ANSI/ISA-88 compliant documents from company existing ones (Phase 3): considering that in pharmaceutical companies recipe development processes are well established by standard operative procedures, one of the most critical tasks is the effective representation of the future recipe development process, together with the transition itself. The proposed method suggests starting the redesign process by transforming existing documents related to a new product development (NPD) process of a specific pharmaceutical product, in order to generate standard ANSI/ISA-88 documents, such as a general and master recipe, to be used as a reference. By creating these new documents, a direct link between the main information of the current and future documentation is possible and the degree of similarity to the ANSI/ISA-88 standard is verified. Once the gaps between processes and documentation are clear, it is possible to outline short term and long term opportunities for improvement. This methodology allows us to identify the misalignments on the main topics of ANSI/ISA-88, concerning recipe structure, process elements, graphical representation and libraries. Int J Eng Bus Manag, 2014, 6:16 \| doi: 10.5772/59025 6 www.intechopen.com • Ease the recipe development to-be process identification with BMPN notation (Phase 3): the recipe development process can be sensibly improved by the adoption of shared documents and the use of ad-hoc tools, such as a process element library or a set of transform components. 4. Proposal for an ANSI/ISA-88 compliance assessment and roadmap identification During the assessment process, a to-be recipe development process has to be identified considering the aforementioned standard documents and tools. BPMN notation and process oriented methodologies have a significant impact on re-designing the recipe development procedures, providing a clear picture of the as-is situation and helping identify steps to reach an ANSI/ISA-88 full compliance. The use of BPMN to map the recipe development process can be considered a critical step of a repeatable approach for assessing the degree of similarity to ANSI/ISA-88 standard models and procedures. template should be shared across the production sites and R&D functions, facilitating their usage and improving the level of standardization of these documents. Standardize the transform components (Phase 4): one of the main difficulties is to define standard transform components, which are a set of equipment parameters and instructions that allow the transformation of process operations, described in the general recipe, into equipment settings and controls. Usually R&D performs the product development process on small-scale pilot plants, which can use completely different technologies from those of production plants. Farther on, the transformation process needs a long time to be performed, including deep characterization studies. Moreover, when product transfers among production sites are planned, the differences between technologies used in each plant become crucial. A consistent part of the transfer process is spent in customizing equipment parameters and working instructions for the different technologies used at the receiving unit. Hence, it is highly recommended to create a modular structure-based transformation components library, in order to standardize the transformation process for the development and transfer of future products. • Establish an education programme on ANSI/ISA-88 (Phase 3/4): in order to raise awareness of ANSI/ISA- 88 benefits across the organization, after providing education on the method, it is advisable to continue the education process even after the assessment phase, especially if the company production processes are distributed into different plants. p p Standardize process elements and share process element libraries (Phase 3/4): the definition of standard elements to describe the recipe development process is only a starting point. These elements, used to represent the recipe flow, should be exclusively selected from a shared library with a strong modular structure. Process actions represent the main building block to start from, in order to define the to-be standard process operations and stages. Margherita De Minicis, Francesco Giordano, Federico Poli and Massimiliano M. Schiraldi: Recipe Development Process Re-Design with ANSI/ISA-88 Batch Control Standard in the Pharmaceutical Industry 7 www.intechopen.com 5. The case study 5. The case study • Outline short term and long-term improvement opportunities, defining the implementation roadmap. A practical experience of how a process-oriented approach can help when analysing and optimizing a recipe development process is now described. The considered company is a leading firm in the healthcare sector and commercializes a large variety of products all around the world, with production plants in more than 50 different countries. The peculiarity of the organizational model is its independent way of managing the production processes distributed on the sites, where operations are locally managed, whereas coordination and control functions are handled by a central committee. In recent years, the number of new product introductions and technical transfers between plants has noticeably increased due to marketing strategies and cost reduction programmes. As a consequence, the company decided to undertake several initiatives aimed at speeding up the time-to-market of new products and improving knowledge management within transfers. The most effective way to reach these goals was identified by top management in the adoption of a recipe management strategy, based on the ANSI/ISA-88 standard, focused on product life-cycle, which uses an end-to-end approach. The ANSI/ISA-88 initiative, discussed here, was then undertaken in the company’s best-in-class manufacturing plant to define the standard recipe management strategy, in view of its worldwide application. 5.1 Main findings on the ANSI/ISA-88 reference model 4. Proposal for an ANSI/ISA-88 compliance assessment and roadmap identification The use of this kind of system may reduce the probability of human errors in the recipe creation and management process, and it is able to guarantee effective communication, capturing all the ANSI/ISA-88 data, including flows and parameters in a single format, and providing a mechanism for content reuse (Recipe Building Blocks), which is critical to recipe normalization. PLM software could also help organizations improve compliance to ANSI/ISA-88 in terms of the standardization of procedures and notations, even allowing the transformation of general and master • Define and adopt the general recipe and master recipe as standard documents (Phase 4): company documents are often not aligned to the ANSI/ISA-88 standard in terms of contents and graphical representation. The creation of specific templates for the general and master recipe, with the intent of providing reference formats for new product recipe development, is recommended. Furthermore, the general recipe • Define and adopt the general recipe and master recipe as standard documents (Phase 4): company documents are often not aligned to the ANSI/ISA-88 standard in terms of contents and graphical representation. The creation of specific templates for the general and master recipe, with the intent of providing reference formats for new product recipe development, is recommended. Furthermore, the general recipe recipe information into equipment-executable recipes. Moreover, the BPMN notation adopted in the proposed method facilitates the implementation of such enterprise management systems. Figure 3 presents an illustrative project Gantt diagram of the industrial case, along with the details of the activities included in each phase. The steps followed during the pilot project were consistent with the proposed approach and concerned the solid oral production process of pharmaceutical drugs performed by the company. The proposed method allowed the following objectives to be reached: To sum up, the proposal combines a series of structured steps to drive the assessment and the definition of a roadmap toward a to-be model, providing a list of practical recommendations to overcome the potential pitfalls and criticalities that companies may be faced during the implementation of this standard. • Map the as-is recipe development process. • Assess the degree of compliance of the analysed processes and documentation to the indications of the standard. The next paragraph describes how effectively the proposed approach helped in deploying a project for a multinational pharmaceutical company. • Identify recipe development process challenges and criticalities. • Define an applicable to-be recipe development process model. 5.1 Main findings on the ANSI/ISA-88 reference model Case s Proces Operatio g Dispensing Solution Prep Mixing Blending Milling Sieving Wet Granulat Dry granulatio Compression Film Coating Membrane Co Caps Filling Laser Drilling Laser Marking Tablet Printin Tablet Drying Suspension Compounding Mould Filling Lyophilization Blister Packag Solid Bottle Packaging Sachet Packag Liquid Bottle F Aerosol Packa Extrusion Film Casting study process el ss on Ch aration Di Di Ag Sie Ho ion He on W Sp Dr oating Dr M Bl g M g Co Co g Co M De n Co ging En Se Dr ging Ca Filling Fr aging Co Bl Bo lements list Process Action harge ischarge issolve gitate eve old eat Weight pray ry ry Mix Mill end Mix ompress Single Lay ompress Bi Layer ompress Tri Layer Metal Check edust oat nteric Coat eal Coat ry Granulate aps Fill reeze Dry ool ister ottle yer Ch Di Di Ag Sie Ho He W Sp Dr Dr M Bl M Co Co Co M De Co En Se Dr Ca Fr Co Bl Bo t Process Action harge ischarge issolve gitate eve old eat Weight pray ry ry Mix Mill end Mix ompress Single Lay ompress Bi Layer ompress Tri Layer Metal Check edust oat nteric Coat eal Coat ry Granulate aps Fill reeze Dry ool ister ottle yer Figu recip ure 4. An exam pe creation proc mple of a BPM cess (illustrative MN2.0 diagram e) for the master r Figu recip ure 4. An exam pe creation proc mple of a BPM cess (illustrative MN2.0 diagram e) for the master Figu plan ure 5 illustrate nts and the AN es the linkage NSI/ISA-88 sta e that emerged andard docum d between the ments. e Figu 5 3 M ure 5. Case study M i fi di y cross referenc i t t ce documentatioon Table 1. Case study process elements list Figuure 5. Case study cross referencce documentatioon By contrast, different from elements, wh process steps elements, w “physically” order to cove layering was of the gene confidentialit procedural el the procedu m the one des hich “theoretic s, had to be li which shoul perform activ er this gap, a s proposed, an eral and ma ty, more d lements canno ral mode app scribed in the cally” describ inked to same ld describe vities on spec conversion of nd implement aster recipe. 5.1 Main findings on the ANSI/ISA-88 reference model As described above, the ANSI/ISA-88 reference model is based on a clear separation of Process, Physical and Procedural Models, with a hierarchical subdivision in different layers. With regard to the Process Model, all elements are divided into processes, stages, operations and actions. In the documentation used within the company, this distinction was not clearly applied; indeed, production processes were mapped using elements that could be connected to both the ANSI/ISA- 88 process operations and actions. This misalignment was not only found on a terminological level but also at a conceptual level. This lack brought a mix of logical information, which misled a correct implementation of the general and master recipes. Consequently, the as-is structure did not allow a complete standardization of the recipe development process. In order to align process representation and mapping, a list of standard elements with a univocal description was edited. This list gave also the input to implement the library of the standard process elements. Even though the analysis took place at a specific production site, the identified building blocks maintained an equipment-independent structure, allowing their application to similar plants. The process stages, operations and actions identified are listed in Table 1. Figure 3. ANSI/ISA-88 assessment project plan (illustrative) Phase 1 1.1 Definition of project boundaries 1.2 Info and data gathering Phase 2 2.1 Identification of ISA 88 applicable points 2.2 Definition of process requirements Phase 3 3.1Analysis of Recipe Development Process AS-IS 3.2 Definition of the degree of similarity 3.3 Recipe & Operations process re-mapping 3.4 Equipment-independent library development Phase 4 4.1 To-Be definition and mapping 4.2 Priority identification and gap analysis 4.3 Identification of action plans to standardization 4.1 1.2 1.1 2.1 2.2 3.1 3.2 3.4 3.3 4.2 4.3 Concerning the physical model, the logical subdivision into the levels used at the site plant was considered coherent with the ANSI/ISA-88 approach. A comprehensive distance in terminology existed, but concept and layering were aligned with the standard guidelines. Figure 3. ANSI/ISA-88 assessment project plan (illustrative) Int J Eng Bus Manag, 2014, 6:16 \| doi: 10.5772/59025 8 www.intechopen.com Process Stage Manufacturing Packaging Table 1. Margherita De Minicis, Francesco Giordano, Federico Poli and Massimiliano M. Schiraldi: Recipe Development Process Re-Design with ANSI/ISA-88 Batch Control Standard in the Pharmaceutical Industry 9 5.1 Main findings on the ANSI/ISA-88 reference model details of th ot be reported peared notice standard. Pro be how to perf e level proced how opera cific equipmen f terminology ted in the exam For reason he physical d in this paper. eably ocess form dural ators nt. In y and mple s of and . 5.3 M Main findings on recipe structures 5.3 M Main findings on recipe structures Even stan asse recip affec tran prov How anot oper was often prod n though all t ndard existed essment brough pe managemen cted the ANS nsfer strategy vided a det wever, this flow ther. Process rations and p s not coherent n, different t duction elemen the relevant in or was genera ht to light the nt in the analys SI/ISA-88 com and the crit tailed produc w chart could d elements wer process actions t with the AN terms were u nt in different d nformation re ated during th absence of a sed case. Three pliance. First, ticality analys ction process differ from one re mapped m s, the resulting NSI-ISA/88 de used to indic documents. quired by the he project, the proper general e main aspects the technical sis documents flow chart. e document to mixing process g terminology efinitions and, ate the same e e l s l s . o s y , e www.intechopen.com 5.2 Main findings from process mapping 5.2 Main findings from process mapping More conside documental f According to main recipes documentatio recipe develo series of files the introduc BPMN diagr decisively he document us erations were flow of the as o ANSI/ISA-88 s (general, s on concernin opment proce s containing a ction of the n ram to map th elped identify sed and the on e dealt with w -is recipe dev 8, the process ite, master a ng the techn esses at the s all the relevan new product he as-is situat the correct lin ne proposed by when analysing velopment pro s is based on and control). ical transfer site consisted nt information t. The use of tion (see Figu nkage between y the standard g the ocess. four The and of a n for f the re 4) n the d. The even repr and crea man ensu equ second point n if ANSI/ISA resentation to suggests pos ation of a pro nagement. Ind ures that only ipment-indep t concerned th A-88 is not n be used, it re sible notation ocess elements deed, the pres y valid eleme pendent recipe he graphical re ormative on equires a logi n solutions, enc s library for g sence of a sta ent definitions es. epresentation: the graphical ical coherence couraging the general recipe ndard library s are used in : l e e e y n Another relevant finding is related to how these documents were produced, and by whom. Initially, documents were created mainly by the collaboration between R&D and the production plant but, as time passed, these documents were continuously updated with more and more details about materials, process characteristics and the equipment, on which the production process was tested and validated. Consequently, documents were added with equipment- dependent information and, then, could not be used as a general recipe. The main consequence is a loss of generality once the recipe is developed and implemented on a specific series of equipment, which implicates difficulties in transferring the information to different plants. In order to overcome this problem, while avoiding a drastic change in the procedure used, the proposed approach consisted of creating a general recipe structure and integrating it into the company’s existing documents. production flow-chart was manually transformed into a series of instructions by using procedure templates or a production version of similar products. 5.2 Main findings from process mapping Critical process parameters were linked to equipment settings and sometimes this connection was not easy: critical parameters, indeed, may be measured in different units or refer to parameters not adjustable on the specific equipment. In order to standardize this transformation process, following the ANSI/ISA-88 guidelines, a set of transform components were created for the pilot project. These transform components consisted of a group of reusable tables, containing process parameters and instructions associated to specific equipment, under a series of conditions. These conditions established the link between the general recipe information and the master recipe contents. Once process operations and process actions are defined in the general recipe, and production equipment are selected at the site level, the production site can automatically define the set of instructions and parameters necessary to produce the product, using the transform components. Fewer considerations emerged concerning the master recipe: from the ANSI/ISA-88 perspective, the master recipe should include all the relevant information to manufacture a product on a specific set of equipment. This information was available, spread over different documents, such as the production version and equipment recipe documents. The main problem concerned the division of recipe contents that did not match the ANSI/ISA-88 categories (introduction, header, formula, equipment requirements, procedure, other information). 6. Conclusions The aim of this paper was to describe how an ANSI/ISA- 88 assessment could be easily performed with a dedicated Business Process Management approach and specific methodologies or notations such as BPMN2.0. This approach has been applied to a pilot project carried out in a large multinational pharmaceutical company. In the project, the authors managed to re-design the recipe development process and create a set of templates and libraries to be used as a reference to the ANSI/ISA-88 standard (general recipe, master recipe, process element library, transform components). The most important result of the initiative was the standardization of the NPI process and related documentation. Recently, the company applied the developed approach to the transfer of different products, obtaining a sensible reduction of time spent for the transfer and, at the same time, reducing the need for process experts travelling between the two plants. These results encouraged the company’s management to introduce the developed recipe management process in similar sites by the end of 2014. With regard to the site recipe and the control recipe documents, an ad-hoc solution was proposed, which is to merge both documents, considering that this action would not affect the advantages of adopting the ANSI/ISA-88 standard. This decision was based on an on- going parallel initiative regarding the standardization of material and suppliers. Int J Eng Bus Manag, 2014, 6:16 \| doi: 10.5772/59025 10 www.intechopen.com 5.4 Main findings on the recipe transformation process More considerations can be made concerning the transformation process, which allows the generation of a master recipe, equipment-dependent and site specific, starting from a general recipe, equipment-independent and valid for all production sites. As mentioned previously, the ANSI/ISA-88 master recipe should contain all the relevant information for the manufacturing process on a specific set of equipment. Thanks to the use of process parameters and procedures included in this recipe, the production site is able to start the manufacturing of the new product for characterization, validation, and registration for commercial purposes, according to the steps of the new product introduction process. Within the project, an important lack was revealed, due to the absence of a formalized and standard process. In the as-is scenario each process element of a Despite these encouraging results, as described in the article, there are different pitfalls and criticalities that companies may face during the implementation of this standard. These can be summarized as follows: • Map the end-to-end as-is recipe development process with a BPM notation. • Start from defining a general recipe and a master recipe as standard documents. • Establish continuous education training on ANSI/ISA-88 contents, leveraging on the assessment phases. Int J Eng Bus Manag, 2014, 6:16 \| doi: 10.5772/59025 10 www.intechopen.com • Standardize and make use of defined process elements. • Standardize and make use of defined process elements. [8] Venkatasubramanian, V., Zhao, C., Joglekar, G., Jain, A., Hailemariam, L., Suresh, P., Reklaitis, G. V. (2006). Ontological informatics infrastructure for pharmaceutical product development and manufacturing. Computers & chemical engineering 30 (10): 1482-1496. • Share process element libraries throughout production plants. • Share process element libraries throughout production plants. • Create a library of standard transform components. • Create a library of standard transform components. Typical misalignments and gaps in an ANSI/ISA-88 compliance are also reported in this article as a result of the experience on the specific case. The practical evidence provided can be considered, on one hand as recurrent criticalities that may arise in any ANSI/ISA-88 assessment, and on the other hand as challenges to deal with, in order to obtain the desired benefits. Most opportunities in the adoption of this standard for recipe management process still reside in the possibility of creating executable recipes, applicable to the same equipment of different plants, through a one-to one correspondence of process actions to equipment phases. 7. References [13] Tommila, T., Ventä, O., Koskinen, K. (2001). 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